Basic Microbiology

Microbiology is the branch of science that deals with microscopic organisms and their interaction with other microscopic
and macroscopic organisms.
 Microorganisms are tiny microscopic organisms that are too small to be seen with naked eyes and thus, can only be seen
with a microscope. Microorganisms include microscopic organisms like bacteria, fungi, archaea, protozoa, and viruses.
 Basic microscopy deals with a diverse group of studies that help in research related to the biochemistry, physiology, cell
biology, ecology, evolution, and clinical aspects of microorganisms, including the host response to these agents.
 Microbiology also deals with the structure, function, classification of such organisms, along with exploiting and controlling
their activities.
 The concept of microbiology began with the discovery of the microscope by Anton Von Leeuwenhoek.
 On the one hand, microbes are used for their unique features which allow the production of antibiotics, amino acids,
hormones, and other therapeutic compounds, and also the production of food and food-related products.
 Microorganisms are also involved in the decomposition of components such as lignocellulosic biomass for second-
generation ethanol or biogas.
 Similarly, certain genetic features and biochemical abilities of microorganisms make them dangerous for industry (food
spoilage) as well as human health.
 Microbiology, initially, was only associated with pathogenic microorganisms that result in different forms of diseases in
different groups of living beings.
 With the establishment of microbiology as a discipline, the application of microorganisms in different areas has also
increased.
 The use of microorganisms in food and pharmaceuticals has given rise to the branching of microbiology into further
disciplines and studies.
 Thus, over the years, the branch has been classified into further groups like agriculture microbiology, food microbiology,
pharmaceutical microbiology, systemic microbiology, etc.
 The studies in microbiology have also increased due to the use of microorganisms in different researches as these are
easy to manipulate and reproduce when compared to other living organisms.
 Studies in microbiology are essential for the discovery of new and advanced methods for the discovery of emerging
microorganisms and associated diseases and applications.
 Microbiology also deals with techniques for the identification of these microorganisms, their classification, and the life
cycle.
 All of this allows for a better understanding of microorganisms and their role in maintaining the ecosystem.
 Microbiology and microorganisms can be applied for the formation of new genetically engineered microorganisms by a
process like genetic recombination.
 Besides, different microorganisms have found their application in the production of food, industrial products, and
antibiotics.

Biosafety Cabinets Definition

Biosafety Cabinets (BSCs) are enclosed workspaces with a ventilated hood that is designed to contain pathogenic microorganisms
during microbiological processes.

 The primary purpose of biosafety cabinets is to protect the laboratory personnel and the environment from the pathogenic
microorganism as aerosols might be formed during the processing of such microorganisms.
 Biosafety cabinets are only used for certain risk group organisms and for processes that might result in aerosol formation.
 These cabinets are provided with HEPA-filters that decontaminate the air moving out of the cabinet.
 Biosafety cabinets might be confused with the laminar hood as both of these pieces of equipment work as enclosed
workspaces. But, laminar hood only provides protection to the sample and not to the personnel and the environment,
whereas biosafety cabinets protect all three.
 The use of biosafety cabinets or other such physical containment is not required in the biosafety level 1, but depending on
the risk assessment, some processes might require such containment.
 BSCs are an essential part of biosafety as they minimize the formation of aerosol, protecting the environment, the pathogen,
and the laboratory personnel.
 Besides, most BSCs also function to sterilize biological materials that are kept inside the cabinets.
Biosafety Cabinet Classes

Figure: Biosafety Cabinets. Image Source: Pro-Lab Diagnostics.

 Biosafety cabinets are classified into three classes by the U.S. Centers for Disease Control and Prevention (CDC), each with
specific performance characteristics and applications.
 Class I and II Biosafety cabinets are used for Biosafety levels I and II but, when used correctly in conjunction with useful
microbiological techniques, these provide an effective containment system for safe manipulation of moderate and high-risk
microorganisms.
 Class III BSCs are most suitable for work with hazardous agents that require Biosafety Level 3 or 4.
1. Biosafety Cabinet Class I
 Class I is the most basic biosafety cabinet that provides protection to the environment and the laboratory personnel.
 It doesn’t, however, provide protection to the product as the unsterilized room air is drawn over the work surface.
 Class I biosafety cabinets are typically used to either enclose specific equipment like centrifuges or for procedures like
aerating cultures that might potentially generate aerosols.
 Biosafety cabinets of this class are either ducted (connected to the building exhaust system) or unducted (recirculating
filtered exhaust back into the laboratory).
 In the Class I BSC, the room air is drawn in through the opening that also allows the entry of the operator’s arm during work.
 The air inside the cabinet then takes in the aerosol particles that may have been generated and moves it away from the
operator towards the HEPA filter.
 The air moving out of the cabinet is thus, sterilized via the HEPA filters before its discharge to the environment.
 In this way, the cabinets protect the operator and the environment from the aerosol but not the sample.

2. Biosafety Cabinet Class II

 BSC-Class II cabinets provide both kinds of protection (of the samples and the environment) since makeup air is also HEPA-
filtered.
 The principle of operation of Class II cabinets involves a fan mounted in the top of the cabinet that draws a curtain of sterile
air over the workstation where the biological products are being handled.
 The air then moves underneath the work station and back up to the top of the cabinet before passing through the HEPA
filters.
 The exhaust that moves out of the facility consists of air being drawn into the front of the cabinet underneath the work
surface.
 The air drawn in acts as a barrier against the potentially contaminated air coming back out to the operator.
  Class II BSCs are further divided into five types depending on the exhaust system and the mechanism of work (recirculation
of the exhaust air); Type A1, Type A2, Type B1, Type B2, and Type C1.
a. Type A1
 The type A1 cabinets have a minimum inflow velocity of 75ft/min where the contaminated divided just above the work station
and mixes with the inflow air.
 The mixed air is then drawn through a duct network so that it reaches the back of the cabinet.
 After this, air might be either recirculated after passing through the HEPA filters or exhausted out of the cabinet, also through
a HEPA filter.
 This type of cabinet is not as widely used as it is not safe to work with hazardous chemical substances.
b. Type A2
 The type A2 cabinets have a minimum inflow velocity of 100 ft/min.
 In Class II, Type A2 BSC air enters the chamber through the front aperture, which provides operator protection.
 The inflow air mixes with the downflow air (from the top of the cabinet)and enters the front intake grille and then passes over
the workstation where the air splits.
 Approximately 60% to 70% of the contaminated air is recycled and pushed back into the workstation in the chamber through
the downflow HEPA filter, while the remaining 30% to 40% is exhausted through the exhaust HEPA filter.
 However, if hazardous, volatile chemicals are to be used within the cabinet, along with the microbiological work, exhaust
must be released into the atmosphere through the direct duct system.
 Because of the chances of the release of hazardous chemicals into the environment, A2 type cabinets are also not
extensively used.
c. Type B1
 Type B cabinets are different from Type A cabinets as they use single-pass airflow to control the flow of hazardous vapors.
 Type B1 cabinets divide the airflow so that the contaminated air is directed towards the exhaust system while the air
between the operator and the workstation mixes with the inflow and is recirculated.
 The exhaust air dispersed out of the facility should be passed through the HEPA filters to provide protection to the
environment.
 These cabinets have a dedicated duct system which allows the release of the contaminated air out of the facility.
 In the case of Type B1 cabinets, 40% of the air is recirculated, whereas the remaining 60% is exhausted out of the facility.
d. Type B2
 For a Type B2 BSC, like in Type A cabinets, the air is drawn in from the front opening creating an air barrier that protects the
operator.
 Air is also drawn in from an opening at the top of the cabinet that supplies the downflow of air in the cabinet.
 The air then passes through a HEPA filter, where 100% of the air is exhausted through a dedicated duct system with an
exhaust fan motor. The air moving out of the facility is thus sterilized before its release into the atmosphere.
 The advantage of this system is the removal of toxic vapors that are generated in the cabinet with no recirculation within the
BSC.
 All of the contaminated airflow (100%) in a Type B2 cabinet is externally exhausted which means the air drawn into the
cabinet is 100% exhausted into the atmosphere.
 As a result, none of the air drawn into the B2 from either inflow or downflow is recycled within the airflow system.
 Because none of the air is recirculated, these cabinets are the best to be used for tasks involving the release of chemical
vapors.
 Type B2 cabinets, however, are expensive, and their use is limited to toxicology laboratories where protection against
hazardous chemicals is imperative.
e. Type C1
 Type C1 cabinets are similar to Type B cabinets in their working mechanism, but these are designed to reduce operating
costs add flexibility to the laboratories.
 These cabinets work by using the single-pass airflow system where the cabinets move the air by mixing it with the downflow
air separated into columns for recirculation.
 The air above the workstation is drawn with a second fan which moves the contaminated air out through the exhaust system
with a HEPA filter.
 In this way, the cabinets provide protection to the environment, the operator, and the workstation or the biological material.
 Type C cabinets are different from Type A cabinets as they use a single-pass airflow mechanism where the air is not
circulated.
 These differ from Type B cabinets in that they don’t require a dedicated ducted exhaust system, can work for an extended
duration to increase operator protection in the case of exhaust failure, and can even run without the exhaust at all.
3. Biosafety Cabinet Class III
 Class III cabinets are leak-tight, totally enclosed but ventilated cabinets, where all air that either enters or leaves through the
facility pass through a HEPA filter.
 The cabinets are provided with rubber gloves that are attached to the system to be used during operations in the cabinet.
This is why these cabinets are also termed ‘glove boxes’.
 The cabinet even has a transfer chamber that facilitates the sterilization of materials before they leave the glove box.
 Even though the gloves restrict the hand movement of the operator inside the cabinet, it prevents direct contact between the
operator and the samples.
 The exhaust air is treated with double HEPA filters or HEPA filters in combination with incineration.
 These cabinets can be used for all four Biosafety levels (1, 2, 3, and 4). But these are the most important for the
manipulation of biological materials in the Biosafety level 4.
 These cabinets are mostly custom-built for specific laboratories with lab equipment built inside the chamber.
 All of these structural and design features provide maximum protection to the operator, the environment, and the sample
against the high-risk group 4 pathogenic organisms.

Definition
A Laminar flow hood/cabinet is an enclosed workstation that is used to create a contamination-free work environment
through filters to capture all the particles entering the cabinet.
 These cabinets are designed to protect the work from the environment and are most useful for the aseptic distribution of
specific media and plate pouring.
 Laminar flow cabinets are similar to biosafety cabinets with the only difference being that in laminar flow cabinets the effluent
air is drawn into the face of the user.
 In a biosafety cabinet, both the sample and user are protected while in the laminar flow cabinet, only the sample is protected
and not the user.

Image Source: Laboratory-supply.net and ProfiLab24. Created with Biorender.com.

Components/ Parts of Laminar flow hood

A laminar flow cabinet consists of the following parts:

1. Cabinet
 The cabinet is made up of stainless steel with less or no gaps or joints preventing the collection of spores.
 The cabinet provides insulation to the inner environment created inside the laminar flow and protects it from the outside
environment.
  The front of the cabinet is provided with a glass shield which in some laminar cabinets opens entirely or in some has two
openings for the user’s hands to enter the cabinet.
2. Working station
 A flat working station is present inside the cabinet for all the processes to be taken place.
 Culture plates, burner and loops are all placed on the working station where the operation takes place.
 The worktop is also made up of stainless steel to prevent rusting.
3. Filter pad/ Pre-filter
 A filter pad is present on the top of the cabinet through which the air passes into the cabinet.
 The filter pad traps dust particles and some microbes from entering the working environment within the cabinet.
4. Fan/ Blower
 A fan is present below the filter pad that sucks in the air and moves it around in the cabinet.
 The fan also allows the movement of air towards the HEPA filter sp that the remaining microbes become trapped while
passing through the filter.
5. UV lamp
 Some laminar flow hoods might have a UV germicidal lamp that sterilizes the interior of the cabinet and contents before the
operation.
 The UV lamp is to be turned on 15 minutes before the operation to prevent the exposure of UV to the body surface of the
user.
6. Fluorescent lamp
 Florescent light is placed inside the cabinet to provide proper light during the operation.
7. HEPA filter
 The High-efficiency particulate air filter is present within the cabinet that makes the environment more sterile for the
operation.
 The pre-filtered air passes through the filter which traps fungi, bacteria and other dust particles.
 The filter ensures a sterile condition inside the cabinet, thus reducing the chances of contamination.

Principle/ Working of Laminar flow hood

Image Source: Laboratory-supply.net
 The principle of laminar flow cabinet is based on the laminar flow of air through the cabinet.
 The device works by the use of inwards flow of air through one or more HEPA filters to create a particulate-free environment.
 The air is taken through a filtration system and then exhausted across the work surface as a part of the laminar flow of the
air.
 The air first passes through the filter pad or pre-filter that allows a streamline flow of air into the cabinet.
 Next, the blower or fan directs the air towards the HEPA filters.
 The HEPA filters then trap the bacteria, fungi and other particulate materials so that the air moving out of it is particulate-free
air.
 Some of the effluent air then passes through perforation present at the bottom rear end of the cabinet, but most of it passes
over the working bench while coming out of the cabinet towards the face of the operator.
  The laminar flow hood is enclosed on the sides, and constant positive air pressure is maintained to prevent the intrusion of
contaminated external air into the cabinet.

Procedure for running the laminar flow cabinet

The procedure to be followed while operating a laminar flow cabinet is given below:
1. Before running the laminar flow cabinet, the cabinet should be checked to ensure that nothing susceptible to UV rays is
present inside the cabinet.
2. The glass shield of the hood is then closed, and the UV light is switched on. The UV light should be kept on for about 15
minutes to ensure the surface sterilization of the working bench.
3. The UV light is then switched off, and a time period of around 10 minutes is spared before the airflow is switched on.
4. About 5 minutes before the operation begins, the airflow is switched on.
5. The glass shield is then opened, and the fluorescent light is also switched on during the operation.
6. To ensure more protection, the working bench of the cabinet can be sterilized with other disinfectants like 70% alcohol.
7. Once the work is completed, the airflow and florescent lamp both are closed and the glass shield is also closed.

Types of laminar flow cabinet

Depending on the direction of movement of air, laminar flow cabinets are divided into two types:

1. Vertical laminar flow cabinet

 In the vertical flow cabinets, the air moves from the top of the cabinet directly towards the bottom of the cabinet.
 A vertical airflow working bench does not require as much depth and floor space as a horizontal airflow hood which makes it
more manageable and decreases the chances of airflow obstruction or movement of contaminated air downstream.
 The vertical laminar flow cabinet is also considered safer as it doesn’t blow the air directly towards the person carrying out
the experiments.
2. Horizontal laminar flow cabinet
 In the horizontal laminar flow cabinets, the surrounding air comes from behind the working bench, which is then projected by
the blower towards the HEPA filters.
 The filtered air is then exhausted in a horizontal direction to the workplace environment.
 One advantage of this cabinet is that airflow parallel to the workplace cleanses the environment with a constant velocity.
 The elluent air directly hits the operator, which might reduce the security level of this type of laminar flow cabinets.

Uses of Laminar flow hood

The following are some common uses of a laminar flow cabinet in the laboratory:
1. Laminar flow cabinets are used in laboratories for contamination sensitive processes like plant tissue culture.
2. Other laboratories processes like media plate preparation and culture of organisms can be performed inside the cabinet.
3. Operations of particle sensitive electronic devices are performed inside the cabinet.
4. In the pharmaceutical industries, drug preparation techniques are also performed inside the cabinet to ensure a particulate-
free environment during the operations.
5. Laminar flow cabinets can be made tailor-made for some specialized works and can also be used for general lab techniques
in the microbiological as well as the industrial sectors.

Precautions
While operating the laminar airflow, the following things should be considered:
1. The laminar flow cabinet should be sterilized with the UV light before and after the operation.
2. The UV light and airflow should not be used at the same time.
3. No operations should be carried out when the UV light is switched on.
4. The operator should be dressed in lab coats and long gloves.
5. The working bench, glass shield, and other components present inside the cabinet should be sterilized before and after the
completion of work.

Definition and Introduction

 Biosafety is the prevention of risk to human health and safety, and the conservation of the environment and the pathogen, as
a result of the use for research and commerce of infectious or genetically modified organisms.
 Biosafety is an important concept in microbiology as bio-related research activities may involve manipulation of microbial,
animal, or plant cells which might potentially be pathogenic.
 The risks associated with the laboratory activities occur either from the samples or the procedural requirements.
 Application to standard microbiological techniques and employing facilities suitable for the risk level of the pathogen helps to
protect the researcher from laboratory-acquired infections.
 The biosafety levels are thus designed to identify various protective measures that are to be taken in a laboratory setting to
protect the researchers, the environment, and the microorganisms.
  These levels are defined by the Central for Disease Control and Prevention (CDC), where each of these levels is outlined
with specific practices and safety requirements.
 Biosafety level designations are based on the combination of the design features, equipment, practices, and procedures
required while working with agents from the various risk groups.
 The allocation of a pathogenic agent to a biosafety level for laboratory work must be based on the risk assessment.
 Such assessments take the risk group as well as other factors into consideration while establishing the appropriate biosafety
level. The biosafety levels, thus, might differ from one region to another.
 As per the CDC, biosafety levels are of four types depending on the risk associated with the microorganism and the facilities
available. The levels of containment range from the biosafety level 1 (BSL-1), which is the lowest to the level 4 (BSL-4),
which is the highest.

1. Biosafety Level -1 (BSL-1)

 Biosafety Level 1 is the level appropriate for work involving well-characterized agents not known to consistently cause
disease in immune-competent adult humans and cause a minimal potential hazard to the laboratory personnel and the
environment.
 Biosafety level 1 is the lowest safety level, and the precautions required for the level are thus limited and not as extensive.
 These laboratories provide general space in which work is done with viable agents that are not associated with disease in
healthy adults.
Requirements
 The BSL-1 laboratories are not necessarily separated from the general traffic in the building.
 Most of the work is typically conducted on open bench tops using general microbiological practices.
 Unique laboratory design or containment equipment are not required but may be used depending on the risk assessment.
 Laboratory personnel must be provided with specific training in the procedures to be conducted in the laboratory, which is
then supervised by a scientist with training in microbiology or related sciences.
The following are the standard practices, safety equipment, and facility requirements required in BSL-1:

Standard microbiological practices

 The laboratory supervisor should implement the policies regarding the access control to the laboratory.
 Laboratory personnel must wash their hands after working with potentially hazardous materials and before leaving the
laboratory.
 Activitttiiies like eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food are not be permitted
in laboratory areas.
 Mouth pipetting is prohibited; mechanical pipetting devices must be employed.
 All procedures to be conducted in the laboratory should be performed while avoiding the creation of splashes and aerosols.
 The work surface like the benchtops should be disinfected after work and after any spill of potentially hazardous biological
material.
  The supervisor must ensure that all the laboratory personnel acquires appropriate training and necessary precautions while
performing their tasks.
Safety practices
 There are no safety specific safety practices required for BSL-1.
Safety equipment
 Special containment devices like the Bio-safety Cabinets are not required for BSL-1.
 In order to prevent the contamination of personal clothing, protective laboratory coats, gowns, or uniforms are
recommended.
 While conducting tests with a high possibility of aerosol formation, protective eyewear can be used.
Uses
 Biosafety Level-1 is commonly used while performing tests on microbial agents that are not known to cause diseases in
immune-compromised individuals.
 These laboratories include the laboratories used for teaching purposes in colleges and training centers.
Organisms
 The common organisms that require Biosafety Level-1 containment include less hazardous organisms like Agrobacterium
radiobacter, Aspergillus niger, Bacillus thuringiensis, Escherichia coli strain K12, Lactobacillus acidophilus, Micrococcus
leuteus, Neurospora crassa, Pseudomonas fluorescens, Serratia marcescens.
 However, the requirement of the biosafety level might differ depending on the risk assessment of the pathogen.

2. Biosafety Level-2 (BSL-2)
 Biosafety level-2 laboratories are the laboratories that are used for the tasks involving microbial agents of moderate potential
hazards to the laboratory personnel, the environment, and the agent.
 However, the infectious agents or the toxins might pose a moderate danger if accidentally inhaled, swallowed, or exposed to
the skin.
 The precautions associated with biosafety level-2 are comparatively more extensive than BSL-1, but BSL-1 and BSL-2
laboratories are generally considered as basic laboratories.
Requirements
 BSL-2 laboratories like BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building.
 However, access into the laboratory is limited while BSL-2 experiments are in progress.
 The annual inspection of the laboratories is also an important part of the BSL-2 requirements. These might include changing
the filters or replacement of some devices.
 The work is mostly conducted on sterilized bench tops except for some processes that might form aerosols. The latter is
conducted in safety cabinets.
 The precautions to be followed in BSL-2 include all the precautions of the BSL-1 and some additional precautions.
Standard microbiological practices
 All the laboratory personnel must wash their hands after using viable microorganisms and before leaving the laboratory.
 Eating, drinking, smoking, and handling contact lenses in the laboratory are strictly prohibited.
 Mechanical pipetting should be done instead of mouth pipetting.
 All contaminated cultures, glassware, plastic ware, and biologically contaminated waste must be treated as bio-hazards and
thus, autoclaved.
 Work surfaces must be decontaminated with disinfectant at the end of the day or after any spills or splashes.
 Used hypodermic syringes and needles, Pasteur pipettes, razor blades, contaminated broken glass, and blood vials are
treated as medical waste and discarded in puncture-resistant sharps disposal containers.
Safety Practices
 People with increased risk of acquiring infections like the immune-compromised and pregnant individuals should not be
allowed to enter the BSL-2 laboratories while the laboratories are at work.
 An annual review of the BSL-2 manual should be done to update the guidelines.
 Documented policies and procedures should be established that limit the entrance to individuals who know of the potential
hazards and are appropriately trained.
 A biohazard symbol is placed on pieces of equipment where biohazardous materials are used or stored.
Safety Equipment
 Protective coats are to be worn while entering the laboratory and then removed and kept in the laboratory post work.
 The laboratory design should be made such that it can be easily cleaned and decontaminated with minimum nooks and
corners.
  The laboratory doors should be closed whenever work with hazardous biomaterials is conducted.
 An autoclave must be available.
Uses
 Biosafety level-2 laboratories are mostly used for routine analysis and culture of moderately hazardous agents.
 Besides, some of the laboratories used for teaching and training purposes are also BSL-2 laboratories.
Organisms
 The organisms that require BSL-2 laboratories include the pathogenic strains of E.
coli, Staphylococcus, Salmonella, Plasmodium falciparum, Toxoplasma, and Herpes Simples Viruses.
 The allocation of organisms to the laboratories, however, might differ depending on the risk assessment.

3. Biosafety Level-3 (BSL-3)
 Biosafety level 3 (BSL-3) is the level where work is performed with agents that may cause severe or potentially lethal
disease through inhalation or aerosol formation, to the personnel, and may even contaminate the environment.
 The tasks performed in the BSL-3 laboratories involve indigenous or exotic agents where the potential for infection by
aerosols is high, and the disease may have lethal consequences.
 Autoinoculation and ingestion present primary hazards to personnel working with these agents at this level.
 Working in such laboratories require laboratory personnel with specific training in handling pathogenic and potentially lethal
agents, along with supervisors scientists competent in handling infectious agents and associated procedures.
Requirements
 Biosafety Level 3 containment laboratories for animals and research are the most challenging containment level facilities to
design and operate.
 These laboratories should be certified for use before initial operation and subsequently on an annual schedule or after a
program change, renovation, or replacement of system components that may affect the operating environment of the
laboratory.
 BSL-3 laboratories are also called the containment laboratory as they require containment equipment to protect the
personnel, the microbial agent, and the environment.
 The requirements for BSL-3 include all the requirements of the BSL-1 and BSL-2 laboratories, along with some additional
design features and special equipment.
Standard Microbiological Practices
 The entry to the BSL-3 laboratories is limited to individuals with appropriate training in handling BSL-3 organisms, all of
whom are selected by the laboratory supervisor.
 Besides the general procedures and laboratory practices, the supervisor also formulates additional policies to limit the entry
to the laboratory.
 All the procedures to be conducted in the BSL-3 must be conducted within a biosafety cabinet to prevent the exposure of the
aerosols to the laboratory personnel.
 Personnel working in the laboratory must wear personal protective equipment before entering the laboratory and then
remove them before leaving.
 The work surfaces and sinks should be decontaminated once every work shift or after any spills or splashes.
 The BSL-3 laboratories should be separated from the general traffic in a building to limit entry into the laboratories at all
times.
Safety Practices
 The doors of the BSL-3 laboratories are closed at all times with appropriate BSL-3 signs outside the suite, along with a
universal biohazard sign and emergency contact information.
 Laboratory personnel must have medical surveillance and offered appropriate immunizations for agents handled or
potentially present in the laboratory.
 Each institution should consider the collection and storage of serum samples from at-risk personnel.
 A laboratory-specific biosafety manual, which is available and accessible to all, must be prepared and adopted as a policy.
 The laboratory supervisor must check for the demonstration of proficiency in standard and special microbiological practices
by all laboratory personnel before working with BSL-3 agents.
 Potentially hazardous materials must be placed in a durable, leak-proof container or vial during collection, processing,
storage, or transport within a facility.
 All laboratory equipment should be routinely decontaminated after work or after any spills or splashes.
 The laboratory biosafety manual must define procedures t be adopted in the case of exposure to infectious materials, and
these should be treated accordingly.
 No work in the BSL-3 laboratories should be conducted on an open bench or an open vessel. All the activities involving the
infectious agents must be conducted within Biosafety cabinets or other physical containment devices.
Safety Equipment
 Biosafety cabinets are to be used for the manipulation of all infectious agents.
 Individual protection gears like personal protective equipment, coats, gloves, and respiratory protection should be worn while
entering the laboratories and then removed before leaving.
 The air flowing in the laboratory shouldn’t be recirculated to any area of the laboratory and should be HEPA-filtered prior to
being discharged to the outside.
 The filters, manuals, equipment, vacuum pipes, autoclaves, etc. should be revised and reviewed annually.
Uses
 BSL-3 laboratories are used for clinical, diagnostic, teaching, research, or production facilities.
 These laboratories are used for the handling and manipulation of highly infectious agents that prose direct severe effects on
the health of the personnel.
 These are used for the studies regarding the effects of infectious agents and various toxins and their effects.
Organisms
 The pathogens that require BSL-3 laboratories include HIV, H1N1 flu, Yersinia pestis, Mycobacterium tuberculosis,
SARS, Rabies Virus, West Nile Virus, Ricketts, etc.
 The placement of the organisms in different Biosafety levels, however, might defer and should also be determined after risk
assessment.

4. Biosafety Level 4 (BSL-4)

 Biosafety level 4 is the highest level that is employed while working with dangerous infectious agents that present a high
individual as well as environmental risk in the form of life-threatening disease, aerosol transmission, or unknown risk of
transmission.
 The BSL-4 laboratories are often used while handling and manipulating Risk Group 4 pathogens that are extremely
dangerous, with no known vaccines or therapies, and require extreme precautions during work.
 The BSL-4 laboratories are of two types; cabinet laboratory where all the work is performed in a Class III biosafety cabinet or
similar physical containment with very carefully formulated precautions and suit laboratory where all the laboratory personnel
are required to wear full-body, air-supplied suits protective gears in the form of PPEs.
Requirements
 The requirements of BSL-4 laboratories are extensive with specific laboratory design, training procedures, and highly
protective equipment and personal gears.
 These laboratories should be certified for use before initial operation and subsequently on an annual schedule or after a
program change, renovation, or replacement of system components that may affect the operating environment of the
laboratory.
 BSL-4 laboratories are also termed the maximum containment laboratories as they have secondary barriers to prevent
hazardous materials from escaping into the environment.
 The BSL-4 laboratories should follow the requirements of all BSL-1, BSL-2, and BSL-3, along with additional specific
precautions.
Standard microbiological practices
 No work conducted within the BSL-4 should be done on an open bench or an open vessel.
 The work stations, equipment, and sinks should be sterilized post work.
 The laboratory personnel should be in protective gear that might include full-body PPEs, gloves, masks, and coats.
 The doors of the laboratories should be closed at all times with the laboratory placed away from the general traffic in the
building.
 Activities like drinking, eating, mouth pipetting should be avoided at all costs.
 Only people that are trained in handling the BSL-4 organisms and the equipment in the laboratory should be allowed into the
laboratory.
Safety Practices
 Viable or intact biological materials to be removed from the Class III cabinet in a BSL-4 are transferred in a nonbreakable,
sealed primary container with a nonbreakable, sealed secondary container.
 No materials, except the biological materials that are to remain in a viable or intact state, are removed from the BSL-4
laboratory unless they have been autoclaved or decontaminated before they leave the facility.
 Only individuals whose presence in the facility is required for microbiological processes or support purposes are authorized
to enter. Individuals that are at increased risk of acquiring an infection or for whom infection may be unusually hazardous are
not allowed in the laboratory.
 Personnel can enter and leave the facility only after the clothing change and through the shower rooms.
 When the BSL-4 laboratory is at work or when infectious materials or infected animals are present in the laboratory, a hazard
warning sign, along with the universal biohazard symbol, is placed on all access doors.
  A system is set up for reporting laboratory accidents, exposures, and the medical surveillance of potential laboratory-
associated illnesses.
Safety equipment
 A Class III biological safety cabinet or Class I or II biological safety cabinets used in conjunction with one-piece personnel
suits ventilated by a life support system are to be present in a BSL-4 while conducting all procedures within the facility.
 Walls, floors, and ceilings of the laboratories must form a sealed internal shell which facilitates fumigation and is animal and
insect-proof.
 A double-doored autoclave is placed for decontaminating materials passing out of the facility.
 The exhaust air from the facility is filtered through HEPA filters before being discharged to the outside so as to prevent its
entry into occupied buildings and air intakes.
Uses
 BSL-4 laboratories are used for diagnostic and research work on easily transmitted pathogens, causing fatal diseases.
 These laboratories are used for new and unknown pathogenic microbes, for which no vaccines or therapies are available.
 They are also used for clinical and production facilities that require highly sophisticated techniques and advanced processes.
Organisms
 The BSL-4 level pathogens include the risk group IV organisms like Ebola virus, SARS-CoV-2, Central European
Encephalitis virus, Hemorrhagic viruses, etc.

Autoclave Definition
An autoclave is a machine that provides a physical method of sterilization by killing bacteria, viruses, and even spores present in
the material put inside of the vessel using steam under pressure.
 Autoclave sterilizes the materials by heating them up to a particular temperature for a specific period of time.
 The autoclave is also called a steam sterilizer that is commonly used in healthcare facilities and industries for various
purposes.
 The autoclave is considered a more effective method of sterilization as it is based on moist heat sterilization.

Autoclave Parts/ Components

The simplest form of the autoclave is the pressure cooker types or laboratory bench autoclaves. The following is the detailed
description of different components/ parts of an autoclave:
Figure: Autoclave Parts or Components. Image Source: pharmawiki.
a. Pressure Chamber
 The pressure chamber is the main component of a steam autoclave consisting of an inner chamber and an outer jacket.
 The inner chamber is made up of stainless steel or gunmetal, which is present inside the out chamber made up of an iron
case.
 The autoclaves used in healthcare laboratories have an outer jacket that is filled with steam to reduce the time taken to
reach the sterilization temperature.
 The inner chamber is the case where the materials to be sterilized are put.
 The size of the pressure chamber ranges from 100 L to 3000 L.
b. Lid/ Door
 The next important component of an autoclave is the lid or door of the autoclave.
 The purpose of the lid is to seal off the outside the atmosphere and create a sterilized condition on ht inside of the autoclave.
 The lid is made airtight via the screw clamps and asbestos washer.
 The lid consists of various other components like:
Pressure gauge
 A pressure gauge is present on the lid of the autoclave to indicate the pressure created in the autoclave during sterilization.
 The pressure gauge is essential as it assures the safety of the autoclave and the working condition of the operation.
Pressure releasing unit/ Whistle
 A whistle is present on the lid of the autoclave is the same as that of the pressure cooker.
 The whistle controls the pressure inside the chamber by releasing a certain amount of vapor by lifting itself.
Safety valve
 A safety valve is present on the lid of autoclave, which is crucial in cases where the autoclave fails to perform its action or
the pressure inside increases uncontrollably.
 The valve has a thin layer of rubber that bursts itself to release the pressure and to avoid the danger of explosion.
c. Steam generator/ Electrical heater
 An electrical steam generator or boiler is present underneath the chamber that uses an electric heating system to heat the
water and generate steam in the inner and the outer chamber.
  The level of water present in the inner chamber is vital as if the water is not sufficient; there are chances of the burning of the
heating system.
 Similarly, if the water is more than necessary, it might interfere with the trays and other components present inside the
chamber.
d. Vacuum generator (if applicable)
 In some types of autoclaves, a separate vacuum generator is present which pulls out the air from the inside of the chamber
to create a vacuum inside the chamber.
 The presence of some air pockets inside the chamber might support the growth of different microorganisms. This is why the
vacuum chamber is an important component of an autoclave.
e. Wastewater cooler
 Many autoclaves are provided with a system to cool the effluent before it enters the draining pipes.
 This system prevents any damage to the drainage pipe due to the boiling water being sent out of the autoclave.
Autoclave Principle/ Working

Figure: Autoclave Principle or Working. Image Source: http://a.2002-acura-tl-radio.info/page-b/autoclave-diagram-48245.html

 The autoclave works on the principle of moist heat sterilization where steam under pressure is used to sterilize the material
present inside the chamber.
 The high pressure increases the boiling point of water and thus helps achieve a higher temperature for sterilization.
 Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg); however, the boiling point of water
increases if the pressure is to be increased.
 Similarly, the high pressure also facilitates the rapid penetration of heat into deeper parts of the material, and moisture
present in the steam causes the coagulation of proteins causing an irreversible loss of function and activity of microbes.
 This principle is employed in an autoclave where the water boils at 121°C at the pressure of 15 psi or 775 mm of Hg.
 When this steam comes in contact on the surface, it kills the microbes by giving off latent heat.
 The condensed liquid ensures the moist killing of the microbes.
 Once the sterilization phase is completed (which depends on the level of contamination of material inside), the pressure is
released from the inside of the chamber through the whistle.
  The pressure inside the chamber is then restored back tot eh ambient pressure while the components inside remain hot for
some time.

Procedure for running an autoclave

In general, an autoclave is run at a temperature of 121° C for at least 30 minutes by using saturated steam under at least 15 psi of
pressure. The following are the steps to be followed while running an autoclave:
1. Before beginning to use the autoclave, it should be checked for any items left from the previous cycle.
2. A sufficient amount of water is then put inside the chamber.
3. Now, the materials to be sterilized are placed inside the chamber.
4. The lid is then closed, and the screws are tightened to ensure an airtight condition, and the electric heater is switched on.
5. The safety valves are adjusted to maintain the required pressure in the chamber.
6. Once the water inside the chamber boils, the air-water mixture is allowed to escape through the discharge tube to let all the
air inside to be displaced. The complete displacement can be ensured once the water bubbles cease to come out from the
pipe.
7. The drainage pipe is then closed, and the steam inside is allowed to reach the desired levels (15 lbs in most cases).
8. Once the pressure is reached, the whistle blows to remove excess pressure from the chamber.
9. After the whistle, the autoclave is run for a holding period, which is 15 minutes in most cases.
10. Now, the electric heater is switched off, and the autoclave is allowed to cool until the pressure gauge indicates the pressure
inside has lowered down to that of the atmospheric pressure.
11. The discharge pipe is then opened to allow the entry of air from the outside into the autoclave.
12. Finally, the lid is opened, and the sterilized materials are taken out of the chamber.

Types of autoclave
There are different types of autoclaves present in the market, some of which are:

Pressure cooker type/ Laboratory bench autoclaves (N-type)

 These, as domestic pressure cookers, are still in use in many parts of the world.
 The more modern type has a metal chamber with a secure metal lid that can be fastened and sealed with a rubber gasket.
 It has an air and steam discharge tap, pressure gauge, and safety valve. There is an electric immersion heater in the bottom
of the chamber.
Gravity displacement type autoclave
 This is the common type of autoclave used in laboratories.
 In this type of autoclave, the steam is created inside the chamber via the heating unit, which then moves around the
chamber for sterilization.
 This type of autoclave is comparatively cheaper than other types.
Positive pressure displacement type (B-type)
 In this type of autoclave, the steam is generated in a separate steam generator which is then passed into the autoclave.
 This autoclave is faster as the steam can be generated within seconds.
 This type of autoclave is an improvement over the gravity displacement type.
Negative pressure displacement type (S-type)
 This is another type of autoclave which contains both the steam generator as well as a vacuum generator.
 Here, the vacuum generator pulls out all the air from inside the autoclave while the steam generator creates steam.
 The steam is then passed into the autoclave.
 This is the most recommended type of autoclave as it is very accurate and achieves a high sterility assurance level.
 This is also the most expensive type of autoclave.
Uses of autoclave
Autoclaves are important devices to ensure the sterilization of materials containing water as they cannot be sterilized by dry heat
sterilization. Besides, autoclaves are used for various other purposes.
1. They are used to decontaminate specific biological waste and sterilize media, instruments, and labware.
2. Regulated medical waste that might contain bacteria, viruses, and other biological materials are recommended to be
inactivated by autoclaving before disposal.
3. In medical labs, autoclaves are used to sterilize medical equipment, glassware, surgical equipment, and medical wastes.
4. Similarly, autoclaves are used for the sterilization of culture media, autoclavable containers, plastic tubes, and pipette tips.

Precautions
Although autoclaves are pretty simple to use, there are certain rules of precautions to be followed while operating an autoclave.
Some of the important precautions to be followed while running an autoclave are:
1. Autoclaves should not be used to sterilize water-proof or water-resistant substances like oil or powders.
2. The autoclave should not be overcrowded, and the materials should be loaded in a way that ensures sufficient penetration of
articles by the steam.
3. TheThe items to be autoclaved should always be placed in a secondary container.
4. Only autoclavable bags are to be used to autoclave packaged waste.
5. To ensure sufficient penetration, articles should be wrapped in something that allows penetration by steam, and materials
like aluminum foils should not be used.
6. The items placed inside the chamber should not touch the sides or top of the chamber.
7. The wastes and clean items should be autoclaved separately.
8. Attempts to open the lid when the autoclave is working should never be made.
9. Liquid components should never be autoclaved in sealed containers.
10. The liquid inside the containers should only be filled 2/3rd of the total volume to prevent the spilling of the liquid.
11. Plastic or polyethylene trays or containers should not be used as they might melt and damage the autoclave.
12. Besides, never autoclave flammable, reactive, corrosive, toxic or radioactive materials, household bleach, or paraffin-
embedded tissue.
13. The paper should not be placed directly inside an autoclave as it is a combustible substance. It should be autoclaved in a
waste bag on a bio bag setting to prevent fire.

Incubator Definition
Incubator, in microbiology, is an insulated and enclosed device that provides an optimal condition of temperature, humidity, and
other environmental conditions required for the growth of organisms.
An incubator is a piece of vital laboratory equipment necessary for the cultivation of microorganisms under artificial conditions.
An incubator can be used for the cultivation of both unicellular and multicellular organisms.

Components/Parts of Incubator

Image Source: McQueen Laboratory.

A microbial incubator is made up of various units, some of which are:

Cabinet
 The cabinet is the main body of the incubator consisting of the double-walled cuboidal enclosure with a capacity ranging
from 20 to 800L.
 The outer wall is made up of stainless steel sheets while the inner wall is made up of aluminum.
 The space between the two walls is filled with glass wool to provide insulation to the incubator.
 The insulation prevents heat loss and in turn, reduces the electric consumption, thereby ensuring the smooth working of the
device.
 The inner wall of the incubator is provided with inward projections that support the shelves present inside the incubator.
Door
 A door is present in all incubators to close the insulated cabinet.
 The door also has insulation of its own. It is also provided with a glass that enables the visualization of the interior of the
incubator during incubation without disturbing the interior environment.
 A handle is present on the outside of the door to help with the maneuvering of the door.
Control Panel
 On the outer wall of the incubator is a control panel with all the switches and indicators that allows the parameters of the
incubator to be controlled.
 The control panel also has a witch to control the thermostat of the device.
Thermostat
 A thermostat is used to set the desired temperature of the incubator.
 After the desired temperature is reached, the thermostat automatically maintains the incubator at that temperature until the
temperature is changed again.
Perforated shelves
 Bound to the inner wall are some perforated shelves onto which the plates with the culture media are placed.
 The perforations on the shelves allow the movement of hot air throughout the inside of the incubator.
 In some incubators, the shelves are removable, which allows the shelves to be cleaned properly.
Asbestos door gasket
 The asbestos door gasket provides an almost airtight seal between the door and the cabinet.
 This seal prevents the outside air from entering the cabinet and thus, creating an isolated hot environment inside the cabinet
without being interrupted by the external environment.
L-shaped thermometer
 A thermometer is placed on the top part of the outer wall of the incubator.
 One end of the thermometer provided with gradations remains outside of the incubator so that temperature can be read
easily.
 The next end with the mercury bulb is protruded slightly into the chamber of the incubator.
HEPA filters
 Some advanced incubators are also provided with HEPA filters to lower the possible contamination created due to airflow.
 AN air-pump with filters creates a closed-loop system so that the air flowing inside the incubator generates less
contamination.
Humidity and gas control
 The CO  incubators are provided with a reservoir underneath the chamber that contains water.
2

 The water is vapourised to maintain the relative humidity inside the chamber.
 Similarly, these incubators are also provided with gas chambers to give the desired concentration of CO  inside the
2

incubator.

Principle/ Working of Incubator

 An incubator is based on the principle that microorganisms require a particular set of parameters for their growth and
development.
 All incubators are based on the concept that when organisms are provided with the optimal condition of temperature,
humidity, oxygen, and carbon dioxide levels, they grow and divide to form more organisms.
 In an incubator, the thermostat maintains a constant temperature that can be read from the outside via the thermometer.
 The temperature is maintained by utilizing the heating and no-heating cycles.
 During the heating cycle, the thermostat heats the incubator, and during the no-heating period, the heating is stopped, and
the incubator is cooled by radiating heat to the surrounding.
 Insulation from the outside creates an isolated condition inside the cabinet, which allows the microbes to grow effectively.
 Similarly, other parameters like humidity and airflow are also maintained through different mechanisms that create an
environment similar to the natural environment of the organisms.
 Similarly, they are provided with adjustments for maintaining the concentration of CO2 to balance the pH and humidity
required for the growth of the organisms.
 Variation of the incubator like a shaking incubator is also available, which allows for the continuous movement of the culture
required for cell aeration and solubility studies.

Procedure for running an incubator

Once the cultures of organisms are created, the culture plates are to be placed inside an incubator at the desired temperature and
required period of time. In most clinical laboratories, the usual temperature to be maintained is 35–37°C for bacteria.
The following are the steps to be followed while running an incubator:
1. Before using the incubator, it should be made sure that no remaining items are present in the incubator from the previous
cycles. However, in some cases, if the same incubator is being used for multiple organisms, and they require the same set of
parameters, they can be placed together in the same incubator.
2. The door of the incubator is then kept closed, and the incubator is switched on. The incubator has to be heated up to the
desired temperature of the growth of the particular organism. The thermometer can be used to see if the temperature has
reached.
3. In the meantime, if the organism requires a particular concentration of CO2 or a specific humidity, those parameters should
also be set in the incubator.
 4. Once all the parameters are met, the petri dish cultures are placed on the perforated shelves upside down, i.e., media
uppermost. This is necessary because if the plates are incubated normally, condensation collects on the surface of the
medium and prevents the formation of isolated colonies.
5. If it is necessary to incubate Petri dish cultures for several days, the plates are sealed with adhesive tapes or are placed in
plastic bags or plastic food containers.
6. Now, the door is locked, and the plates are kept inside for the required time before taking them out.

Types of incubators

Figure: Some Incubators used in Microbiology Lab. Image created using bioredner.com

On the basis of the presence of a particular parameter or the purpose of the incubator, incubators are divided into the following
types:

Benchtop incubators
 This is the most common type of incubator used in most of the laboratories.
 These incubators are the basic types of incubators with temperature control and insulation.
CO2 incubators
 CO2 incubators are the special kinds of incubators that are provided with automatic control of CO2 and humidity.
 This type of incubator is used for the growth of the cultivation of different bacteria requiring 5-10% of CO2 concentration.
 For humidity control, water is kept underneath the cabinet of the incubator.
Cooled incubators
 For incubation at temperatures below the ambient, incubators are fitted with modified refrigeration systems with heating and
cooling controls.
 This type of incubator is called the cooling incubator.
 In the cooling incubator, the heating and cooling controls should be appropriately balanced.
Shaker incubator
 A thermostatically controlled shaker incubator is another piece of apparatus used to cultivate microorganisms.
 Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased
aeration, resulting in acceleration of growth.
 This incubator, however, can only be used for broth or liquid culture media.
Portable incubator
 Portable incubators are smaller in size and are used in fieldwork, e.g. environmental microbiology and water examination.

Uses of Incubator
Incubators have a wide range of applications in various areas including cell culture, pharmaceutical studies, hematological studies,
and biochemical studies.
Some of the uses of incubators are given below:
1. Incubators are used to grow microbial culture or cell cultures.
2. Incubators can also be used to maintain the culture of organisms to be used later.
3. Some incubators are used to increase the growth rate of organisms, having a prolonged growth rate in the natural
environment.
4. Specific incubators are used for the reproduction of microbial colonies and subsequent determination of biochemical oxygen
demand.
5. These are also used for breeding of insects and hatching of eggs in zoology.
6. Incubators also provide a controlled condition for sample storage before they can be processed in the laboratories.

Precautions
The following precautions are to be followed while running an incubator:
1. As microorganisms are susceptible to temperature change, the fluctuations in temperature of the cabinet by repeatedly
opening the door should be avoided.
2. The required parameters growth of the organism should be met before the culture plates are placed inside the cabinet.
3. The plates should be placed upside down with the lid at the bottom to prevent the condensation of water on to the media.
4. The inside of the incubators should be cleaned regularly to prevent the organisms from settling on the shelves or the corners
of the incubator.
5. While running the incubator for an extended period of time, sterile water should be placed underneath the shelves to prevent
the culture media from drying out.

Serial dilution Definition

Serial dilution, as the name suggests, is a series of sequential dilutions that are performed to convert a dense solution into a more
usable concentration.

 In simple words, serial dilution is the process of stepwise dilution of a solution with an associated dilution factor.
 In biology, serial dilution is often associated with reducing the concentration of cells in a culture to simplify the operation.

Objectives of Serial dilution

 The objective of the serial dilution method is to estimate the concentration (number of organisms, bacteria, viruses, or
colonies) of an unknown sample by enumeration of the number of colonies cultured from serial dilutions of the sample.
 In serial dilution, the density of cells is reduced in each step so that it is easier to calculate the concentration of the cells in
the original solution by calculating the total dilution over the entire series.
 Serial dilutions are commonly performed to avoid having to pipette very small volumes (1-10 µl) to make a dilution of a
solution.
 By diluting a sample in a controlled way, it is possible to obtain incubated culture plates with an easily countable number of
colonies (around 30–100) and calculate the number of microbes present in the sample.

Serial dilution formula/calculations

 Serial dilution involves the process of taking a sample and diluting it through a series of standard volumes of sterile diluent,
which can either be distilled water or 0.9 % saline.
 Then, a small measured volume of each dilution is used to make a series of pour or spread plates.
 Depending on the estimated concentration of cells/organisms in a sample, the extent of dilution is determined. For e.g., if a
water sample is taken from an extremely polluted environment, the dilution factor is increased. In contrast, for a less
contaminated sample, a low dilution factor might be sufficient.
 Serial two-fold and ten-fold dilutions are commonly used to titer antibodies or prepare diluted analytes in the laboratory.
 The dilution factor in a serial dilution can be determined either for an individual test tube or can be calculated as a total
dilution factor in the entire series.
 The dilution factor of each tube in a set:

 For a ten-fold dilution, 1 ml of sample is added to 9 ml of diluent. In this case, the dilution factor for that test tube will be:

 After the first tube, each tube is the dilution of the previous dilution tube.
Now, for total dilution factor,

 Total dilution factor for the second tube = dilution of first tube × dilution of the second tube.
Example:
For the first tube, dilution factor = 10-1 (1 ml added to 9 ml)
For the second tube, dilution factor = 10-1 (1ml added to 9 ml)
Total dilution factor = previous dilution × dilution of next tube
= total dilution of 10-1 × 10-1 = 10-2

Procedure of Serial dilution

Image Source: Chromoscience
The following is the procedure for a ten-fold dilution of a sample to a dilution factor of 10 -6:
1. The sample/culture is taken in a test tube and six test tubes, each with 9 ml of sterile diluents, which can either be distilled
water or 0.9% saline, are taken.
2. A sterile pipette is taken.
3. 1 ml of properly mixed sample/culture is drawn into the pipette.
4. The sample is then added to the first tube to make the total volume of 10 ml. This provides an initial dilution of 10 -1.
5. The dilution is thoroughly mixed by emptying and filling the pipette several times.
6. The pipette tip is discarded, and a new pipette tip is attached to the pipette.
7. Now, 1 ml of mixture is taken from the 10-1 dilution and is emptied into the second tube. The second tube now has a total
dilution factor of 10-2.
8. The same process is then repeated for the remaining tube, taking 1 ml from the previous tube and adding it to the next 9 ml
diluents.
9. As six tubes are used, the final dilution for the bacteria/cells will be 10-6 (1 in 1,000,000).

Applications/Uses
Serial dilution is performed in a number of experimental sciences like biochemistry, pharmacology, physics, and homeopathy.
1. Serial dilution is used in microbiology to estimate the concentration or number of cells/organisms in a sample to obtain an
incubated plate with an easily countable number of colonies.
2. In biochemistry, serial dilution is used to obtain the desired concentration of reagents and chemicals from a higher
concentration.
3. In pharmaceutical laboratories, serial dilution is performed to receive the necessary concentration of chemicals and
compounds as this method is more effective than individual dilutions.
4. In homeopathy, homeopathic dilutions are used where a substance is diluted in distilled water or alcohol. It is believed than
dilution increases the potency of the diluted substance by activating its vital energy.

Limitation/Problems
Even though serial dilution is a useful technique in laboratories, it faces some challenges. Some of which are:
1. An error might occur during the propagation of the sample, and the transfer inaccuracies lead to less accurate and less
precise transfer. This results in the highest dilution to have the most inaccuracies and the least accuracy.
2. Because serial dilution is performed in a stepwise manner, it requires a more extended period of time which limits the
efficiency of the method.
3. Serial dilution only allows the reduction of bacteria/cells but not the separation of bacteria/cells like in other techniques like
flow cytometry.
 4. This technique also requires highly trained microbiologists and experts in aseptic techniques.

Examples
 A simple example of serial dilution performed in our daily life is tea or coffee. In coffee, we add a certain amount of cold
press coffee and add water over it so obtain a desired concentration of coffee.
 Another example of serial dilution is the dilution of acids and bases in chemistry to obtain a required concentration.
 Serial dilution of culture to determine the number of bacteria in a given sample through a plating technique is also an
essential example of serial dilution.

What is Chemical Sterilization?

 Chemical Sterilization is the process of removal of microorganisms by the use of chemical bactericidal agents.
 Even if physical methods of sterilization are more appropriate for effective sterilization, it is not always appropriate to use for
heat-sensitive materials like plastics, fiber optics, and biological specimens.
 Under such conditions, chemical either in liquid or gaseous state can be used for sterilization. However, it is crucial to ensure
that the materials undergoing sterilization are compatible with the chemical being used.
 Besides, it is important to adopt safety rules in the workplace safety during the use of chemical agents.
 The chemical method of sterilization can be categorized as liquid and gaseous sterilization.
Read also: Physical methods of sterilization

1. Gaseous Sterilization
 Gaseous sterilization involves the process of exposing equipment or devices to different gases in a closed heated or
pressurized chamber.
 Gaseous sterilization is a more effective technique as gases can pass through a tiny orifice and provide more effective
results.
 Besides, gases are commonly used along with heat treatment which also facilitates the functioning of the gases.
 However, there is an issue of release of some toxic gases during the process which needs to be removed regularly from the
system.
 The mechanism of action is different for different types of gases.
 Some of the common gases used for gaseous sterilization are explained below:

i. Ethylene oxide
 Ethylene oxide (EO) gas is a common gas used for chemical treatment applied to sterilize, pasteurize, or disinfect different
types of equipment and surfaces because of its wide range of compatibility with different materials.
 EO treatment often replaces other sterilization techniques like heat, radiation, and even chemicals in cases where the
objects are sensitive to these techniques.
 This method is a widespread method used for almost 70% of all sterilizations and around 50% for disposable medical
devices.
  The mechanism of antimicrobial action of this gas is assumed to be through the alkylation of sulphydryl, amino, hydroxyl, and
carboxyl groups on proteins and imino groups of nucleic acids.
 EO treatment is usually conducted at the temperature range of 30-60°C for several hours which aids in the activity of the
gas.
 The efficacy of the gas depends on the concentration of gas available for each article which is greatly assisted by the good
penetrating nature of the gas, which diffuses readily into many packaging materials including rubber, plastics, fabric, and
paper.
 Ethylene oxide kills all known microorganisms, such as bacteria (including spores), viruses, and fungi (including yeasts and
molds), and is compatible with almost all materials even when repeatedly applied.
 This process, however, is not without drawbacks as the level of gas in the sterilizer goes on decreasing due to absorption,
and the treated articles need to undergo a process of desorption to remove the toxic residual wastes.
 Organisms are more resistant to ethylene oxide treatment in a dried state, as are those protected from the gas by inclusion
in crystalline or dried organic deposits.

ii. Formaldehyde
 Formaldehyde is another important highly reactive gas which is used for sterilization.
 This gas is obtained by heating formalin (37%w/v) to a temperature of 70-80°C.
 It possesses broad-spectrum biocidal activity and has found application in the sterilization of reusable surgical instruments,
specific medical, diagnostic and electrical equipment, and the surface sterilization of powders.
 Formaldehyde doesn’t have the same penetrating power of ethylene oxide but works on the same principle of modification of
protein and nucleic acid.
 As a result of the low penetrating power, its use is often limited to paper and cotton fabrics.
 Formaldehyde can generally be detected by smell at concentrations lower than those permitted in the atmosphere and thus
can be detected during leakage or other such accidents.

iii. Nitrogen dioxide (NO2)

 Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of common bacteria, fungi, and even
spores.
 NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard temperature.
 This property of NO2 enables the use of the gas at standard temperature and pressure.
 The biocidal action of this gas involves the degradation of DNA by the nitration of phosphate backbone, which results in
lethal effects on the exposed organism as it absorbs NO2.
 An advantage of this gas is that no condensation of the gas occurs on the surface of the devices because of the low level of
gas used and the high vapor pressure. This avoids the need for direct aeration after the process of sterilization.

iv. Ozone
 Ozone is a highly reactive industrial gas that is commonly used to sterilize air and water and as a disinfectant for surfaces.
 Ozone is a potent oxidizing property that is capable of destroying a wide range of organisms including prions, without the use
of hazardous chemicals as ozone is usually generated from medical-grade oxygen.
 Similarly, the high reactivity of ozone allows the removal of waste ozone by converting the ozone into oxygen by passing it
through a simple catalyst.
 However, because ozone is an unstable and reactive gas, it has to be produced on-site, which limits the use of ozone in
different settings.
 It is also very hazardous and thus only be used at a concentration of 5ppm, which is 160 times less than that of ethylene
oxide.

2. Liquid Sterilization
 Liquid sterilization is the process of sterilization which involves the submerging of equipment in the liquid sterilant to kill all
viable microorganisms and their spores.
 Although liquid sterilization is not as effective as gaseous sterilization, it is appropriate in conditions where a low level of
contamination is present.
 Different liquid chemicals used for liquid sterilization includes the following:

i. Hydrogen peroxide
 Hydrogen peroxide is a liquid chemical sterilizing agent which is a strong oxidant and can destroy a wide range of
microorganisms.
 It is useful in the sterilization of heat or temperature-sensitive equipment like endoscopes. In medical applications, a higher
concentration (35-90%) is used.
 H2O2 has a short sterilization cycle time as these cycles are as short as 28 minutes where ethylene oxide has cycles that as
long as 10-12 hours.
  However, hydrogen peroxide has drawbacks like low material compatibility, lower capacity of penetration, and associated
health risks.
 Vaporized hydrogen peroxide (VHP) is used to sterilize largely enclosed and sealed areas, such as entire rooms and aircraft
interiors.

ii. Glutaraldehyde
 Glutaraldehyde is an accepted liquid sterilizing agent which requires comparatively long immersion time. For the removal of
all spores, it requires as long as 22 hours of immersion time.
 The presence of solid particles further increases the immersion time.
 The penetration power is also meager as it takes hours to penetrate a block of tissues.
 The use of glutaraldehyde is thus limited to certain surfaces with less contamination.

iii. Hypochlorite
 Hypochlorite solution, which is also called liquid bleach, is another liquid chemical that can be used as a disinfectant, even
though sterilization is difficult to obtain with this chemical.
 Submerging devices for a short period in liquid bleach might kill some pathogenic organisms but to reach sterilization
submersion for 20-24 hours is required.
 It is an oxidizing agent and thus acts by oxidizing organic compounds which results in the modification of proteins in
microbes which might ultimately lead to death.
 Appropriate concentrations of hypochlorite can be used for the disinfection of workstations and even surfaces to clean blood
spills and other liquids.

What is Sterilization?
 Sterilization is the complete removal of microorganisms from an object or surfaces.
 Sterilization is obtained when microorganisms are subjected to antimicrobial agents for sufficient time and at optimum
conditions.

Figure: Physical methods of sterilization. Image created using biorender.com

Some physical methods associated with sterilization are explained below:

Heat Sterilization
 Heat sterilization is the most effective and widely used method of sterilization, where the bactericidal activity results through
the destruction of enzymes and other essential cell constituents.
 The effects of heat sterilization occur more rapidly in a fully hydrated state, as it requires a lower heat input, with low
temperature and less time, under high humidity conditions where the denaturation and hydrolysis reactions are predominant,
rather than in the dry state where oxidative changes take place.
 Under circumstances where thermal degradation of a product is possible, it can usually be minimized by adopting a higher
temperature range, as the shorter exposure times generally result in a lower partial degradation.
  This method of sterilization is applicable to thermostable products. Still, it can be applied to both moisture-sensitive and
moisture-resistant products, for which dry (160–180°C) and moist (121–134°C) heat sterilization procedures are respectively
used.

Moist Heat Sterilization

 Moist heat sterilization is one of the most effective methods of sterilization where the steam under pressure acts as a
bactericidal agent.
 Moist heat sterilization usually involves the use of steam at temperatures in the range 121–134°C.
 High pressure increases the boiling point of water and thus helps achieve a higher temperature for sterilization.
 High pressure also facilitates the rapid penetration of heat into deeper parts of material and moisture present in the steam
causes the coagulation of proteins causing an irreversible loss of function and activity of microbes.
 The high temperature-short time cycles not only often result in lower fractional degradation, but they also provide the
advantage of achieving higher levels of sterility assurance due to more significant inactivation factors.
 The most commonly used standard temperature-time cycles for clinical porous specimens (e.g. surgical dressings) and
bottled fluids are 134°C for 3 minutes and 121°C for 15 minutes, respectively.
 An autoclave is a device that works on the principle of moist heat sterilization through the generation of steam under
pressure.
 In this method, the microorganisms are killed by coagulating their proteins, and this method is much more effective than dry
heat sterilization where microbes are killed through oxidation.
 In the pharmaceutical and medical sectors, it is used in the sterilization of dressings, sheets, surgical and diagnostic
equipment, containers, and aqueous injections, ophthalmic preparations, and irrigation fluids, in addition to the processing of
soiled and contaminated items.
 Moist heat can be used in sterilization at different temperatures:
At temperatures below 100°C
 The sterilization technique employed at a temperature below 100°C involves pasteurization.
 In this process, all non-spore forming microbes are killed in milk by subjecting the milk to a temperature of 63°C for 30
minutes (the holder method) or 73°C for 20 seconds (the flash method).
 In pasteurization, however, not all the pathogenic organisms are killed. The principle of pasteurization is the logarithmic
reduction in the number of viable microbes so that they can no longer cause diseases.
 All mesophilic non-sporing bacteria can be killed by exposure to a moist heat at 60C for half an hour with the exception of
some organisms which require different temperature-time cycles.
 The milk is not heated above its boiling point as the milk might curdle, and its nutritional value might be destroyed.
 Besides milk, other fluids and equipment like vaccines of non-sporing bacteria are also pasteurized at 60°C for 1 hour in
special water baths.
 Similarly, serum and body fluids with congealable proteins are also sterilized at 56°C for 1 hour in water baths.
At a temperature of 100°C
 Boiling at 100°C is a moist heat sterilization technique that doesn’t ensure complete sterility, but is enough for the removal of
pathogenic vegetative microbes and some spores.
 In this case, the items to be sterilized are immersed in boiling distilled water for 30-40 minutes.
 Distilled water is preferred because hard water might result in the formation of a film of calcium salts on the instruments.
 Tyndallization is a method that is used for sterilization of media with sugar and gelatin at 100°C for 30 minutes on three
successive days so as to preserve sugar which might be decomposed at a higher temperature.
 Moist heat at 100°C is applicable for contaminated dishes, beddings, pipettes, and other instruments that are not soiled or
contaminated as well as for objects that are temperature sensitive.
At temperatures above 100°C
 Moist heat sterilization above 100°C involves sterilization by steam under pressure.
 Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg); however, the boiling point of water
increases if the pressure is to be increased.
 This principle is employed in an autoclave where the water boils at 121°C at the pressure of 15 psi or 775 mm of Hg.
 As a result, the steam under pressure has a higher penetrating power. When this steam comes in contact on the surface, it
kills the microbes by giving off latent heat.
 The condensed liquid ensures the moist killing of the microbes.
 Autoclaves are used for the sterilization of contaminated instruments along with different culture media as it ensures
complete sterility.

Dry heat sterilization

 Dry sterilization is the process of removing microorganisms by applying moisture-free heat which is appropriate for moisture-
sensitive substances.
  The dry heat sterilization process is based on the principle of conduction; that is the heat is absorbed by the outer surface of
an item and then passed onward to the next layer. Ultimately, the entire item reaches the proper temperature needed to
achieve sterilization.
 Dry moisture-less heat destroys microorganisms by causing denaturation of proteins and also lyses the proteins in many
organisms, causes oxidative free radical damage, causes drying of cells, and can even burn them to ashes, as in
incineration
 Dry heat sterilization is used for the sterilization of materials which are difficult to sterilize by moist heat sterilization for
several reasons.
 Substances like oil, powder, and related products cannot be sterilized by moist heat because moisture cannot penetrate into
deeper parts of oily materials, and powders are destroyed by moisture.
 Similarly, laboratory equipment like Petri dishes and pipettes are challenging to sterilize by moist heat due to the penetration
problem.
 The lethal effects of dry heat on microorganisms are primarily due to oxidative processes which are less effective when
compared to the hydrolytic damage that results from exposure to steam in moist heat sterilization.
 Thus, in dry heat sterilization usually higher temperatures in the range 160–180°C are employed and also require exposure
times of up to 2 hours depending upon the temperature employed.
 This principle is used in instruments like hot air oven and incineration, which generates very hot moisture-free air.
 The primary industrial application of dry heat sterilization is in the sterilization of glass bottles which are to be filled
aseptically.
 In addition to the fact that this method achieves an adequate sterility assurance level, this method also destroys bacterial
endotoxins (which are the products of Gram-negative bacteria also called pyrogens, which cause fever when injected into
the body) which are difficult to eliminate through other sterilization techniques.
 For the purposes of depyrogenation of glass, temperatures of approximately 250°C are used.
 There are different types of dry heat sterilization which are explained below:
Red Heat
 Rest heat sterilization is the process of instant sterilization by holding the instruments in a Bunsen flame till they become red
hot.
 This method is based on dry heat sterilization is commonly used for sterilization of instruments like incubation loops, wires,
and points of forceps.
 This process ensures effective sterilization; however, it is only limited to substances that can endure heating until redness in
flame.
Flaming
 Flaming is a type of dry sterilization that involves exposure of metallic objects to flame for some time where the flame burns
microbes and other dust presents in the instrument.
 In the case of flaming, the instrument is dipped in alcohol or spirit before burning it in a gas flame.
 This process doesn’t ensure sterility and is not as effective as red hot sterilization.
Incineration
 Incineration is the process of sterilization along with a significant reduction in the volume of the wastes. It is usually
conducted during the final disposal of the hospital or other residues.
 The scraps are heated till they become ash which is then disposed of later.
 This process is conducted in a device called incinerator.
Infrared radiation
 Infrared radiation (IR) is a method of thermal sterilization in which the radiation is absorbed and then converted into heat
energy.
 For this purpose, a tunnel containing an IR source is used. The instruments and glassware to be sterilized are kept in a tray
are then passed through the tunnel on a conveyer belt, moving at a controlled speed.
 During this movement, the instruments will be exposed to the radiation, which will result in a temperature of about 180°C for
about 17 minutes.
 IR is applicable for mass sterilization of packaged items like syringes and catheters.
Hot air oven
 Hot air oven is a method of dry heat sterilization which allows the sterilization of objects that cannot be sterilized by moist
heat.
 It uses the principle of conduction in which the heat is first absorbed by the outer surface and is then passed into the inner
layer.
 A hot air oven consists of an insulated chamber that contains a fan, thermocouples, temperature sensor, shelves and door
locking controls.
  The commonly-used temperatures and time that hot air ovens need to sterilize materials are 170°C for 30 minutes, 160°C for
60 minutes, and 150°C for 150 minutes.
 These ovens have applications in the sterilization of glassware, Petri plates, and even powder samples.

Filtration
 The process of filtration is unique among sterilization techniques in that it removes, rather than destroys, microorganisms.
 Further, it is capable of preventing the passage of both viable and nonviable particles and can thus be used for both the
clarification and sterilization of liquids and gases.
 The primary mechanisms involved in filtration are sieving, adsorption, and trapping within the matrix of the filter material.
 Filtration uses membranous filters that have tiny pores that let the liquid pass through but prevent bigger particles such as
bacteria from passing through the filter. Therefore, the smaller the pore, the more likely the filter is to stop more things from
going through it.
 Certain types of filter (membrane filters) also have an essential role in sterility testing, where they can be employed to trap
and concentrate contaminating organisms from solutions under test.
 These filters are then placed in a liquid nutrient medium and incubated to encourage growth and turbidity.
 The principal application of sterilizing-grade filters is the treatment of heat-sensitive injections and ophthalmic solutions,
biological products, air, and other gases for supply to aseptic areas.
 They may also be required in industrial applications where they become part of venting systems on fermenters, centrifuges,
autoclaves, and freeze dryers.
Filtration sterilization of liquids
 Membrane filters, in the form of discs, can be assembled into pressure-operated filter holders for syringe mounting and in-
line use or vacuum filtration tower devices for filtration of liquid.
 Filtration under pressure is generally considered most suitable, as filling at high flow rates directly into the final containers is
possible without problems of foaming, solvent evaporation, or air leaks.
 Membrane filters are often used in combination with a coarse-grade fiberglass depth prefilter to improve their dirt-handling
capacity.
Filtration sterilization of gases
 Filters employed for this generally consist of pleated sheets of glass microfibres separated and supported by corrugated
sheets of Kraft paper or aluminum which are employed in ducts, wall or ceiling panels, or laminar air flow cabinets.
 These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of particles >0.3mm in diameter and thus are
acting as depth filters.
 In practice, their microorganism removal efficiency is rather better as the majority of bacteria are found associated with dust
particles.
 Other applications of filters include sterilization of venting or displacement air in tissue and microbiological culture (carbon
filters and hydrophobic membrane filters); decontamination of air in mechanical ventilators (glass fiber filters); treatment of
exhausting air from microbiological safety cabinets (HEPA filters); and the clarification and sterilization of medical gases
(glass wool depth filters and hydrophobic membrane filters).

Irradiation
 Irradiation is the process of exposing surfaces and objects to different kinds of radiation for sterilization.
 Mainly electromagnetic radiation is used for sterilization.
 The major target for these radiations is considered to be microbial DNA, where damage occurs as a result of ionization and
free radical production (gamma-rays and electrons) or excitation (UV light).
Ultraviolet (non-ionizing) radiation
 Ultraviolet radiation includes light rays from 150-3900 Å, of which 2600 Å has the highest bactericidal effect.
 Non-ionizing waves have a very little penetration power, so microorganisms only on the surface are killed.
 Upon exposure, these waves are absorbed by many materials, particularly nucleic acids.
 The waves, as a result, cause the formation of pyrimidine dimers which bring error in DNA replication and cause the death of
microbes by mutation.
 UV radiation owing to its poor penetrability of conventional packaging materials is unsuitable for sterilization of
pharmaceutical dosage forms.
 It is, however, applied in the sterilization of air, for the surface sterilization of aseptic work areas, and the treatment of
manufacturing-grade water.
Ionizing Radiation
 X-ray and gamma rays are the commonly used ionizing radiation for sterilization.
 These are high energy radiation which causes ionization of various substances along with water.
  The ionization results in the formation of a large number of toxic O2 metabolites like hydroxyl radical, superoxide ion, and
H2O2 through ionization of water.
 These metabolites are highly oxidizing agents and kill microorganisms by oxidizing various cellular components.
 With ionizing radiation, microbial resistance decreases with the presence of moisture or dissolved oxygen (as a result of
increased free radical production) and also with elevated temperatures.
 Radiation sterilization is generally exposed to items in the dried state which include surgical instruments, sutures,
prostheses, unit-dose ointments, plastic syringes, and dry pharmaceutical products.

Sound (sonic) waves Vibration

 Sonic waves can be used as bactericidal agents which employ ultrasound (usually from 20–40 kHz) to vibrate a fluid.
 The ultrasound can be used with just water, but the use of a solvent appropriate for the object to be cleaned and the type of
soiling present enhances the effect.
 The explanation for the antibacterial activity of airborne sound waves on a physical is based on the possible transformation
of acoustic energy into heat.
 The impact of airborne sound differs with the medium as the absorption of acoustic energy is greatly affected by the nature
of the medium through which it is transmitted.
 As the sound waves propagate through a detergent solution, these ultrasonic waves produce alternating tensile and
compressive forces that oscillate, and these oscillating forces cause millions of microscopically-sized cavities to form in the
detergent solution.
 Once they reach a maximum size, these cavities violently collapse, causing submicroscopic voids to form which induce the
formation of high-energy hydraulic shock waves.
 These shock waves, which may reach temperatures as high as 10,000°F and hydrodynamic pressures as low as 10,000
PSI, physically loosen and remove microorganisms and other adhering debris from even the most inaccessible surfaces of a
contaminated instrument.
 The antibacterial activity of airborne sound waves is directly related to the sound intensity, the period of irradiation, and the
distance of the sample from the sound source.
 These sonic waves are found to be effective against bacteria spores depending on the chemical composition of these
spores.
 The lipoidal substances within the microbes readily absorb ultrasonic frequencies, and any changes in the lipid level of the
test organism directly affect the amount of acoustic energy taken up by the spore as well as the removal of those spores.
 This method is routinely used by healthcare facilities to clean surgical and dental instruments before the terminal sterilization.

Pressure (Pascalization)
 Pascalization or High-Pressure Processing (HPP) is a method employed for preservation and sterilization of food, in which
products are processed under very high pressure (hundreds of megapascal), leading to the death of
specific microorganisms and inactivation of enzymes in the food.
 HHP treatments may be applied at room temperature, and with the exception of some vegetables, shape, color, and
nutrients of most foods are not affected.
 Hydrostatic pressures are nonthermal, and covalent bonds are not broken, so that flavor is unaffected.
 At 400-600 MPa, proteins are easily denatured, and cell morphology is altered, and ribosomes are destroyed.
 Changes occur in the lipid-protein complex of cell membranes, and increased membrane fluidity is also observed, which
causes leakage of nucleic acids.
 Pascalization is not particularly effective against spores, but combined treatment with heat is found to be effective in the
inactivation of spores.
 Pascalization is especially useful on acidic foods, such as yogurts and fruits, because spores which are pressure-tolerant
don’t have the ability to live in environments with low pH.
 The treatment works equally well for both solid and liquid products.

Sunlight (Solar Disinfection)

 Solar disinfection is a process used for the removal of microorganisms with the help of sunlight.
 This process is commonly used to purify or disinfect drinking water.
 Solar disinfection is based on the inactivation of pathogenic organisms as a result of the UV-A (wavelength 320–400 nm)
part of the sunlight, which reacts with oxygen dissolved in the water and releases highly reactive forms of oxygen (oxygen
free radicals and hydrogen peroxides).
 These metabolites damage pathogens, while it also interferes with metabolism and destroys bacterial cell structures, and
simultaneously the full band of solar energy (from infrared to UV) heats the surface.
 The principle of solar disinfection is similar to that of radiation sterilization; however, the efficacy of solar disinfection is
significantly low as it requires a long period of exposure.
 However, this process is economical and an environment-friendly option.
Instruments used in Microbiology Lab with Principle and Uses
The instruments used in the microbiology labs include a bunch of different kinds of instruments required for a lot of different
processes conducted within those laboratories.

1. Analytical Balance
 An analytical balance is a type of balance that is commonly used for the measurement of mass in the sub-milligram range.
Working Principle
 These types of balances are made with a measuring pan enclosed in a transparent covering that prevents smalls particles or
air currents from getting collected on the pan.
 An electric analytical balance uses the force necessary to counteract the mass rather than measuring the mass itself.
 An electromagnet is used to create a force required to achieve a balance with the mass of the substance, and the resulting
force is displayed.
Uses
 As they are highly precise and based on advanced technology, analytical balances are explicitly used in laboratories for the
effective completion of tasks like weighing test materials and sampling amounts, formulation, density determination, purity
analysis, quality control testing, and material and conformance testing.

2. Autoclave
 An autoclave is a pressurized chamber used for the process of sterilization and disinfection by combining three factors: time,
pressure and steam
Working Principle
 Autoclaves use steam as their sterilization agent. The basic principle of an autoclave is that all the items within the autoclave
come in direct contact with the steam for a particular period irrespective of the nature of the material- whether it is liquid,
plastic ware, or glassware.
 The amount of time and the temperature depends on the type of material being sterilized and the increase in temperature of
the cycle allows for shorter periods.
  Uses
 Autoclaves are mostly used for the sterilization of medical or laboratory equipment with the capacity of sterilizing a large
number of materials at once.
 They are commonly used for the preparation of culture media during laboratory applications.

3. Bunsen burner
 Bunsen burner is a standard tool used in laboratories, named after Robert Bunsen.
 It is a gas-fueled single open flame.
Working Principle
 This burner is made with a metal tube on a flat base with a gas inlet at the bottom of the tube, which may have an adjustable
valve. On the sides of the tube are openings which can be adjusted with a collar to control the amount of air that can enter.
 Once the burner is connected to a gas source, the gas is forced by the gas pressure so that the gas reaches the top where
the flame is ignited with a match or a lighter.
Uses
 It is commonly used for processes like sterilization, combustion, and heating. In medical or microbiology laboratories, it is
commonly used for micro-loop sterilization.

4. Centrifuge
 A centrifuge is a device that allows the rotation of an object about a single axis, where an outward force is applied
perpendicularly to the axis.
 A laboratory centrifuge is motor-based and allows the rotation of a liquid sample resulting in the separation of the
components of the mixture.
Working Principle
 A centrifuge works on the principle of sedimentation, where the high speed of the rotation causes the denser particles to
move away from the center while smaller, less dense particles are forced towards the center.
 Thus, the denser particles settle at the bottom while the lighter particles are collected at the top.
 In a laboratory tabletop centrifuge, the sample tubes are aligned at an angle so that the particles have to travel a shorter
distance before they hit the bottom.
Uses
 The primary application of a centrifuge is the separation of particles suspended in a suspension. It can be used for the
separation of cell organelles, nucleic acid, blood components, and separation of isotopes.

5. Colony Counter
 A colony counter is used to estimate the density of a liquid culture by counting the number of CFU (colony forming units) on
an agar or culture plates.
Working Principle
 This instrument can accommodate different sizes of plates which are scanned on top with UV, white light and/or fluorescent
illumination.
 One can accomplish the counting either manually with the touch pressure or with a digital counter.
Uses
 A colony counter is primarily used for counting the number of colonies present on a culture plate to estimate the
concentration of microorganisms in liquid culture.

6. Deep Freezer
Working  Principle
 Deep freezers are based on the principle that under extremely low temperatures, there is minimum microbial growth which
allows for the protection and preservation of different substances.
 Based on this principle, we can even preserve cultures over a long period of time without any change in the concentration of
the microorganisms.
Uses
 A deep freeze can be used for the preservation of different things used in the laboratories for a very long period of time.
Deep freezers are used in laboratories to store and preserve medical equipment, food items, blood samples, medicines, and
injections, etc. for a more extended period of time.

7. Homogenizer
 Homogenizer is a device used in laboratories for the mixing of various liquids and materials like tissue, plant, food, soil, and
many others.
Working Principle
 This instrument is based on the principle that when large globules in coarse emulsion are passed under high pressure
through a narrow orifice, they break down into smaller particles giving a more uniform and stable mixture.
 A homogenizer has a metal rod with narrow parallel openings in the form of a comb at the end which acts as the orifice for
the homogenization process.
Uses
 A homogenizer is primarily used to disrupt cells to acquire cell organelles for different microbiological processes.
  It is used in the preparation step before the extraction and purification of different macromolecules like proteins, nucleic
acids, and lipids.

8. Hot plate
 A hot plate is a stand-alone appliance used in microbiology laboratories as a tabletop heating system.
Working Principle
 Unlike the traditional ways of producing heat through the fire, a hot plate produces heat by the flow of electricity.
 On a hot plate, electricity runs through the coils which have a high level of electrical resistance. The resistance in the coils
converts the electrical energy into heat energy which causes the coils to release heat.
Uses
 In a laboratory, hot plates are used to heat glassware and their components.
 They are used over water baths as in water baths might be hazardous in case of any spills or overheat.

9. Hot air oven

 A hot air oven is an electrical device that is used for sterilization of medical equipment or samples using dry heat.
Working Principle
 Hot air oven is a type of dry heat sterilization which is performed on dry materials and on substances that do not melt or
catch fire under high temperature.
 There are two types of hot air oven based on the working principle
 Forced air hot air oven: In this type of hot air oven, the heated air inside the oven is distributed throughout the oven with a
fan. This prevents the rising of hot air towards the top while keeping the cold air at the bottom. This allows for the
adequate heating of materials inside the oven.
 Static air hot air oven: In this type of oven, the heat is produced by coils present at the bottom of the oven with no fan. The
hot air rises and doesn’t allow the effective sterilization of the materials.
 The equipment inside the oven acquire heat and pass the heat towards the center, one layer at a time which allows for
effective dry heat sterilization.
Uses
 Hot air oven can be used to sterilize materials like glassware, metal equipment, powders, etc.
 It allows for the destruction of microorganisms as well as bacterial spores.

10. Incubator
 An incubator is a device that is used in the laboratories for the growth and maintenance of microorganisms and cultures.
 Incubator provides an optimal temperature, pressure, moisture, among other things required for the growth of
microorganisms.
Working Principle
 The incubator is based on the principle of maintaining a proper atmosphere for the growth of microorganisms.
 Incubators have a heating system that allows for the temperature within the incubator to be adjusted according to the type of
organism cultivated inside.
 Similarly, they are provided with adjustments for maintaining the concentration of CO2 to balance the pH and humidity
required for the growth of the organisms.
 Variation of the incubator like a shaking incubator is also available, which allows for the continuous movement of the culture
required for cell aeration and solubility studies.
Uses
 Incubators have a wide range of applications including cell culture, pharmaceutical studies, hematological studies, and
biochemical studies.
 Incubators can also be used in the steam cell research area.

11. Laminar Air Flow/ Laminar Hood

 Laminar Hood is a closed device primarily for processes or instruments sensitive to microbial contamination.
Working Principle
 A Laminar Hood is made up of stainless steel, avoiding joints and corners to prevent the accumulation of bacterial spores.
 This device creates a sterile environment with the flow of sterile air through a High-Efficiency Particulate Air (HEPA) filter and
shortwave ultraviolet germicidal lamp that sterilizes the workstation.
 Laminar Air Flow has to turn on 15 minutes before to ensure complete sterilization and the workstation should be cleaned
with ethanol before and after use.
Uses
 Laminar Hood is commonly used to conduct processes that are sensitive to contamination.
 It is used for experiments related to plant tissue culture and for the experiments of genetic transformation.

12. Magnetic Stirrer

 Magnetic Stirrer is a device commonly used in microbiology laboratories for the purpose of mixing liquids.
Working Principle
 This device consists of a rotating magnetic or an electromagnet creating a rotating magnetic field that allows the stir bar (a
piece of heavy metal) to move around in the vessel.
 It is coupled with a heating system to heat the liquid while it mixed.
Uses
 It is usually used for mixing various liquid components in a mixture in a chemical or microbiology laboratory.
 This device is used in place of other stirrers as it is noise-free and because the size of the stir bar is so tiny, there is less
chance of contamination.

13. Microscope
 Microscopes are devices that allow the observer to an exceedingly close view of minute particles.
Working Principle
 There are many different types of microscopes, each of which works on their respective principles. However, there is some
commonality in them.
 The basic principle in a microscope is magnification. Based on the relative position of the object from the lens or
electromagnets, different positions, nature, and magnification of the image can be achieved.
 Different types of microscopes are developed to cater to the specific needs of the observation. However, the common theme
is magnification.
Uses
 Based on the type of microscopes, different microscopes are used for different purposes.
 They are primarily used for the observation of minute particles which cannot be observed with naked eyes.

14. pH Meter
 pH meter is a device used in laboratories that measure the H-ion concentration in water-based solutions to determine the
acidity or alkalinity of the solution.
 A pH meter is often termed as “potentiometric pH meter” as it measures the difference in electric potential between the
reference and a pH electrode.
Working Principle
 In a potentiometric pH meter, single or multiple glass electrodes, connected to a bulb selective to hydrogen ions, are
attached to a metal rod.
 When the bulb with the electrodes is dipped into a solution, hydrogen ions in the solution exchange with positive charges on
the electrode generating an electrochemical potential which is displayed in terms of pH units on display.
Uses
 A pH meter is primarily used to measure the acidity of pharmaceutical chemicals, cultures, soil, and water treatment plant.
 It can be used to measure the acidity level in wine and cheese during their production.

15. Spectrophotometer
 The spectrophotometer is an optical instrument for measuring the intensity of light in relation to the wavelength.
 Based on the amount of light absorbed by a colored solution, a quantitative analysis of the solution can be done.
Working Principle
 Spectrophotometry is based on the Beer-Lambert Law, which states the absorbance of light by a solution (of a particular
wavelength) is directly proportional to the concentration of the substance.
 Different wavelengths of lights are passed through a solution as different substances have better absorbance at different
wavelengths. Based on the absorbance of a particular wavelength, the quantitative analysis of a solution can be done.
Uses
 In a microbiology laboratory, a spectrophotometer is applied for the measurement of substance concentration of protein,
nucleic acids, bacterial growth, and enzymatic reactions.
16. Vortex Mixture/ Vortexer
 A vortex mixture is one of the basic technologies used for the mixing of samples in glass tubes or flasks in laboratories.
Working Principle
 It is based on the simple principle of causing reactions and homogenization by agitating the mixture.
 Motorized draft shafts present on the mixer oscillates and transfers the movement to the sample tubes causing the sample
fluids to undergo turbulent flow.
Uses
 Vortex mixer is mostly used for the mixing of various sample fluids in the sample tubes and also allows for the
homogenization of cells and cell organelles.

17. Water Bath

 Water Bath is a conventional device that is used for chemical reactions that required a controlled environment at a constant
temperature.
Working Principle
 A sensor in the device transfers water temperature to a reference value which is then amplified and a control system
generates a signal for the heating system which heats the water to the desired temperature.
Uses
 Water baths are primarily used for heating samples under a controlled temperature.
 These are suitable for heating chemicals that might be flammable under direct ignition.

18. Water Distiller

 A water distiller is a device that purifies water by the process of distillation.
 This instrument is commonly used in medical laboratories, microbiology laboratories, organic chemistry laboratories and
medical industries.
Working Principle
 A water distiller is based on the principle of distillation.
 According to this process, water is first brought to a boil and then condensed into liquid form to obtain pure distilled water.
Uses
 It is used to obtain distilled water required for many lab tests as well as for the preparation of culture media.

Bacteria vs Fungi- Definition, 21 Major Differences, Examples

Bacteria Definition
Bacteria are single-celled microscopic organisms that are characterized by the presence of incipient nucleus and few
membrane-less cell organelles.
 Bacteria are diverse in shape, size, and color, and their habitats also vary ranging from soil, water, to the insides of living
organisms.
 Bacteria exist in various shapes like cocci, bacillus, or spirilla where the cells are arranged in either chains or clusters.
 There are different groups of bacteria where some are pathogenic while the rest are harmless or even beneficial.
 Pathogenic bacteria have a capsid as the outermost covering which serves the function of protection.
 Based on the staining techniques, bacteria are divided into Gram-positive and Gram-negative bacteria.
 The difference in staining is due to the structural differences in the cell wall of different bacteria.
 The cell wall is made up of peptidoglycan that protects the cell membrane and other organelles inside.
 Inside the cell wall is the cell membrane, which is composed of a phospholipid bilayer with globular proteins.
 The cytoplasm has a membrane-less nucleus and some ribosomes. The genetic material in bacteria is mostly DNA which is
not associated with histone proteins.
 Extrachromosomal DNA is also present in some bacteria in the form of a plasmid.
 Reproduction takes place through binary fission, budding, and fragmentation but different methods
like transformation, transduction, and conjugation are available for the transfer of genetic materials.
 Bacteria can be either producer (chemoautotrophs) or consumers (heterotrophs) and even decomposers.

Fungi Definition
Fungi, singular fungus, are eukaryotes that are characterized by the presence of chitin in the cell wall.
 Common fungi include microscopic organisms like molds and yeasts and macroscopic organisms like mushrooms.
 Fungi are heterotrophs that depend on autotrophs for their food and energy indirectly. These organisms absorb their
nutrients from secreting digestive enzymes to the environment.
 Fungi are the principal decomposers in the ecosystem which convert complex organic compounds into inorganic
compounds.
 Fungi can either be free-living or might exist in a parasitic or symbiotic relationship with other organisms.
 Because fungi are eukaryotic organisms, they have a distinct nucleus surrounded by a nuclear membrane. These are
separated from plants on the basis of the presence of chitin in the cell wall and the absence of chlorophyll.
 They also have multiple cell organelles like mitochondria, vesicles, and 80S ribosomes.
 Most fungi grow in the form of a long elongated thread-like structure called hyphae, which can be either septate or aseptate.
 Macroscopic fungi might appear in the form of colonies in culture media which are different in shape size, texture, and color.
 Reproduction in fungi mostly occurs via budding and sporulation. Sporulating fungi form dry spores that are dispersed by
wind or other factors.
 Sexual reproduction is possible in fungi that occur through reproductive spores. The haploid spores combine via cell fusion
to form a diploid zygote.
 Fungi are further divided into six groups on the basis of the appearance of the spore; Glomeromycota, Ascomycota,
Basidiomycota, Chytridiomycota, Blastocladiomycota, and Zygomycota.
 Fungi are essential organisms as most of them are used for the extraction of antimicrobial products for pharmaceutical
industries. Similarly, some fungi are edible and can be used as a source of food.

Key Differences (Bacteria vs Fungi)

Basis for
Bacteria Fungi
Comparison
Bacteria are single-celled
Fungi, singular fungus, are
microscopic organisms that are
eukaryotes that are
Definition characterized by the presence
characterized by the presence
of incipient nucleus and few
of chitin in the cell wall.
membrane-less cell organelles.

Cell Type All bacteria are prokaryotes. All fungi are eukaryotes.

No. of cells Bacteria are unicellular Most fungi are multicellular

organisms with simpler cellular with complex cellular
 structures. Some fungi like
structure. yeast might be unicellular.

The size of bacteria ranges The size of the fungi ranges

Size
from 0.5 to 5 µm. from 2 to 10 µm.

The cell wall of bacteria is The cell wall of fungi is made

made up of peptidoglycan up of chitin.
Cell wall
under which a cell membrane is
present.  

Fungi are found to have

Bacteria are found to have
varying shapes, but most of
three distinct shapes viz round
Morphology them are spotted in the form
(cocci), spiral (Spirilla), and rod-
of a thread-like structure
shaped (bacillus).
called hyphae.

Bacteria grow best in the Fungi mostly prefer a slightly

pH neutral environment of pH acidic environment with pH
range 6.5-7. value 4-6.

Fungi are immobile

Some bacteria are motile with organisms.
Mobility
flagella.
 

The genetic material in fungi

The genetic material in bacteria is localized in the nuclear
Nucleus is localized in the nuclear region.
region of the cytoplasm.
 

Bacteria have few membrane- Fungi contain several

Cell organelles
less organelles. membrane-bound organelles.

Bacteria like all prokaryotes Fungi, like all eukaryotes,

contain 70S ribosomes. 70S contain 80S ribosomes. The
Ribosomes
ribosomes consist of 50S and 80S ribosome is composed of
30S subunits. two subunits 60S and 40S.

Fungi reproduce through both

Bacteria reproduce by an
asexual and sexual methods.
Reproduction asexual method like binary
Sexual reproduction takes
fission.
place through fungal spores.

Bacteria can be autotrophs or Fungi are mostly heterotrophs

Nutrition heterotrophs. that feed on dead and
decaying matter.
 

Source of energy Bacteria derive their energy Fungi obtain their energy from
from inorganic matter or
 organic matter like sugar, pre-existing organic matter.
protein, or fat.
 

Most fungi like yeast perform

Bacteria perform aerobic and
Respiration ethanol fermentation or
anaerobic respiration.
anaerobic respiration.

Some bacteria might have pili. Fungi don’t have pili.

Pili
   

Bacteria do not have Fungi have both microtubules

Cytoskeleton cytoskeletons like microtubules and microfilaments.
or microfilaments.
 

Bacteria have shorter cell

Fungi have longer cell cycles
Cell cycle cycles ranging from 20 to 60
ranging from 12 to 24 hours.
minutes.

Diseases like skin infections,

Diseases like tuberculosis,
Aspergillosis, Aspergilloma,
Diseases tetanus, leprosy, typhoid,
Histoplasmosis are caused by
cholera are caused by bacteria.
fungi.

Beneficial uses of bacteria Beneficial uses of fungi

Use include the production of include the production of
antibiotics and other chemicals. beer, bread, and antibiotics.

E. coli, Staphylococcus aureus, Saccharomyces cerevisiae,

Examples Salmonella Typhi, Histoplasma, Aspergillus
Lactobacillus spp., etc. niger, Agaricus boirus, etc.

Examples of Bacteria
Escherichia coli (E. coli)
 E. coli is a model microorganism used for various research studies. These organisms are found in multiple environments,
and many are found in the lower intestine of human beings and other warm-blooded animals.
 Most varieties of E. coli are harmless, but few might cause mild to severe diarrhea. Some microbes even produce Vitamin K
and Vitamin B-12.
 E. coli is a Gram-negative and facultative anaerobe that flourishes at room temperature.
 It is rod-shaped and has short lifecycles which makes it ideal for research studies.
 E. coli are non-sporing and have peritrichous flagella.
Salmonella Typhi
 Salmonella Typhi is a pathogenic organism that infects the intestinal tract and blood of humans and other organisms.
 This organism results in the disease ranging from mild typhoid fever to life-threatening septic shock.
 S. Typhi is Gram-negative organisms that are rod-shaped and non-sporing with peritrichous flagella.
 Salmonella enterica serotype Typhi is usually contracted by the ingestion of food or water that is contaminated with the feces
of those that carry the organism.
 These are chemotrophs and can obtain energy through oxidation reactions. These are facultative anaerobes that can utilize
oxygen to produce energy when available. When oxygen is not available, they perform anaerobic respiration.
Examples of Fungi
Yeast
 Yeasts are single-celled eukaryotic organisms, most of which are economically important or pathogenic.
 These are mostly found in soil, on plant surfaces and in the sugar-rich fruits and flowers.
 Saccharomyces cerevisiae is the most commonly known and studied yeast that is commonly used as Baker’s yeast in
various baking recipes.
 Some are found as microflora on our body like Candida albicans in the vagina. Other pathogenic fungi
include Histoplasma and Blastomyces.
 Yeast infections are one of the most common infections in women.
 Yeasts reproduce by budding where the chains of daughter cells mature and detach later.
 Some yeasts can reproduce via fission and Torula is a wild yeast that reproduces by sexual spores.
Mushroom
 Mushrooms are the spore-bearing fruiting body of fungi mostly found on soil or cultivated on its food source.
 The term mushroom is used for fungi with a stem, cap, and gills, but Agaricus bisporus is the standard mushroom.
 The gills of these fungi form spores that help spread fungi for reproduction.
 Edible mushroom has nutritional value and thus are consumed as a source of Vitamin D. Some mushrooms are toxic as they
produce toxins as secondary metabolites.
 Some mushrooms are also being studied as possible treatments for diseases and the extraction of polysaccharides,
glycoproteins, and proteoglycans having medical properties.

Archaea vs Bacteria- Definition, 15 Major Differences, Examples

Archaea Definition
Archaea is a group of primitive prokaryotes that based on their distinct characteristics form a separate domain from
bacteria and eukaryotes.
 The term ‘Archaea’ is derived from a Greek word, ‘archaios’ which means primitive or ancient, indicating the primitive
structure of these organisms.
 These organisms usually inhabit extreme environments like deep-sea vents, saline waters, hot springs, and even below
petroleum deposits.
 These are mostly anaerobic and live in low-oxygen environments. Most of the archaea cannot be cultured in laboratories and
thus, have to be identified through culture-independent techniques.
  Organisms in this domain might share some characteristics with both bacteria and eukaryotes. They have an incipient
membrane-less nucleus like bacteria but share some genes, metabolic pathways, and enzymes that are also observed in
eukaryotes.
 However, these organisms also have some unique characteristics. The membrane lipids of archaea contain fatty acid linked
to glycerol molecule by ether bond instead of ester bond as in bacteria and eukaryotes.
 Because archaea inhabit many extreme environments, they tend to have distinct metabolic pathways as well as genes that
support their survival. Halophilic archaea have a unique set of genes that limit the extent of osmosis, facilitating their survival.
 Reproduction in archaea is asexual by budding, fission, and fragmentation. The usual division process of mitosis and
meiosis does not take place.
 Most archaea aid the process of biogeochemical cycles for various elements like carbon, nitrogen, and sulfur.
 Many archaea are methanogens that utilize anaerobic cellular respiration to produce methane as a by-product.
 Even though oxygen-generating photosynthesis doesn’t occur in these organisms, some of them (phototrophs) use sunlight
as a source of energy.

Bacteria Definition
Bacteria are single-celled primitive organisms that form a domain of organisms diverse in shape, size, structure, and even
habitats.
 Bacteria are prokaryotes that have a membrane-less nucleus and lack many cell organelles, which make them simple in
structure and function.
 The domain Bacteria includes organisms that are found in many different forms of life from high mountains to inside the body
of other organisms.
 Some bacteria are beneficial that help in various purposes like antibiotics production, industrial use, and biogeochemical
cycles. However, some are pathogenic organisms that result in mild to severe diseases.
 Bacteria are the smallest living entities in the world and are microscopic. These organisms are observed under a microscope
by performing a number of staining techniques.
 Based on the staining techniques, bacteria are divided into Gram-positive and Gram-negative bacteria.
 Almost all bacteria have a cell wall made up of peptidoglycan that protects the bacteria against harmful chemicals. The
cytoplasm has few ribosomes and a membrane-less incipient nucleus containing the genetic material.
 The membrane lipids in bacteria are composed of fatty acids bound to glycerol by ester bonds.
 Bacteria also have a unique RNA called transfer-messenger RNA (tmRNA).
 The genetic material in bacteria is DNA which is transferred to their offsprings via asexual reproduction.
 Reproduction takes place through binary fission, budding, and fragmentation but different methods like transformation,
transduction, and conjugation are available for the transfer of genetic materials.

Key Differences (Archaea vs Bacteria)

Basis for
Archaea Bacteria
Comparison
Archaea is a group of primitive Bacteria are single-celled
prokaryotes that based on their primitive organisms that form
Definition distinct characteristics form a a domain of organisms diverse
separate domain from bacteria and in shape, size, structure, and
eukaryotes. even habitats.

Most archaea are extremophiles

Bacteria reside in different
and are found in extreme
habitats ranging from soil,
Habitat environments like the deep sea,
water to inside living, and non-
mountains, hot springs, salt brine,
living organisms.
etc.

The archaeal cell wall is made up The bacterial cell wall is made
of pseudopeptidoglycan and lack up of peptidoglycan consisting
Cell wall
D-aminoacids and N- of N-acetylmuramic acid and
acetylmuramic acid. D-amino acids.
 The fatty acids in membrane lipids The fatty acids in membrane
Membrane lipid of archaea are bound to glycerol by lipids of bacteria are bound to
ether bonds. glycerol by ester bonds.

Archaea do not use glycolysis or Glycolysis and Kreb’s cycle

Kreb’s cycle for glucose oxidation are important metabolic
Glucose oxidation
but follow metabolic pathways pathways in bacteria for
similar to these. glucose oxidation.

Archaea do not perform oxygen- Many bacteria containing

generating photosynthesis but are photosynthetic pigments can
Photosynthesis
phototrophs, that use sunlight as a perform photosynthesis to
source of energy. prepare their own food.

Archaea are divided into different Bacteria are divided as Gram-

groups like Methanogens, negative and Gram-positive
Types
Thermophiles, and Halophiles on based on their response to
the basis of their characteristics. Gram staining.

Bacterial flagella are hollow

Archaeal flagella, also termed and are assembled by adding
Flagella archaella, are synthesized by subunits moving from the
adding subunits at the base. central pore towards the tip of
the flagella.

Archaea reproduce by fission, Some bacteria are capable of

budding, and fragmentation. forming spores that help them
Reproduction
Sporulation doesn’t occur in survive extreme conditions for
archaea. a particular period of time.

Thymine is absent in the t-RNA of Thymine is present in the t-

tRNA
archaea. RNA of bacteria.

tmRNA (transfer messenger RNA)

tmRNA tmRNA is found in bacteria.
is found in archaea.

Introns are present in the Introns are absent in the

Chromosomes
chromosomes of archaea. chromosomes of bacteria.

RNA polymerase in archaea is

complex with more than eight Bacterial RNA polymerase is
RNA polymerase
polypeptides. They might even simple, with four polypeptides.
have multiple RNA polymerases.

Bacteria might be pathogenic

Pathogenicity Archaea are non-pathogenic.
or non-pathogenic.

Pseudomonas aeruginosa,
Thermosphaera aggregans,
Bacillus subtilis,
Examples Staphylothermus
Staphylococcus aureus,
marinus, Sulfolobus tokodaii.
Salmonella Typhi.
Examples of Archaea
Sulfolobus
 Sulfolobus is a genus of organisms that belong in the domain Archaea and are both acidophilic and thermophilic in nature.
 They grow at a pH of 2-3 and a temperature of about 80°C. These are mostly found in volcanic springs.
 The proteins found in Sulfolobus are particularly important in biotechnology as they are thermostable and also can function
at low pH.
 These microorganisms are also special because they utilize sulfur as the final electron acceptor during cellular respiration.
 These are thus, dependent on sulfur for the autotrophic or heterotrophic mode of nutrition.
 Sulfolobus was also used as a model for the study of DNA replication. Multiple sites of origin of replication were identified
during studies on these organisms.
 Some species belonging to this genus are Sulfolobus tokodaii and Sulfolobus metallicus.
Methanogens
 Methanogens are prokaryotes belonging to the domain Archaea which are named so because they produce methane as a
by-product during metabolic activities.
 These are found mostly in wetlands and inside the gastrointestinal tracts of various ruminants and even human beings.
Some methanogens are extremophiles and are found in hot springs and deep-sea vents.
 There are more than 50 species of methanogens known so far, many of which produce methane through different metabolic
pathways.
 Some methanogens reduce carbon dioxide in the presence of hydrogen to produce methane. However, others produce
methanol via anaerobic respiration.
 Methanogens are mostly used in the treatment of wastewater via bio composition, which is a cost-effective and faster
wastewater treatment process.
 Some common species of methanogens are Methanosarcina bakeri, Methanosarcina acetivorans, and Methanococcus
maripaludis.

Examples of Bacteria
Escherichia coli (E. coli)
 E. coli is a model microorganism used for various research studies. These organisms are found in multiple environments,
and many are found in the lower intestine of human beings and other warm-blooded animals.
 Most varieties of E. coli are harmless, but few might cause mild to severe diarrhea. Some microbes even produce Vitamin K
and Vitamin B-12.
 E. coli is a Gram-negative and facultative anaerobe that flourishes at room temperature.
 It is rod-shaped and has short lifecycles which makes it ideal for research studies.
 E. coli are non-sporing and have peritrichous flagella.
Lactobacilli
 Lactobacillus is a group of rod-shaped, Gram-positive, non-spore-forming microorganisms belonging to the family
 The term Lactobacilli is given to indicate their ability to produce lactose as a by-product of glucose metabolism.
 These organisms are mostly found in milk and milk products.
 Many varieties of lactobacillus are used commercially to produce fermented products of milk and different vegetables.
 Some commonly used species of this genus are Lactobacillus brevis, Lactobacillus casei, and Lactobacillus plantarum.
 These organisms are even found within the body of living beings like in the intestine and vagina of human beings.

Prokaryotic cells- characteristics, structure, division, examples

Definition of prokaryotic cells
Prokaryotic cells are single-celled entities that are primitive in structure and function as they lack a membrane-bound nucleus and
other organelles. The term “prokaryote” is derived from two Greek words, ‘pro’ meaning ‘before’ and ‘karyon’ meaning ‘nucleus’.
Prokaryotes are considered to be the first living organisms of the earth as they are the simplest form of life.
Characteristics of prokaryotic cells
The general characteristics of prokaryotic cells are listed below:

 In general, prokaryotic cells range in size from 0.1 to 5.0 µm and are considerably smaller than eukaryotic cells.
 The shape of prokaryotic cells ranges from cocci, bacilli, spirilla, and vibrio. However, prokaryotic cells with modifications of
these shapes are also found in nature.
 The cellular organization of prokaryotic cells is primitive as they lack a membrane-bound nucleus and other membrane-
bound cell organelles.
 The genetic material of prokaryotic cells in a single chromosome is made up of a single strand of DNA.
 A critical protein, histone protein, that is found bound in the chromosomes of eukaryotes is absent in prokaryotic cells.
 Prokaryotic cells also lack the nucleolus and the mitotic apparatus.
 The cell wall of prokaryotic cells is non-cellulosic and is made up of carbohydrates and lipids.
 Prokaryotic cells are asexual and thus, reproduce via asexual means without the formation of gametes.

Structure (Components/ Parts) of a prokaryotic cell

The structure of a prokaryotic cell is not as complex as eukaryotic cells as they have primitive cell organelles. Generally, most
prokaryotic cells have the following components/ parts:

1. Capsule
 This is an additional outer covering in some prokaryotic cells that serve to protect the cell against foreign invaders.
 The capsule is made up of polysaccharides, that allows the cells to cling to various surfaces and preserves the moisture in
the cell.
2. Cell wall
 The cell wall is a tough coring of prokaryotic cells present inside the capsule.
 The cell wall of most prokaryotes is made up of polymer of carbohydrates and lipids termed, peptidoglycan.
 In Archaeal cells, however, the cell wall doesn’t contain peptidoglycan but some other structure called pseudopeptidoglycan.
It Is made up of proteins and other polymers.
 The cell wall provides shape to the cell while protecting the cell organelles present in the cytoplasm of the cell.
3. Cell membrane/ Plasma membrane/ Cytoplasmic membrane
 Underneath the cell wall is a cell membrane that is made up of phospholipid.
 The phospholipid forms a bilayer consisting of lipid composed of glycerol attached to a hydrophobic phosphate head and two
hydrophilic fatty acid tails.
 In archaea, the phospholipid tails are usually connected, forming a monolayer instead of the bilayer structure.
 The plasma membrane in prokaryotic cells provides protection to the cell while allowing the transport of essential molecules
in and out of the cell.
 4. Cytoplasm
 The cytoplasm is the entire space of cells present inside of the cell membrane.
 It contains a gel-like cytosol and water-based solution that contains minerals and other ions essential for the cell.
 Besides, the cytoplasm also contains other cellular structures like the chromosomes and ribosomes.
5. Ribosomes
 All prokaryotic cells have 70S ribosomes. The 70S ribosomes are made up of two subunits, 30S, and 50S.
 Here, the 50S subunit contains 23S, and 5S rRNA and the 30S subunit contains 16S rRNA.
 The ribosome is the most commonly observed internal structure in prokaryotic cells.
 The size and number of ribosomes differ in different prokaryotic cells.
 The ribosome is responsible for the formation of polypeptides and in turn, proteins.
6. Nucleoid region
 The nucleoid region of cytoplasm in prokaryotic cells contains a single circular chromosome and small rings of
extrachromosomal DNA called plasmids.
 The single circular chromosome is present as a single copy of genetic material in contrast to the two copies of DNA in
eukaryotes.
 The prokaryotic genomes are also smaller in size than the eukaryotic genomes.
 The plasmids, in turn, are copied independently copied outside of the chromosomes. These plasmids might carry some non-
essential genes.
7. Appendages
 Many prokaryotic cells have cell appendages that protrude out from the cell surface as flagella, pili, and fimbriae.
 Flagella are the most common appendages in many prokaryotic cells.
 These are tail-like structures that assist the cell in moving around.
 Fimbriae are thin filamentous structures that are used to stick the cells to various surfaces.
 Pilli, in turn, are longer filaments that have different roles in different cells. One example of this is the sex pilli that holds two
cells together as they transfer the DNA molecules by the process of conjugation.

Division of prokaryotic cells (Reproduction)

As mentioned earlier, prokaryotic cells reproduce asexually without the formation of gametes. Some asexual modes of reproduction
in prokaryotes are:

Binary fission
 Binary fission is a type of asexual reproduction where a single living cell or an organelle grows twice its size and then splits
into two identical daughter cells, where each of these daughter cells has the potential to grow into the size of the original cell
or organelle.
 Binary fission is the mode of reproduction in many prokaryotes including, archaea, cyanobacteria, and eubacteria.
 During this process, the genetic material of the parent cell is equally divided into two daughter cells. As a result, no genetic
variation is observed in the newly formed prokaryotic cells.
Steps of binary fission
1. The DNA of the cell divides to form two identical DNA molecules, both of which are moved towards the cell membrane.
2. The cell then doubles its size, and the cell membrane slowly starts to divide with each having a copy of the DNA.
3. Once the division of the cell membrane is completed, the cell wall is formed between the two strands of DNA dividing the
parent cell into two identical daughter cells.
Recombination
 Another asexual mode of reproduction in prokaryotic cells is via recombination.
 In this case, the genetic material of one cell is incorporated into the cell of another prokaryote via transduction,
transformation, and conjugation.
 In conjugation, two cells are connected via sex pilli where genes are transferred through the pilli.
 In transformation, the prokaryotic cell takes up the genetic material from the environment and incorporates it into the
bacterial chromosome.
 In transduction, the exchange of genes occurs via viral infection. The bacteriophage first infects one bacterium and takes up
the targeted gene and transfers it to another cell.

Prokaryotic cell examples

Bacterial cells
 Bacteria are the single-celled organisms that are found in all ecosystems throughout the world.
 The cell wall of the bacterial cell is formed of peptidoglycan that makes it tough and thick.
  Capsules are unique to some bacteria and thus might not be present in other prokaryotic cells.
 The genetic material of bacteria is present in the form of circular coils of chromosomes.
 Examples of bacterial cells are E. coli, Streptomyces spp, Pseudomonas spp, etc.
Archaeal cell (Archaea)
 Archaeal cells are similar to bacterial cells as they too are primitive unicellular organisms.
 Archaeal cells are mostly found in extreme environments like hot springs, oceans, and marshlands.
 The capsule is not present in archaeal cells, and the cell wall is made up of pseudopeptidoglycan, composed of proteins.
 Similarly, the cell membrane of archaeal cells has a monolayer of phospholipid that protects the cell against harsh
environments.
 Examples of archaeal cells are Halobacterium spp, Thermoplasma spp, Sulfolobus spp, etc.

FAQs / Revision Question Answers

What are three examples of prokaryotic cells?
Any three examples of prokaryotic cells are blue-green algae, E. coli, and mycoplasma.
Do prokaryotic cells have ribosomes?
Yes, prokaryotic cells have ribosomes. The ribosome is of 70S type.
Do prokaryotic cells have a nucleus?
No, prokaryotic cells do not have a membrane-bound nucleus, but they do have a nucleoid region in the cytoplasm that contains the
genetic material.
Do prokaryotic cells have mitochondria?
No, prokaryotic cells do not have mitochondria.
Is DNA found in prokaryotic cells?
Yes, DNA is found as genetic material and extrachromosomal plastids in prokaryotic cells.
How do prokaryotic cells divide?
Prokaryotic cells divide through asexual methods like binary fission and conjugation.

Morphology of Bacteria- Sizes, Shapes, Arrangements, Examples

 Bacteria are a type of biological cell that is prokaryotic and unicellular. Due to the lack of a membrane-bound nucleus, these
are simpler than other types of living organisms.
 Although only some of them can be seen by naked eyes while the rest are microscopic, they display a wide range of shapes,
sizes, and structures.
Bacterial Size

Figure: The relative sizes of various microscopic and nonmicroscopic objects. Note that a typical virus measures about 100 nm, 10
times smaller than a typical bacterium (~1 µm), which is at least 10 times smaller than a typical plant or animal cell (~10–100 µm).
An object must measure about 100 µm to be visible without a microscope. Image Source: Lumen Learning.
 The unit of measurement used in bacteriology is the micron (micrometer) which is one-thousandth of a millimeter.
 Bacteria are, in general one-tenth the size of the eukaryotic cell. On average, the size of bacteria ranges from 0.5 to 5 µm.
 However, they can be as tiny as 0.3 µm and as large as 0.7mm.
 The limit of resolution with the unaided eye is about 200 microns, and as many bacteria are smaller than this size, they are
not visible with naked eyes.
Among the largest bacteria is Thiomargarita namibiensis, which is up to
half a millimeter long and Epulopiscium fishelsoni which is 0.75 mm long.
The smallest bacteria are members of genus Mycoplasma which are
only 0.3 µm, as small as the largest viruses.
 The size of common bacteria like Escherichia coli ranges in size from 1.1 to 1.5 µm in diameter.
 It has been observed that the size of bacteria has a significant role in the survival of the organisms.
 Owing to their tiny size, they are capable of surviving and even thriving in various unlikely environments like the vertical
sediments in the marine environment.
 Since other organisms are absent in such an environment, bacteria can utilize the available resources.
 Besides, the small size of bacteria favors parasitism and the ability to survive in areas with low nutrition.
 The high surface area-volume ratio also allows the bacteria to take up all the nutrients required for survival while allowing the
steady growth and reproduction.

Other Shapes and Arrangements

Appendaged Bacteria
 The bacteria that produce a unique structure like pillus or fimbriae are called appendaged bacteria.
 These bacteria are more virulent than other bacteria that do not form these appendages.
 Example: Neisseria gonorrheae, the agent of Gonorrhea.
Box-shaped/ Rectangular Bacteria
 Box-shaped bacteria are rectangular in shape and resemble a box.
  Example: Haloarcula marismortui.
Club-shaped Rod Bacteria
 These bacteria are thinner on one side than the other.
 One of the classic examples of this group is Corynebacterium.
Filamentous Bacteria
 These are bacteria that are long, thin, and filament-shaped.
 They, sometimes, divide to form branches resembling strands of hair or spaghetti called mycelium.
 Example: Actinomycetes.
Triangular-shaped Bacteria
 This group includes bacteria that are triangular in shape.
 Example: Haloarcula.
Pleomorphic Bacteria
 The bacteria that do not have a specified shape are included in this group.
 They can change their shape, but in pure culture, they appear to have a definite form.
 Examples: Mycoplasma pneumoniae, M. genitalium.
Stalked Bacteria
 These are the bacteria that possess a stalk on one end of the cell.
 Examples: Caulobacter crescentus.
Star-shaped Bacteria
 The bacteria that look like stars or are star-shaped are included in this group.
 Examples: Stella humosa.
Bacterial Shape

Image created using biorender.com

 Most of the bacteria have a rigid cell wall that provides a definite shape to the bacteria while protecting the internal
components.
 Even though this characteristic is valid for the majority of bacteria, they vary in shape that allows them to be classified into
different groups based on their forms.
 This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton.
 Even though bacteria have a wide variety of shapes, anyone genus typically exhibits a limited subset of morphologies,
indicating that, with a universe of shapes to choose from, individual bacteria adopt only those that are adaptive.
 Bacteria with different shapes present different physical features to the outside world, and these features help cells cope with
and adapt to external conditions.
 It has been observed that bacterial shape contributes a measure of survival value in the face of nutrient acquisition, cell
division, predators, attachment to surfaces, passive dispersal, active motility, and internal or external differentiation.
The common categories of bacteria based on their shapes are:
Cocci
 The bacteria that are oval or spherical in shape are included called cocci bacteria.
 These may either remain single or attached to one another in groups. They appear flattened when placed in groups.
 It is assumed that coccoid forms were derived from rod-shaped organisms through evolutionary time.
Bacilli (Rod-shaped)
 These are rod-shaped cells that also like cocci, remain either single or attached to other cells.
 Bacilli bacteria are among the first bacteria to have arisen, and this shape is said to be not as advantageous as other
shapes. This has been assumed upon the observation of the behavior of filamentous  E. coli cells which, though motile and
chemotactic, move slowly and cannot tumble to change direction.
Spiral
 This group includes bacteria that are either helical-shaped or curved (comma-shaped).
 The bacteria can range from slightly curved to corkscrew-like spiral.
Arrangements of Cocci
 Cocci bacteria can be arranged either singly, in pairs, in groups of four, in chains, in clusters or cubes consisting of eight
cells.
 These cells remain attached during cell division.
Coccus

 This group includes bacteria that are present as a single cell.

Diplococci

 This arrangement results when two bacterial cells occur as a pair (joined together).
 Some of the cells in this arrangement might remain spherical while some might appear flattened, elongated, or bean-shaped.
 Examples: Streptococcus pneumonia, Moraxella catarrhalis, Enterococcus spp, Neisseria gonorrhea.
Tetrad

 Tetrad bacteria are arranged in a group of four cells that remain attached and grow in the attachment after cell division.
 This arrangement results when the cells divide into two planes.
 Examples: Aerococcus, Pediococcus, and Tetragenococcus.
Sarcina

 In this arrangement, the bacterial cells form a group of eight cells.

 This happens when the cells divide in a perpendicular plane.
 The common characteristic associated with these organisms is being strict anaerobe.
 Examples: Sarcina aurantiaca, Sarcina lutea, Sarcina ventriculi.
Streptococci

 Here, the bacteria are arranged in long chains.

 These bacteria are present in family Streptococcaceae, which is characterized by a lack of motility and Gram-positive
bacteria.
 Examples: Streptococcus pyogenes, Streptococcus pneumonia, Streptococcus mutans.
Staphylococci

 This type includes bacteria that are arranged in grape-like clusters.

 This results from cell division in both the planes and are characterized by organisms which are immotile and Gram-positive.
 Examples: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus aureus, Staphylococcus capitis.
Arrangement of Bacilli
Bacillus

 Bacilli are the bacteria which are rod-shaped and are present as single cells.
 These bacteria can form endospores and are facultative anaerobes.
 Examples: Salmonella enterica subsp, Bacillus cereus, and Salmonella choleraesuis.
Diplobacilli

 As in Diplococci, Diplobacilli also exists in pairs.

 After cell division, the two cells do not divide and grow in an attached arrangement.
 Examples: Coxiella burnetii, Klebsiella rhinoscleromatis, Moraxella bovis.
Streptobacilli

 In this group, bacteria are arranged in chains.

 This results from cell division in a single chain.
 Examples: Streptobacillus moniliformis, Streptobacillus Levaditi, Streptobacillus felis, Streptobacillus hongkongensis.
Coccobacilli

 As the name suggests, coccobacilli resemble both cocci as well as bacilli.

 These are shorter in size and thus, appear stumpy.
 Examples: Chlamydia trachomatis, Haemophilus influenza, Gardnerella vaginalis.
Pallisades

 Pallisades are the type of bacilli bacteria that resemble a picket fence structure as a result of the bent at the point of division
during cell division.
 They appear similar to Chinese letters.
 Example: Corynebacterium diphtheria that causes diphtheria.

Arrangement of Spiral
Vibrio

 These are the slightly curved bacteria resembling a comma shape.

 Examples: Vibrio mytili, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio cholera.
Spirochetes

 Spirochetes are spiral bacteria having a helical shape.

 These are flexible and have an axial filament which helps in motility. These filaments are essential distinguishing character
between spirochetes and other bacteria.
 These filaments run throughout the length of the bacteria and thus, help in twisting the motion of the bacteria.
 Examples: Leptospiraspecies (Leptospira interrogans), Treponema pallidum, Borrelia recurrentis.
Spirilla (Helical-shaped/Corkscrew form)

 These bacteria are similar in structure with spirochetes but are more rigid.
 They, too, have a flagellum but lack the endoflagella like in spirochetes.
 Examples: Campylobacter jejuni, Helicobacter pylori, Spirillum winogradskyi.

What is Sensitivity, Specificity, False positive, False negative?

  These are terms that are fundamental to understanding the utility of clinical tests. They are the most essential variables
when designing diagnostic tests, in determining how reliable the tests are and also how reliable the results obtained from
the results are. When using known samples of a disease, sensitivity and specificity variables can be calculated for
evaluation of the test and its results.
 Sensitivity and specificity are independent of the population of interest subject to the tests while Positive predictive value
(PPV) and negative predictive value (NPV) is used when considering the value of a test to a clinician and are dependent
on the prevalence of the disease in the population of interest.
 The positive predictive value (PPV) is the probability that a subject/sample that returns a positive result really is positive
and the negative predictive value (NPV) is the probability that a subject/sample that returns a negative result really is
negative
 To evaluate a diagnostic test, it is very important to calculate its sensitivity and specificity in order to determine its
effectiveness.
 Positive and Negative terms do not refer to the value of the condition of interest but the presence or absence of the
condition. The condition could be a disease, therefore positive means diseased and negative means healthy.

What is Sensitivity?
 Sensitivity is the ability of a test to correctly identify those patients with the disease. It is also known as the True Positive
Rate (TPR), i.e. the percentage of sick persons who are correctly identified as having the condition. Therefore sensitivity is
the extent to which actual positives are not overlooked.
 For example, a test that correctly identifies all positive samples in a panel is a very sensitive test while a test that only
detects 80 % of the true positive samples and 20% of the samples are undetected, hence false negatives in the panel. This
test will be termed to have a lower sensitivity because it is missing positives and having a false-negative rate (FPR). This
type of error is known as type II errors, false negatives are the failure to reject a false null hypothesis where the sample is
negative.
 High sensitivity is very important when detecting a very serious type of infection, for example, the ongoing COVID-
19 pandemic, for proper management and treatment. Screening tests should have a very limited type-2 error, if not none for
accurate identification of the disease. The COVID-19 IgG/IgM diagnostic test is a screening test for COVID-19 with a 95%
sensitivity rate.
 To determine the sensitivity of a test probability (percentage) that a sample tests positive given that the patient has the
disease,
 Calculation:
What is Specificity?
 This is the ability of a clinical test to correctly identify those patients without the disease. It is also known as the True
Negative Rate (TNR), i.e the percentage of healthy people who are correctly identified as not having the condition. A test
that can identify all sample tests from healthy individuals to be negative is very specific.
 Therefore, a test with 100% specificity correctly identifies all patients without the disease, while a test with 80% specificity
correctly reports 80% of patients without the disease as test negative (true negatives) but 20% patients without the disease
are incorrectly identified as to test positive (false positives).
 A test that has a high sensitivity but low specificity causes many patients who do not have the disease being told of a
possibility that they have a disease, subjecting them to further testing. Ideally, the test should be 100% accurate but this is
an unrealistic scenario. Alternatively, subjecting patients to a test with high sensitivity and low specificity and a second test
with low sensitivity and high specificity can identify all false positives and false negatives.

Sensitivity vs Specificity mnemonic

 SnNouts and SpPins is a mnemonic to help you remember the difference between sensitivity and specificity.
 SnNout: A test with a high sensitivity value (Sn) that, when negative (N), helps to rule out a disease (out).
 SpPin: A test with a high specificity value (Sp) that, when positive (P) helps to rule in a disease (in).

What is False positive and False negative?

 The true/false refers to the assigned classification being correct or incorrect while positive/negative refers to the assignment
to a positive or negative category of results.
 These terminologies are dependent on the population subject to the test. Normally, when there is a disease outbreak,
diagnostic tests are done to determine if an individual has the disease or not.
 For persons who are sick, the test outcome is positive while those without the disease, the test outcome will be negative. But
in some circumstances, the test results may not match the individual’s status, therefore they can be defined as:
 False-positive: Healthy people incorrectly identified as sick
 False-negative: Sick people incorrectly identified as healthy.
 A true-positive means that the individual who is sick has been correctly identified to have the disease while an individual who
is a true-negative, means the individual who does not have the disease has been correctly diagnosed to not having the
disease.
Example of Sensitivity and specificity
Assumption: You have a new rapid diagnostic test being evaluated for the screening of COVID-19, on the specific antibodies
produces against the virus, SARS-CoV-2. You have a sample size of 600 people and by validity, there are samples that you know
definitely have the disease (480) and/or healthy individual samples from the disease in question (120). After running the test, then
you compare the results to their know disease staus and find that:
 True positive (test positive and are correctly positive) = 480
 False-positive (test positive but are actually negative) = 15
 True negative (test negative and are genuinely negative) = 100
 False-negative (test negative but are actually positive) =5

Tabulated Results
  Positive Negative Total

True Positive 480 – 480

False Positive 15 – 15

True Negative – 100 100

False Negative – 5 5

Total 495 105 600

  Sensitivity = 480/(480+5)= 0.98

 Therefore, the test has a 98% sensitivity.
 Specificity = 100/(100+15)=0.87
 Therefore, the test has 87% specificity.

Positive Predictive Value (PPV) and Negative Predictive Value (NPV)

 Positive predictive value (PPV) is a probability value that a sample test positive is really positive while the negative predictive
value (NPV) is the probability value that a sample test is really negative.
 They are useful indexes in evaluating sample results and they can be calculated like sensitivity and specificity. These values
can also be used to calculate sensitivity and specificity.
 Therefore, it is correct to say that Sensitivity and specificity evaluate the test while PPV and NPV evaluate the results.

Calculation
 Positive Predictive Value (PPV)=(Number of true positive )/(Number of true positives+number of false positives)
OR
 Positive Predictive Value (PPV)=(Number of True positive)/(Total of positive results)
 

 Negative Predictive Value (NPV)=(Number of True Negative)/(Number of true negative+Number of False-negative)

OR
 Negative Predictive Value=(Number of True Negative)/(Total of negative results)
 Using the table above, we can calculate PPV and NPV as follows:
 PPV=480/(480+15)=0.97….97%
 NPV=100/(100+5)= 0.95…95%
 Therefore, if a test is positive, there is a 97% chance that it is correct and if the result is negative, there is a 95% chance it is
correct.
 The opposite values are False Discovery Rate (FPV) for Positive Predictive Value (PPV) and False Omission Rate (FOR) for
Negative Predictive Value (NPV).
Sensitivity and Specificity
 When developing diagnostic tests or evaluating results, it is important to understand how reliable those tests and therefore
the results obtained are.
 By using samples of known disease status, values such as sensitivity and specificity can be calculated that allow its
evaluation.
 Therefore, when evaluating diagnostic tests, it is important to calculate the sensitivity and specificity for that test to determine
its effectiveness.

Image Source: Wikipedia

Sensitivity
 The term sensitivity was introduced by Yerushalmy in the 1940s as a statistical index of diagnostic accuracy.
 It is also called the true positive rate, the recall, or probability of detection.
 It has been defined as the ability of a test to identify correctly all those who have the disease, which is “true-positive”.
 A 90 percent sensitivity means that 90 percent of the diseased people screened by the test will give a “true-positive” result
and the remaining 10 percent a “false-negative” result.
 Thus, a highly sensitive test rarely overlooks an actual positive (for example, showing “nothing bad” despite something bad
existing).

Calculating Sensitivity
 The sensitivity of a diagnostic test is expressed as the probability (as a percentage) that a sample tests positive given that
the patient has the disease.
 The following equation is used to calculate a test’s sensitivity:
Specificity
 It is defined as the ability of a test to identify correctly those who do not have the disease, that is, “true-negatives”.
 It is also called as the true negative rate.
 A 90 percent specificity means that 90 percent of the non-diseased persons will give a “true-negative” result, 10 percent of
non-diseased people screened by the test will be wrongly classified as “diseased” when they are not.
 Thus, a highly specific test rarely registers a positive classification for anything that is not the target of testing.

Calculating Specificity
The specificity of a test is expressed as the probability (as a percentage) that a test returns a negative result given that that patient
does not have the disease.
The following equation is used to calculate a test’s specificity:

Relationship between Sensitivity and Specificity

 In medical tests, sensitivity is the extent to which actual positives are not overlooked (so false negatives are few), and
specificity is the extent to which actual negatives are classified as such (so false positives are few). 
 Although a screening test ideally is both highly sensitive and highly specific, we need to strike a balance between these
characteristics, because most tests cannot do both.
 We determine this balance by an arbitrary cut-off point between normal and abnormal.
 If we want to increase sensitivity and to include all true positives, we are obliged to increase the number of false positives,
which means decreasing specificity.
 Reducing the strictness of the criteria for a positive test can increase sensitivity, but by doing this the test’s specificity is
reduced.
 Likewise, increasing the strictness of the criteria increases specificity but decreases sensitivity.

Biochemical Test of Bacteria

Biochemical reactions are very important in the identification of bacterial isolates and in the identification of different bacterial
species. These tests depend on the presence of certain enzymes, such as catalase, oxidase, urease, gelatinase, etc., produced by
the bacteria.
Different bacteria produce varying spectra of enzymes. For example, some enzymes are necessary for the bacterium’s individual
metabolism, and some facilitate the bacterium’s ability to compete with other bacteria or establish an infection. Tests that measure
single bacterial enzymes are simple, rapid, and generally easy to interpret. They can be performed on organisms already grown in
culture and often provide presumptive identification.
Click on the link below to know the biochemical reactions of specific bacteria.
1. Biochemical Test of Acinetobacter baumannii
2. Biochemical Test of Actinomyces israelii
3. Biochemical Test of Aeromonas caviae
4. Biochemical Test of Aeromonas hydrophila
5. Biochemical Test of Alcaligenes faecalis subsp. faecalis
6. Biochemical Test of Bacillus anthracis
7. Biochemical Test of Bacillus cereus
8. Biochemical Test of Bacillus subtilis
9. Biochemical Test of Bacteroides fragilis
10. Biochemical Test of Bifidobacterium bifidum
11. Biochemical Test of Bordetella pertussis
12. Biochemical Test of Brucella melitensis
13. Biochemical Test of Burkholderia cepacia
14. Biochemical Test of Burkholderia pseudomallei
15. Biochemical Test of Campylobacter fetus subsp. fetus
16. Biochemical Test of Campylobacter jejuni
17. Biochemical Test of Chlamydia trachomatis
18. Biochemical test of Citrobacter freundii
19. Biochemical Test of Clostridium botulinum
20. Biochemical Test of Clostridium difficile
21. Biochemical Test of Clostridium perfringens
22. Biochemical Test of Clostridium tetani
23. Biochemical Test of Corynebacterium diphtheriae
 24. Biochemical Test of Edwardsiella tarda
25. Biochemical Test of Enterobacter aerogenes
26. Biochemical Test of Enterobacter cloacae
27. Biochemical Test of Enterococcus faecalis
28. Biochemical Test of Enterococcus faecium
29. Biochemical Test of Escherichia coli (E. coli)
30. Biochemical Test of Francisella tularensis subsp. tularensis
31. Biochemical Test of Fusobacterium necrophorum
32. Biochemical Test of Gardnerella vaginalis
33. Biochemical Test of Haemophilus aegyptius
34. Biochemical Test of Haemophilus influenzae
35. Biochemical Test of Haemophilus parainfluenzae
36. Biochemical Test of Hafnia alvei
37. Biochemical Test of Helicobacter pylori
38. Biochemical Test of Kingella kingae
39. Biochemical Test of Klebsiella granulomatis
40. Biochemical Test of Klebsiella oxytoca
41. Biochemical Test of Klebsiella pneumoniae
42. Biochemical Test of Lactobacillus spp.
43. Biochemical Test of Listeria monocytogenes
44. Biochemical Test of Mycobacterium tuberculosis
45. Biochemical Test of Neisseria gonorrhoeae
46. Biochemical Test of Proteus mirabilis
47. Biochemical Test of Providencia stuartii
48. Biochemical Test of Pseudomonas aeruginosa
49. Biochemical Test of Salmonella Typhi
50. Biochemical Test of Serratia marcescens
51. Biochemical Test of Shigella flexneri
52. Biochemical Test of Staphylococcus aureus
53. Biochemical Test of Staphylococcus epidermidis
54. Biochemical Test of Streptococcus agalactiae
55. Biochemical Test of Streptococcus canis
56. Biochemical Test of Streptococcus mutans
57. Biochemical Test of Streptococcus pneumoniae
58. Biochemical Test of Streptococcus pyogenes
59. Biochemical Test of Vibrio cholerae
60. Biochemical Test of Yersinia pestis

Read Also ...

 28 Differences Between Bacteria and Virus (Bacteria vs Virus)
 31 Differences Between Gram Positive and Gram Negative Bacteria
  Classification of Bacteria
 Bacterial Growth and Factors Affecting Growth of Bacteria
 Classification of Bacteria on the basis of Nutrition
 Morphology of Bacteria
 Gram-positive bacteria- cell wall, examples, diseases, antibiotics
 Gram-negative bacteria- cell wall, examples, diseases, antibiotics
 Morphology of Bacteria- Sizes, Shapes, Arrangements, Examples
 Archaea vs Bacteria- Definition, 15 Major Differences, Examples

Peptone Water
Peptone water is formulated as per Shread, Donovan, and Lee. It is a broth medium used for the growth of the organism and a base
for determining carbohydrate fermentation patterns of non-fastidious organisms. In addition, it is also used for the detection of
indole production by the organism.

Composition
Ingredients Gms / L

Peptone 10.0

Sodium chloride 5.0

Final pH (at 25°C) 7.2±0.2

Preparation
 Suspend 15.0 grams in 1000 ml distilled water.
 Mix thoroughly and distribute into the final containers.
 Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Principle
The two basic components of peptone water are peptone and sodium chloride. Peptone provides nitrogenous and carbonaceous
compounds, long-chain amino acids, vitamins provide essential nutrients. Sodium chloride provides the necessary electrolyte and
maintains the osmotic balance of the medium.
Peptone Water is particularly suitable as a substrate in the study of indole production. Peptone used in Peptone Water is rich in
tryptophan content that aid in the detection of the indole using either Kovacs or Ehlrich reagent. Peptone Water is also utilized as a
base for carbohydrate fermentation studies with the addition of sugar and indicators such as bromocresol purple, phenol red or
bromothymol blue. Peptone Water is recommended for studying the ability of an organism to ferment a specific carbohydrate which
aid in the differentiation of genera and species. The acidity formed during fermentation can be detected by addition of phenol red
indicator, which shows a color change of the medium from red to yellow under acidic conditions. In order to detect gas production if
produced by the organism, the Durhams tube is inserted into the medium.

Result
Positive result (inoculated medium): Growth, turbidity seen
Negative result (uninoculated medium): No growth

Uses
 Peptone Water is used as a growth medium and as a base for carbohydrate fermentation media.
 It is a broth medium used for the detection of indole.
 It is minimal growth medium which is used for determining carbohydrate fermentation patterns of non-fastidious organisms.
 It is useful as a diluent or for making suspensions of non-fastidious microorganisms for microbial enumeration procedures.
 Peptone Water with pH adjusted to 8.4 (alkaline condition) is suitable for the cultivation and enrichment of Vibrio species.

Limitations
 It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from
pure culture for complete identification.
 The medium is not well suited for the growth or maintenance of fastidious microorganisms due to its low nutrient content.
 Due to nutritional variations, some strains may show poor growth.
 Some sugar solutions may affect the pH of the Peptone Water, hence checking must be done.
 Sub-cultures may be necessary to ensure the purity of the inoculant. Mixed or contaminated cultures will give false reactions.

Koch’s postulates and its limitations

Koch’s postulate forms the very basis of the pathogenic microbiology. The causality of almost all infectious diseases is based on the
postulate and theories developed by Robert Koch, who is rightly called the “father of pathogenic microbiology,” and his
contemporaries. Developed in the late 19th century, it has stood the test of time.
The postulate can be summarized as follows:
1. A microbe suspected as the causal agent of a particular disease must be found in all subjects suffering from a similar
disease but must be absent in clinical specimens from healthy individuals.
2. The suspected microorganism can be isolated from the diseased individual and grown in pure culture.
3. When this isolated suspect microbe is injected into healthy, susceptible animals (some human volunteers were also
reportedly used by Robert Koch), signs and symptoms of a disease similar to the disease under investigation must develop
in the infected animal.
4. The microbe cultured from the infected animal must be morphologically and physiologically identical to the strain initially
isolated from the patient (in point 1).
However, Koch’s postulates have their limitations and so may not always be the last word.

They may not hold if:

 The particular bacteria (such as the one that causes leprosy) cannot be “grown in pure culture” in the laboratory.
 There is no animal model of infection with that particular bacteria.

Harmless bacteria may cause disease if:

 It has acquired extra virulence factors making it pathogenic.
 It gains access to deep tissues via trauma, surgery, an IV line, etc.
 It infects an immunocompromised patient.
 Not all people infected by a bacteria may develop a disease-subclinical infection is usually more common than clinically
obvious infection.
Despite such limitations, Koch’s postulates are still a useful benchmark in judging whether there is a cause-and-effect relationship
between a bacteria (or any other type of microorganism) and clinical disease.

Here are Koch’s postulates for the 21st century as suggested by Fredricks and Relman:
1. A nucleic acid sequence belonging to a putative pathogen should be present in most cases of an infectious disease.
Microbial nucleic acids should be found preferentially in those organs or gross anatomic sites known to be diseased, and not
in those organs that lack pathology.
2. Fewer, or no, copy numbers of pathogen-associated nucleic acid sequences should occur in hosts or tissues without the
disease.
3. With the resolution of disease, the copy number of pathogen-associated nucleic acid sequences should decrease or become
undetectable. With clinical relapse, the opposite should occur.
4. When sequence detection predates disease or sequence copy number correlates with severity of disease or pathology, the
sequence-disease association is more likely to be a causal relationship.
5. The nature of the microorganism inferred from the available sequence should be consistent with the known biological
characteristics of that group of organisms.
6. Tissue-sequence correlates should be sought at the cellular level: efforts should be made to demonstrate specific in situ
hybridization of microbial sequence to areas of tissue pathology and to visible microorganisms or to areas where
microorganisms are presumed to be located.
7. These sequence-based forms of evidence for microbial causation should be reproducible.

Degradation and Deterioration
 Nature has always provided, in considerable amounts and variety, polymeric materials with interesting compositions and
structures.
 These natural polymers include polysaccharides, such as cellulose, chitin, starch, alginate, galactan, hyaluronic acid,
dextran, and gellan, obtained from plants, animals, and microbial sources and polyesters, such as poly(b-hydroxybutyrate)
and poly(b-hydroxybutyrate-co-b–hydroxyvalerate), produced by numerous bacteria.
 In biological systems, polysaccharides and their derivatives are found as energy reservoirs, as components of the cell wall of
plants, in bacteria, and in the connective tissues.
Both degradation and deterioration processes involve the breakdown of materials including polymers. However, there is a distinction
between ‘biodegradation’ and ‘biodeterioration’.
Whereas ‘biodegradation’ is concerned with the use of microorganisms to modify materials with a positive or useful
purpose, ‘biodeterioration’ refers to the ‘negative impact of live-organisms activity’. Biological process/(es) are the primary
cause of deterioration in biodeterioration.

Degradation
 The reduction of a chemical compound to one less complex, as by splitting off one or more groups is called degradation.
 Biological degradation of natural and some synthetic polymers occurs when polymer depolymerization takes place as the
result of enzyme secretions by some microorganisms.
 These enzymes either oxidize or hydrolyze the polymer by attacking the polymer chain’s main functional chain and end
groups so that polymer chains with either oxygen or nitrogen or both with low levels of crystallinity are most susceptible.
  Natural fibers fitting most of these criteria are therefore highly biodegradable whereas synthetic fibers such as polypropylene
and polyethylene are least likely to be biologically attacked and then degrade.
 Aromatic polyesters and polyamides, despite having oxygen and nitrogen in their respective molecular chains, also resist
biodegradation largely because of their molecular chain rigidity and high levels of crystallinity.
 Material degradation results from the complex interaction between physical, chemical and biological degradation processes.
 The term biodegradation is often used to denote degradation occurring in a biological environment. It may be defined as the
“gradual breakdown of a material mediated by a specific biological activity”.
 Degradation is an important process to transform or accumulate environmental pollutants, including hydrocarbons (e.g.
oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), heterocyclic compounds (such
as pyridine or quinoline), pharmaceutical substances, radionuclides, and metals.
 Moreover, it also ensures proper nutrient cycling in the environment.

Deterioration
 Deterioration refers to the action or process of becoming impaired or inferior in quality, functioning, or condition.
 Deterioration is defined as a loss of structural capacity with time as a result of the action of the external agents or material
leaching (Sanchez-Silva et al. 2008).
 Biodeterioration in its widely accepted form of definition is: “any undesirable change in the properties of a material caused by
the vital activities of organisms” (Hueck, 1968).
 Another definition is: “the process by which biological agents (i.e. live organisms) are the cause of the (structural) lowering in
quality or value”
 The process of deterioration of materials induced by outdoor environmental factors is a complex interplay of the effects of
climate and local meteorological characteristics, of biological processes and frequently of complicated chemical processes
resulting from the impact of pollutants and natural constituents from the surrounding environment.
 Except for the decay caused by cataclysmic events, the principal natural environmental factors affecting the deterioration of
materials include, but are not limited to, moisture, temperature, solar radiation, air movement and pressure, precipitation,
chemical and biochemical attack, and intrusion by micro and macro-organisms.
 Natural factors, together with those expected to be a part of the pollution processes, continuously promote weathering and
material decay, including metal corrosion.
 Biological deterioration caused by insect attack and/or fungal growth and the other form of deterioration is caused
by adverse environmental conditions such as extremes of dampness or wide fluctuations in relative humidity associated with
large variations in day and night temperatures, light and atmospheric pollutants. 

Biological Factors of Deterioration

 Where there is condensation or moisture due to high humidity, there is always the presence of biological growths such as
molds or fungi, insects and rodents causing an infestation.
 Biological agents attack paper and other organic materials when both temperature and humidity are uncontrolled.
 Mold spores remain suspended in the air until they find suitable conditions for their growth. If mold is observed in the
collection yet environmental conditions are not altered to halt its proliferation, the mold will digest the material on which it has
begun to grow.
 This results in the staining and deterioration of materials attacked and in rapid loss of strength of organic materials.

General Aseptic Techniques in Microbiology Laboratory

 Aseptic technique is a set of routine measures that are taken to prevent cultures, sterile media stocks, and other solutions
from being contaminated by unwanted microorganisms (i.e., sepsis).
 While such actions are sometimes called “sterile technique,” that terminology is appropriate only in reference to preventing
the introduction of any organisms to the laboratory or medical equipment and reagents (e.g., during surgery).
 Since the goal of a biologist is to grow microorganisms or eukaryotic cells without the introduction of extraneous organisms,
aseptic techniques are crucial for accurate and meaningful experimentation.
 One should always remember that a completely sterile working environment does not exist.
 However, there are a number of simple, common sense procedures that will reduce the risk of culture contaminations.
 The aseptic techniques control the opportunities for contamination of cultures by microorganisms from the environment, or
contamination of the environment by the microorganisms being handled.
Examples of aseptic technique are:
 Cleaning and disinfecting lab surfaces prior to use
 limiting the duration that cultures or media are uncapped and exposed to the air
 keeping petri dishes closed whenever possible
 effectively sterilizing inoculating loops and other equipment that comes into contact with cultures or media, and
 avoiding breathing on cultures or sterile instruments.
There are some general rules to follow for any aseptic technique.
  Close windows and doors to reduce draughts and prevent sudden movements which might disturb the air.
 Make transfers over a disinfected surface. Ethanol disinfection is recommended because of its rapid action. If the bench
surface is difficult to clean, cover the bench with a sheet of tough material which is more easily disinfected.
 Start the operations only when all apparatus and materials are within immediate reach.
 Complete all operations as quickly as possible, but without any hurry.
 Vessels must be open for the minimum amount of time possible.
 While vessels are open, all work must be done close to a Bunsen burner flame where air currents are drawn upwards.
 On opening a test tube or bottle, the neck must be immediately warmed by flaming (see below) with the vessel held as near
to horizontal as possible and so that any movement of air is outwards from the vessel.
 During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to contamination from the air.
 The parts of sterile pipettes which will be put into cultures or sterile vessels must not be touched or allowed to come into
contact with other non-sterile surfaces, such as clothing, the surface of the working area, or the outside of bottles/ test tubes.
 All items which come into contact with microorganisms must be sterilised before and after each such exposure. This could
be either by the technical team preparing for and clearing up after a piece of practical work (for example, in the case of
glassware to be used), or by the worker during the course of the practical (for example, in flaming a wire loop).

Specific Aseptic Techniques

Sterile Handling
 Always wipe your hands and work area with 70% ethanol.
 It is recommended to wear gloves. This will prevent any foreign contaminants from coming in contact with the customers and
sample during testing. If gloves are not used, it is necessary to wash hands before and after testing.
 Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood.
 Avoid pouring media and reagents directly from bottles or flasks.
 Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to
avoid cross contamination.  Do not unwrap sterile pipettes until they are to be used.  Keep pipettes at the work area.
 Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resalable bags to prevent
microorganisms and airborne contaminants from gaining entry.
 Never uncover a sterile flask, bottle, Petri dish, etc. until the instant you are ready to use it and never leave it open to the
environment.  Return the cover as soon as you are finished.
 If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down.
 Use only sterile glassware and other equipment.
 Be careful not to talk, sing, or whistle while performing sterile procedures.
 Perform experiments as rapidly as possible to minimize contamination.
Inoculating agar plates, slopes and cultures
 Carry out the transfer of cultures as quickly as possible, with tubes and plates open to the air for the minimum length of time.
 Normal practice is to open agar plates away from the body and without removing the lid completely from the base.
 In instances when the lid of the Petri dish may be removed for longer periods than normal, work very close to the Bunsen
burner flame to reduce the chances of contamination.
 If you experience frequent contamination of plates with fungal spores, reduce the chance of draughts further, and consider
inoculating plates from below with the agar surface facing downwards. In this way there is perhaps less chance of spores
settling onto the plate from the air.
Using a wire loop
 While using a wire loop, hold the handle of the wire loop close to the top, as you would hold a pen, at an angle that is almost
vertical. This leaves the little finger free to take hold of the screw cap/ cotton wool plug of the bottle/ test tube. It also ensures
that any liquid culture on the loop will run down into the flame.
 Sterilise a wire loop by heating to red hot in a roaring blue Bunsen burner flame before and after use. This ensures that
contaminating bacterial spores are destroyed.
 The flaming procedure should heat the tip of the loop gradually. This is because after use it will contain culture, which may
splutter on rapid heating and possibly release small particles of culture, forming an aerosol.
 Position the handle end of the wire in the light blue cone of the flame. This is the coolest area of the flame.
 Draw the rest of the wire upwards slowly into the hottest region of the flame – immediately above the blue cone.
 Hold there until it is red hot.
 Ensure the full length of the wire receives adequate heating.
 Allow to cool for a few seconds in the air, then use immediately.
 Do not put the loop down, or wave it around.
 Re-sterilise the loop immediately after use.
Using a pipette
 Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and sterile solutions.
  Remove the pipette from its container/ wrapper by the end containing a cotton wool plug, taking care to touch as little of the
pipette as you need to take a firm hold.
 Fit the teat. It is sometimes helpful to dip the teat first in sterile liquid to lubricate it.
 Hold the pipette barrel as you would a pen, but do not grasp the teat. This leaves your little finger free to take hold of the cap/
cotton wool plug of a bottle/ test tube and your thumb free to control the teat.
 Depress the teat cautiously and take up an amount of fluid which is adequate for the amount required, but does not reach
and wet the cotton wool plug.
 Squeezing the teat with the pipette tip beneath the liquid surface introduces air bubbles which may cause ‘spitting’ and,
consequently, aerosol formation. Avoid this by squeezing the teat before placing the tip into the liquid.
 Then gently release the pressure until the required amount of liquid is drawn up, and lift the pipette tip out of the liquid.
 Return any excess gently.
 Immediately after use put the contaminated pipette into a nearby discard pot of disinfectant.
 Remove the teat only once the pipette is within the discard pot otherwise drops of culture will contaminate the working
surface.
Flaming the neck of bottles and test tubes
This ensures that no microorganisms enter the mouth of the vessel to contaminate the culture or the medium. Passing the mouth of
the bottle through a flame produces a convection current away from the opening, and helps to prevent contamination. The hot part
of the flame is above the inner bright blue ‘cone’ and the vessel needs to be moved through the flame, not held in place.
 Loosen the cap of the bottle so that it can be removed easily.
 Lift the bottle/ test tube with your left hand.
 Remove the cap/ cotton wool plug of the bottle/ test tube with the little finger curled towards the palm of your right hand.
(Turn the bottle, not the cap.)
 Do not put down the cap/ cotton wool plug.
 Flame the neck of the bottle/ test tube by passing the neck forwards and back through a hot Bunsen burner flame.
 After carrying out the procedure required, for example, withdrawing culture, replace the cap/ cotton wool plug on the bottle/
test tube using your little finger. Take care! The bottle will be hot. (Turn the bottle, not the cap.)
 If cotton wool plugs have partly lost their shape, they can be more easily guided back into the neck of the vessel by slowly
twisting the mouth of the vessel as the plug is pushed down.
Disinfecting surfaces
 For technicians, ethanol disinfection is recommended because of its rapid action (around 5 minutes). Technicians will be
more experienced and able to deal with the associated fire hazards of working with ethanol.

Tools Used for Maintaining Aseptic Conditions

The Bunsen Burner
 Probably the easiest way to create a relatively sterile environment on the laboratory bench is by using a simple gaspowered
burner.
 This common piece of equipment burns a continuous stream of a flammable gas—usually natural gas (methane)—based
upon a design made almost 150 years ago by the German chemist Robert Wilhelm Bunsen (1811-1899).
 A major purpose of the open flame in aseptic technique is to create a cone of hot air above and around the laboratory bench
to reduce the viability of organisms on suspended dust particles.
 The ability of the Bunsen burner flame to heat things very quickly also makes it an ideal choice for sterilizing inoculating
loops, warming glass bottle necks, or igniting alcohol on culture spreaders.
 A Bunsen burner is not practical in some situations, e.g., within a laminar flow unit where the heat will disrupt airflow.
 A microincinerator may be used as an alternative. This consists of a circular heating element. Placing an inoculating loop or
needle within the ring will quickly heat and sterilize the loop/needle.
 Note that a microincinerator does not provide other aseptic technique benefits of a lit Bunsen burner.
The Laminar Flow Unit
 A laminar flow unit (or hood) is a sophisticated appliance that can further help prevent contamination of reagents and
biological cultures.
 Used correctly, it provides the work space with clean, ultrafiltered air.
 It also keeps room air from entering the work area and both suspends and removes airborne contaminants introduced into
the work area by personnel.
 The most important part of a laminar flow hood is a high-efficiency bacteria-retentive filter, i.e., the HEPA (high-efficiency
particulate air) filter.
 A certified HEPA filter must capture a minimum of 99.97% of dust, pollen, mold, bacteria, and any airborne particles with a
size of >0.3 μm at 85 liters/min.
 The first HEPA filters were developed in the 1940s by the U.S.A.Atomic Energy Commission as part of the Manhattan
Project (the development of the atomic bomb) to provide an efficient, effective way to filter radioactive particulate
contaminants.
  HEPA filter technology was declassified after World War II, allowing extensive research and commercial use.
 Laminar flow hoods are essential components of many biosafety level (BSL)-2 laboratories, where they help
prevent spread of viruses and some bacteria.

Direct Microscopic Counts

 Studies involving the analysis of materials including food, water, milk, and, in some cases, air require quantitative
enumeration of microorganisms in the substances.
 Many methods have been devised to accomplish this, including direct microscopic counts, use of an electronic cell counter
such as the Coulter Counter, chemical methods for estimating cell mass or cellular constituents, turbidometric
measurements for increases in cell mass, and the serial dilution–agar plate method.

 Direct microscopic counts require the use of a specialized slide called the Petroff-Hausser counting chamber, in which an
aliquot of a eukaryotic cell suspension is counted and the total number of cells is determined mathematically.
 The Petroff-Hausser counting chamber is a thick glass microscope slide with a chamber 0.02 mm (1/50 mm) deep in the
center.
 The chamber contains an etched grid and has improved Neubauer rulings (1/400 square mm).
 The rulings cover 9 mm2. The boundary lines (Neubauer rulings) are the center lines of the groups of three.
 The center square millimeter is ruled into groups of 16 small squares, and each group is separated by triple lines, the middle
one of which is the boundary.
 The ruled surface is 0.02 mm below the cover glass, which makes the volume over a square millimeter 0.02 mm3 (cubic
mm). All cells are counted in this square millimeter.
 The number of cells counted is calculated as follows:
Number of cells per mm = number of cells counted * dilution * 50,000
[The factor of 50,000 is used in order to determine the cell count for 1 ml: 1 ml = 1000 mm3 = (50 times the chamber depth of 0.02
mm) * 1000.]
A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In
order to detect and prove that thermophilic bacteria were growing in boiling hot springs, T.D. Brock immersed microscope slides in
the springs and withdrew them periodically for microscopic observation. The bacteria in the boiling water attached to the
glass slides naturally and grew as microcolonies on the surface.

Advantages of Direct Microscopic Count

 Rapid, Simple and easy method requiring minimum equipment.
 Morphology of the bacteria can be observed as they counted.
 Very dense suspensions can be counted if they are diluted appropriately.

Limitations of Direct Microscopic Count

 Although rapid, a direct count has the disadvantages that both living and dead cells are counted.
 Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration
to increase sensitivity.
  It is not sensitive to populations of fewer than 1 million cells.
 Small cells are difficult to see under the microscope, and some cells are probably missed.
 Precision is difficult to achieve
 A phase contrast microscope is required when the sample is not stained.

Classification of Protozoa
Protozoa Definition
Protozoa may be defined as  “microscopic acellular animalcules existing
singly or in colonies, without tissue and organs, having one or more
nuclei”.

Some of the characteristics are:

1. There are about 50,000 known species of Phylum Protozoa.
2. Protozoans exhibit mainly two forms of life; free-living (aquatic, freshwater, seawater) and parasitic (ectoparasites or
endoparasites). They are also commensal in habitat.
3. They are small, usually microscopic, not visualize without a microscope.
4. They are the simplest and primitive of all animals.
5. They have a simple body organization. i.e. with a protoplasmic grade of organization.
6. The body is unicellular (without tissue and organs).
7. They have one or more nuclei which are monomorphic or dimorphic.
8. Body naked or bounded by a pellicle, but in some forms may be covered with shells and often provided with an internal
skeleton.
9. They are solitary (existing alone/single) or colonial (individuals are alike and independent).
10. Body shape variables may be spherical, oval, elongated or flattened.
11. Body symmetry either none or bilateral or radial or spherical.
12. Body form usually constant, varied in some, while changing with environment or age in many.
13. Body protoplasm is differentiated into an outer ectoplasm and inner endoplasm.
14. The single-cell body performs all the essential and vital activities, which characterize the animal body; hence
only subcellular physiological division of labor.
15. Locomotory organs are fingers like pseudopodia, whip-like flagella, hair-like cilia or none.
16. Nutrition may be holozoic (animal-like), holophytic (plant-like), saprozoic or parasitic.
17. Digestion occurs intracellularly which takes place inside the food vacuoles.
18. Respiration occurs by diffusion through the general body surface.
19. Excretion occurs through the general body surface, but in some forms through a temporary opening in the ectoplasm or
through a permanent pore called cytopyge.
 20. Contractile vacuoles perform osmoregulation in freshwater forms and also help in removing excretory products.
21. Reproduction asexual (binary or multiple fission, budding, sporulation) or sexual (conjugation (hologamy), game formation
(syngamy)).
22. The life cycle often complicated with alternation of asexual and sexual phases (alternation of generation).
23. Encystment commonly occurs to resist unfavorable conditions of food, temperature, and moisture, and also helps in
dispersal.
24. The single-celled individual not differentiated into somatoplasm and germplasm; therefore, exempt from natural death which
is the price paid for the body.
25. Protozoans exhibit mainly two forms of life; free-living (aquatic, freshwater, seawater) and parasitic (ectoparasites or
endoparasites). They are also commensal in habitat.
26. Examples: Euglena, Amoeba, Plasmodium, Paramecium, Podophyra, etc.

Classification of Phylum Protozoa

Phylum protozoa is a large and varied group and possess a complication in its classification.

The conventional scheme followed by Hyman (1940), Hickman (1961) and Storer (1965), etc. recognizes two subphyla on the basis
of organs of locomotion and 5 classes as follows:

Sub Phylum A: Plasmodroma

 Locomotory organelles are flagella, pseudopodia, or none.
 Nuclei is of one kind.
Class 1: Mastigophora
 Move by one to many flagella.
 Example: Euglena.
Class 2: Sarcodina
 Move and capture food by pseudopodia.
 Example: Amoeba.
Class 3: Sporozoa
 No locomotory organs.
 All parasitic.
 Spore-formation is common.
 Example: Plasmodium.
ub Phylum B: Plasmodroma
 Locomotory organelles are cilia or sucking tentacles.
 Nuclei of two kinds.
Class 4: Ciliate
 Move by cilia.
  Example: Paramecium.
Class 5: Suctoria
 Move by cilia as young and by tentacles as an adult.
 Example: Podophyra.
Another classification is based on the scheme given by the Committee on Taxonomy and Taxonomic Problems of the
Society of Protozoologists, and mainly proposed by BM Honigberg and others (1964).
It divides protozoa into four subphyla.
Subphylum I: Sarcomastigophora
Subphylum II: Sporozoa
Subphylum III: Cnidospora
Subphylum IV: Ciliophora

Subphylum I: Sarcomastigophora
 Locomotor organelles are pseudopodia or flagella.
 The nucleus is of a single type (monomorphic).
 There is no spore formation.
 Syngamy occurs in reproduction.

Superclass A: Mastigophora
 They are commonly called flagellates.
 Locomotory organelles are flagella in adults.
 The body is covered by a pellicle.
 Binary fission is longitudinal.
 They are mostly free-living though some are parasitic.
 Nutrition is autotrophic or heterotrophic or both.
Class 1: Phytomastigophorea
 Chlorophyll-bearing chromatophores present.
 Nutrition mainly holophytic by phototrophy.
 Reserve food is starch or paramylon.
 They have usually only one or two flagella.
 The nucleus is vesicular.
Order 1: Chrysomonadina.
 Examples: Chromulina, Ochromonas, Dinobryon, Synura, Chrysamoeba, etc.
Order 2: Coccolithophorida.
 Examples: Coccolithus, Rhabdosphaera, etc.
Order 3: Heterochloride.
 Examples: Heterochloris, Myxochloris, etc.
Order 4: Cryptomonadida.
 Examples: Chilomonas, Cryptomonas, etc.
Order 5: Dinoflagellida.
 Examples: Noctiluca, Ceratium, etc.
Order 6: Euglenida.
 Examples: Euglena, Phacus, Copromonas, Peranema, etc.
Order 7: Volvocida (Phytomonadida).
 Examples: Volvox, Chlamydomonas, Eudorina, etc.
Order 8: Chloromonadida.
 Examples: Vacularia, Coelomonas, Gonyostomum, etc.
Class 2: Zoomastigophorea
 Chlorophyll or chromatophores absent.
 Mostly parasitic.
 Reserve food as glycogen.
 Flagella one to many.
 There is an undulating membrane.
Order 1: Choanoflagellida.
 Example: Proterospongia.
Order 2: Rhizomastigida.
 Examples: Mastigoamoeba, Dimorpha, etc.
Order 3: Hypermastigida.
 Examples: Trichonympha, Lophomonas, Leptomonas, etc.
Order 4: Diplomonadida.
 Examples: Giardia, Hexamita, etc.
Order 5: Kinetoplastida.
Suborder 1: Bodonina.
 Examples: Bodo.
Suborder 2: Trypanosomatina.
 Examples: Trypanosoma, Leishmania, etc.
Order 6: Bicosoecida
 Examples: Salpingoeca, Poteriodendron, etc.
Order 7: Retortamonadida.
 Example: Chilomonas.
Order 8: Oxymonadida.
 Example: Oxymonas, Pyrsonympha, etc.
Order 9: Trichomonadida.
 Example: Trichomonas.
Superclass B: Opalinata
 They have numerous cilia like organelles in oblique rows over the entire body surface.
 There is no cytostome.
 Two or more monomorphic nuclei are present.
 Binary fission is interkinetal.
 There is syngamy with flagellated anisogametes.
 All are parasitic, mainly in frogs and toads.
 Examples: Opalina, Protoopalina, Zelleriella, Protozelleriella, and Cepedea.

Superclass C: Sarcodina
 Locomotory organelles are pseudopodia.
 The amoeboid form is predominant.
 Some have a hard shell.
 They generally do not form spores.
 The formation of gametes and flagellated young ones are common.
 Nutrition holozoic or saprozoic.
Class 1: Rhizopodea
 Locomotory organelles are pseudopodia (lobopodian or filopodia but never axopodia).
 They are generally creeping forms.
Subclass a: Lobosia
 Pseudopodia as lobopodian.
Order 1: Amoebida.
 Examples: Amoeba, Entamoeba, Pelomyxa, etc.
Order 2: Arcellinida.
 Examples: Arcella, Diffugia, Euglypha, etc.
Subclass b: Filosia
 They have tapering and branching filopodia.
 Examples: Gromia, Allogromia, Penardia (naked).
Subclass c: Granuloreticulosia
 They have finely granular reticulose rhizopodia (reticulopodia).
Order 1: Foraminiferida
 Examples: Globigerina, Elphidium, etc.
Subclass d: Mycetozoia
 The amoeboid trophic stage develops either into a multicellular aggregation or into a true multinucleate plasmodium.
 The life cycle is complex and has sexual reproduction.
 Usually, sporangia are formed which liberate spores.
 Nutrition is phagocytic.
 Example: Plasmodiophora.
Class 2: Actinopodea
 Pseudopodia mainly axopodia with axial filaments, radiating from a spherical body.
 They are primarily sessile or floating forms.
 Gametes are usually flagellated.
 Reproduction is both sexual and asexual.
Subclass a: Radiolaria
 The central capsule is perforated by one to many pores.
 They have spicules or siliceous skeleton.
 Filopodia or axopodia are present.
 The capsule separates the protoplasm into ectoplasm and endoplasm.
 All are marine.
 Examples: Thalassicola, Collozoum, Lithocircus, etc.
Subclass b: Acantharia
 Imperforate, non-chitinoid central capsule without pores.
 The anisotropic skeleton of strontium sulfate.
 Axopodia present.
 Marine
 Example: Acanthometra.
Subclass c: Heliozoia
 There is no central capsule.
 Rounded body with radiating axopodia.
 Usually naked, if a skeleton is present it is made of siliceous scales and spines.
 They have axopodia or filopodia.
 There may be more than one nucleus, mostly in freshwater.
 Examples: Actinophrys, Actinosphaerium, Clathrulina, etc.
Subclass d: Proteomyxidia
 Largely marine and freshwater parasites of algae and higher plants.
 Filopodia and reticulopodia in some species.
 Examples: Vampyrella, Pseudospora, etc.
Class 3: Piroplasmea
 Small, round-shaped or amoeboid parasites in vertebrate red blood cells.
 Example: Babesia.
Subphylum II: Sporozoa
 Locomotory organelles absent.
 Spores usually present.
 Exclusively endoparasites.
 Cilia or flagella may be present in gametes.
 Syngamy takes place after which many spores are formed.
 The spores are simple and contain one to many sporozoites.
 Sporozoites are the infective phase.
 The nucleus is of the single type.

Class 1: Telosporea
 Pseudopodia are generally absent.
 Locomotion by gliding or body flexion.
 Spores are formed and there are flagellated microgametes in some.
 Spores are without polar capsules and filaments, naked or encysted.
 Reproduction by both sexual and asexual methods.
Subclass a: Gregarinia
 Mature trophozoites are large and extracellular.
 Reproduction is entirely sexual with sporogony.
 The spores contain eight sporozoites.
  They are parasites of the digestive tract and body cavity of invertebrates.
 Examples: Gregarina, Monocystis, Nematocystis, etc.
Subclass b: Coccidia
 Mature trophozoites are small and typically intracellular.
 Each oocyst produces many sporozoites.
 They are parasites of the digestive tract or blood of vertebrates.
 Gametocytes are dimorphic.
 Sporozoites multiply by schizogony in tissue cells.
 Examples: Eimeria, Isospora, Plasmodium, etc.
Order 1: Eucoccida
 Schizogony takes place.
 Both sexual and asexual phases take place.
 They are parasitic in epithelial and blood cells of invertebrates and vertebrates.
Suborder 1: Eimeriina
 Macrogamete and microgametocyte develop independently.
 There is no syzygy.
 Macrogametocyte produces many microgametes.
 The zygote is non-motile.
 Oocyst does not increase the size during sporogony.
 Sporozoites are encased in sporocyst.
 Example: Eimeria.
Suborder 2: Haemosporina
 Macrogamete and microgametocyte develop independently.
 There is no syzygy.
 Microgametocyte produces only a few microgametes.
 Zygote of often motile.
 Oocyst increases size during sporogony.
 Sporozoites are naked.
 Schizogony takes place in vertebrates and sporogony in an invertebrate host.
 Hemoglobin of host cells forms pigment.
 Example: Plasmodium.

Class 2: Toxoplasmea
 Spores are absent.
 There are no flagella or pseudopodia at any stage.
 Reproduction by asexual reproduction (binary fission).
 Cysts are formed which have many naked sporozoites.
 Examples: Sarcocystis, Toxoplasma, etc.

Class 3: Haplosporea
 Spores are present.
 Pseudopodia may be present but flagella are absent.
 Reproduction only by an asexual method.
 Schizogony takes place.
 Examples: Caelosporidium, Ichthyosporidium, etc.

Subphylum III: Cnidospora

 Spores have several cells having one or more polar filaments which are coiled threads and can be shot out, and one or more
sarcoplasms or sporoplasms (analogous to sporozoites).
 All are parasitic.
 Zygote gives rise to one or more trophozoites without sporogony.

Class 1: Myxosporidea
 Spores are of multicellular origin and large.
 There are one or more sporoplasms with two or three valves.
  They are parasites of fish.
 Examples: Myxobolus, Myxidium, Ceratomyxa, etc.

Class 2: Microsporidea
 Spores are of unicellular origin and small.
 There is one long tubular polar filament through which the sporoplasms emerges one valve only.
 They are cytozoic (intracellular parasites) in arthropods and vertebrates.
 Example: Nosema.

Subphylum IV: Ciliophora

 They possess simple ciliary organelles for locomotion, infraciliature is subpeculiar.
 They have two nuclei, a trophic macronucleus, and a reproductive micronucleus.
 Binary fission is perkinetal.
 Conjugation takes place with the fusion of nuclei, autogamy and cytogamy also occur.
 There are never any free gametes.
 Nutrition is mixotrophic or heterotrophic.
 They usually have a cytostome.

Class 1: Ciliata
 They possess cilia or compound ciliary structure as locomotory or food acquiring organelles.
 There is the presence of an infraciliary system, composed of basal granules below the cell surface and interconnected by
longitudinal fibrils.
 Most ciliates possess a cell mouth or cytostome.
 Anal aperture (cytopyge) permanent.
 Two types of nuclei, one vegetative (macronucleus) and the other reproductive (micronucleus).
 Fission is transverse.
 Sexual reproduction never involves the formation of free gametes.
 One or more contractile vacuoles present even in marine and parasitic types.
Subclass 1: Holotricha
 Body cilia simple and uniform.
 Buccal cilia mostly absent.
Order 1: Gymnostomatida.
 Examples: Coleps, Dileptus, Didinium, Prorodon, Nassula, etc.
Order 2: Trichostomatida.
 Examples: Colpoda, Balantidium, etc.
Order 3: Chonotrichida.
 Examples: Spirochona, Lobochona, Chilodochona, etc.
Order 4: Apostomatida.
 Example: Hyalophysa.
Order 5: Astomatida.
 Examples: Anoplophyra, Maupasella, Hoplitophyra, etc
Order 6: Hymenostomatida.
 Examples: Colpidium, Tetrahymena, Paramecium, etc.
Order 7: Thigmotrichida.
 Examples: Thigmophyra, Boveria, etc.
Subclass 2: Peritricha
 Adults without body cilia.
 Apical end with buccal cilia.
Order 1: Peritrichida.
 Examples: Vorticella, Carchesium, Trichodina, etc.
Subclass 3: Suctoria
 Sessile and stalked body.
 Young with cilia, and adult with suctorial tentacles.
Order 1: Suctorida.
 Examples: Acineta, Ephelota, Podophyra, etc.
Subclass 4: Spirotrichia
 Reduced body cilia.
 Buccal cilia are well marked.
Order 1: Heterotrichida.
 Examples: Stentor, Bursaria, Spirostomum, Nyctotherus, etc.
Order 2: Oligotrichida.
 Examples: Halteria, Strombidium.
Order 3: Tintinnida.
 Examples: Codonella, Favella, etc.
Order 4: Entodinomorphida.
 Examples: Entodinium, Cycloposthium, etc.
Order 5: Odontostomatida.
 Example: Saprodinium.
Order 6: Hypotrichida.
 Examples: Euplotes, Stylonychia, Urostyla, Oxytricha, etc.

Bacterial and Fungal Preservation Methods

 Biopreservation is the process of preserving the integrity and functionality of cells.
 Most bacteriological laboratories maintain stock cultures of microorganisms for educational, research, bioassay, industrial, or
other purposes.
 A wide variety of techniques are available for the preservation of bacteria and it may be difficult to choose a method for a
particular strain, which not only assures survival, but which also makes certain that the genotype and hence the unique
characteristics do not change. 
 The primary aim of culture preservation is to maintain the organism alive, uncontaminated, and without variation or mutation,
that is, to preserve the culture in a condition that is as close as possible to the original isolate.

Agar Slant Cultures

 All microbiology laboratories preserve micro-organisms on agar slant.
 The agar slants are inoculated and incubated until good growth appears.
 They are then covered with sterile mineral oil to a depth of 1 cm above the tip of slant surface.
 The slants are incubated for 24hr or more and are then stored in a refrigerator.
 These cultures are periodically transferred to fresh media.
 Transfers are made by removing a loop full of the growth, touching the loop to the glass surface to drain off excess oil,
inoculating a fresh medium and then preserving the initial stock culture.
 Time intervals at which the transfers are made which varies with the origin and condition of growth.
  This is a simple and most economical method of preserving bacteria and fungi where they remain viable for several years at
room temperature.

Refrigeration
 Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms.
 This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities
of the microorganisms are greatly slowed down but not stopped.
 Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the
death of the microbes after sometime.

Paraffin Method
 This is a simple and most economical method of maintaining pure cultures of bacteria and fungi.
 In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature.
 The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium.
 This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for
several years.

Saline Suspension
 Sodium chloride in high concentration is frequently an inhibitor of bacterial growth.
 Bacteria are suspended in 1% salt solution (sublethal concentration in screw cap tubes to prevent evaporation).
 The tubes are stored at room temperature. Whenever needed the transfer is made on agar slant.

Cryopreservation
 Cryopreservation (i.e., freezing in liquid nitrogen at-196°C) helps survival of pure cultures for long storage times.
 In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing
agents such as glycerol, that prevent the formation of ice crystals and promote cell survival.

Lyophilisation (Freeze-Drying)
 In this method, the culture is rapidly frozen at a very low temperature (around -70°C) and then dehydrated by vacuum.
 Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the
microbes go into dormant state and retain viability for years.
 Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators.
 Freeze- drying method is the most frequently used technique by culture collection centres.
The Lyophilisation Process
 In this process the microbial suspension is placed in small vials.
 A thin film is frozen over the inside surface of the vial by rotating it in mixture of dry ice (solid carbon dioxide) and alcohol, or
acetone at a temperature of −78oC .
 The vials are immediately connected to a high vacuum line. This dries the organism while still frozen.
 Finally, the ampules are sealed off in a vacuum with small flame.
 These cultures can be stored for several years at 40°C.
 This method is also employed for preservation of toxins, sera, enzymes and other biological material.
 To revive microbial cultures it is merely necessary to break open the vial aseptically, add a suitable stale medium, and after
incubation make further transfers.
 The process permits the maintenance of longer number of culture without variation in characteristics of the culture and
greatly reduces the danger of contamination.
Preservation at Very Low Temperature
 The organisms are suspended in nutrient broth containing 15% glycerol.
 The suspension is frozen and stored at -15°C to -30°C.

Preservation by Drying in Vacuum

 The organisms are dried over calcium chloride in the vacuum and are stored in the refrigerator.

Spread Plate Technique- Principle, Procedure, Advantages

Spread Plate Technique
 A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or
from the sample cultures.
 The spread plate method is a technique to plate a liquid sample containing bacteria so that the bacteria are easy to count
and isolate.
 A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate.
 Spread plate culture technique is among the most widely used culture technique for isolating the bacteria.

Principle
In this technique, a serially diluted specimen containing 2 or more bacteria or microbe (Mixed culture) is used which is spread over
the solidified agar media plates as a thin layer with the help of a sterile L-shape glass rod (Spreader) while the media plate is spun
on a turntable.
The principle behind this method is that when the Media plate is spun, at some stage, single cells will be deposited with the bent
glass rod (Spreader) onto the surface of the Agar media. Some of the cells present in the specimen / diluted specimen will be
separated from each other by a distance sufficient to allow the colonies that develop to be free from each other.

Procedure
1. Make a dilution series from a sample.
2. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate.
3. Dip the L-shaped glass spreader into alcohol.
4. Flame the glass spreader (hockey stick) over a Bunsen burner.
5. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petri
dish underneath at the same time.
6. Incubate the plate at 37°C for 24 hours.
7. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine
the number of CFU/mL in the original sample.
Applications
 The Spread Plate Technique, in conjunction with serial dilutions, is a valuable research tool.
 To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size, elevation, etc.).
 To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium.
 For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances.
 To obtain the sufficient growth of the bacterium for various biochemical and other tests.
 To estimate the viable counts of the bacteria in the specimen.
 To maintain the stock cultures.
 To transport or short-term storage of the specimen (e.g. stab culture).

Advantages
1. Heat sensitive microbes are not affected.
2. No subsurface colonies appear in spread plate so isolation of the organism is easy.

Limitations
 Strict aerobes are favored while microaerophilic tends to glow slower.
 Crowding of the colonies makes the enumeration difficult.
 Accurate dilutions using pipettes should be made.
 Volume no greater than 0.1 ml can be spread on the nutrient agar plate because it would not soak well and may result in
colonies to coalesce as they form.

Classification of Bacteria on the basis of Nutrition

 Nutrition is substances used in biosynthesis and energy production and therefore are required for all living things.
 Bacteria, like all living cells, require energy and nutrients to build proteins and structural membranes and drive biochemical
processes.
 Bacteria require sources of carbon, nitrogen, phosphorous, iron and a large number of other molecules.
 Carbon, nitrogen, and water are used in the highest quantities.
 The nutritional requirements for bacteria can be grouped according to the carbon source and the energy source.
 Some types of bacteria must consume pre-formed organic molecules to obtain energy, while other bacteria can generate
their own energy from inorganic sources.
Nutritional Types of Bacteria
On the basis of energy source organisms are designated as:
Phototrophs:
 The organisms which can utilize light as an energy source are known as phototrophs. These bacteria gain energy from light.
Chemotrophs:
 These bacteria gain energy from chemical compounds. They cannot carry out photosynthesis.
On the basis of electron source organisms are designated as:
Lithotrophs: 
 Some organisms can use reduced organic compounds as electron donors and are termed as Lithotrophs.
 They can be Chemolithotrophs and Photolithotrophs
Organotrophs: 
 Some organisms can use organic compounds as electron donors and are termed as organotrophs.
 Some can be Chemoorganotrophs and Photoorganotrophs.
Thus, bacteria may be either:
 Photo-lithotrops: These bacteria gain energy from light and use reduced inorganic compounds such as H2S as a source of
electrons. eg: Chromatium okeinii.
 Photo-organotrophs: These bacteria gain energy from light an d use organic compounds such as Succinate as a source of
electrons.eg; Rhodospirillum.
 Chemo-lithotrophs: These bacteria gain energy from reduced inorganic compounds such as NH3 as a source of electron
eg; Nitrosomonas.
 Chemo-organotrophs: These bacteria gain energy from organic compounds such as glucose and ammino acids as a
source of electrons.eg; Pseudomonas pseudoflora.
 Some bacteria can live ether chemo-lithotrophs or chemo-organotrophs like Pseudomonas pseudoflora as they can use
either glucose or H2S as electron source.
On the basis of carbon source bacteria may be:
 All organisms require carbon in some form for use in synthesizing cell components.
 All organisms require at least a small amount of CO2.
 However, some can use CO2 as their major or even sole source of carbon; such organisms are termed as Autotrophs
(Autotrophic bacteria).
 Others require organic compounds as their carbon source and are known as Heterotrophs (Heterotrophic bacteria).

Autotrophic Bacteria
These bacteria synthesize all their food from inorganic substances (H2O, C02, H2S salts).
The autotrophic bacteria are of two types:
(i)  Photoautotrophs
 These bacteria capture the energy of sunlight and transform it into the chemical energy.
 In this process, CO2 is reduced to carbohydrates.
 The hydrogen donor is water and the process produce free oxygen.
 Photoautotroph has Chlorophyll pigment in the cell and its main function is to capture sunlight e.g., Cyanobacteria.
 Some photoautotrophic bacteria are anaerobes and have bacteriochlorophyll and bacteriovirdin pigments respectively.
Purple Sulphur Bacteria:
These bacteria have the pigment bacteriochlorophyll located on the intracytoplasmic membrane i.e., thylakoids. These bacteria
obtain energy from sulfur compounds e.g., Chromatiiun. Theopedia rosea, Thiospirilium.
Green Sulphur Bacteria:
These bacteria use hydrogen sulfide (H2S) as hydrogen donor. The reaction takes place in the presence of light and pigment
termed as bacteriovirdin or bacteriopheophytin or chlorobium chlorophyll e.g., Chlorobium limicola, Chlorobacterium etc.
These bacteria take hydrogen from inorganic sources like sulphides and thiosulphates. Therefore, these bacteria are also known
as photolithographs.
(ii) Chemoautotrophs
 These bacteria do not require light (lack the light phase but have the dark phase of photosynthesis) and pigment for their
nutrition.
 These bacteria oxidize certain inorganic substances with the help of atmospheric oxygen.
 This reaction releases the energy (exothermic) which is used to drive the synthetic processes of the cell.
Sulphomonas (Sulphur bacteria):
These bacteria obtain energy by oxidation of elemental sulphur or H2S, e.g., Thiobacillus, Beggiatoa.
 Elemental Sulphur Oxidising Bacteria: Denitrifying sulphur bacteria oxidize elemental sulphur to sulphuric acid
e.g., Thiobacillus denitrificans
2S + 2H2O + 3O2 → 2H2SO4 + 126 kcal.
  Sulphide Oxidizing Bacteria: These bacteria oxidizes H2S and release the sulphur e.g., Beggiatoa.
2H2S +4O2 → 2H2O + 2S + 141.8 cal
Hydromonas (Hydrogen bacteria)
 These convert hydrogen into water, e.g., Bacillus pantotrophus, Hydrogenomonas.
2H2 + O2 → 2H2O + 55 kcal.
4H2 + CO2 → 2H2O + CH4 + Energy
Ferromonas (Iron bacteria):
 These bacteria inhabit water and obtain energy by oxidation of ferrous compounds into ferric forms.
e.g., Thiobacillus ferroxidans, Ferro bacillus, Leptothrix.
4FeCo3 + 6H2O + O2 → 4Fe (OH)3 + 4CO2 + 81 kcal.
Methanomonas (Methane bacteria):
 These bacteria get their energy by oxidation of methane into water and carbon dioxide.
Nitrosomonas (Nitrifying bacteria):
 These bacteria get their energy by oxidation of ammonia and nitrogen compounds into nitrates.
 Nitrosomonas oxidises NH3 to nitrites. NH3 + ½O2 ® H2O + HNO2 + Energy
 Nitrobacter converts nitrites to nitrates. NO2 + ½O2 ® NO2 + Energy
Carbon Bacteria:
These bacteria oxidizes CO into CO2 e.g., Bacillus oligocarbophillous, Oligotropha carboxydovorans
2CO + O2 → 2CO2 + Energy

Heterotrophic Bacteria
 The heterotrophic bacteria obtain their-ready made food from organic substances, living or dead.
 Most of pathogenic bacteria of human beings, other plants and animals are heterotrophs.
 Some heterotrops have simple nutritional requirement while some of them require large amount of vitamin and other growth
promoting substance. Such organisms are called fastidious heterotrophs.
 Heterotrophic bacteria are of three types:
a. Photoheterotrophs
 These bacteria can utilize light energy but cannot use CO2 as their sole source of carbon.
 They obtain energy from organic compounds to satisfy their carbon and electron requirements. Bacteriochlorophyll pigment
is found in these bacteria.
 g., Purple non-sulphur bacteria (Rhodospirillum, Rhodomicrobium, Rhodopseudomonas palustris).
b. Chemoheterotrophs
 Chemoheterotrophs obtain both carbon and energy from organic compounds such as carbohydrates, lipids and proteins.
Glucose or Monosaccharide [(CH2O)n] + O2 → CO2 + H2O + Energy
There are three main categories that differ in how chemohetrotrophs obtain their organic nutrients:(i) Saprophytic bacteria.
(ii) Parasitic bacteria.
(iii) Symbiotic bacteria.
i) Saprophytic bacteria
 Saprophytic bacteria obtain their food from the dead and organic decaying matter such as leaves, fruits, vegetables, meat,
animal feces, leather, humus etc.
 These bacteria secrete enzymes to digest the food and absorb it.
 The enzymes secreted to break down the complex compounds such as carbohydrate and protein, into simpler soluble
compounds, which are easily absorbed.
 Examples are Bacillus mycoides, B. ramosus, Acetobacter etc.
ii) Parasitic bacteria
 These bacteria obtain their nutrition from the tissues of the hosts on which they grow.
 They may be harmless or may cause serious diseases.
 Parasitic bacteria which cause various diseases in plants and animals are known as pathogens, e.g., Bacillus typhosus,
B. anthracis, B.tetani. B.diplheriae, B.tuberculosis, B. pneumoniae, Vibrio cholerae, Pseudomonas citri etc.
iii) Symbiotic bacteria
 Symbiotic bacteria live in close association with other organisms as symbionts.
 They are beneficial to the organisms.
 The common examples are the nitrogen-fixing bacteria, e.g., Bacillus radicicola, B. azotobacter, Rhizobium,
Clostridium, Rhizobium spp., B. radicicolaand B. azotobacter.
 These bacteria live inside the roots of leguminous plants.
 These bacteria fix free atmospheric nitrogen into nitrogenous compounds which are utilized by the plants. In return, the plant
provides nutrients and protection to the bacteria.
Classification of Fungi
Classification of Fungi
 Fungi are eukaryotic microorganisms. They can occur as yeasts, molds, or as a combination of both forms.
 Some fungi are capable of causing superficial, cutaneous, subcutaneous, systemic or allergic diseases.
 Yeasts are microscopic fungi consisting of solitary cells that reproduce by budding. Molds, in contrast, occur in long filaments
known as hyphae, which grow by apical extension.
 Regardless of their shape or size, fungi are all heterotrophic and digest their food externally by releasing hydrolytic enzymes
into their immediate surroundings (absorptive nutrition).
 Other characteristics of fungi are the ability to synthesize lysine by the L-α-adipic acid biosynthetic pathway and possession
of a chitinous cell wall, plasma membranes containing the sterol ergosterol, 80S rRNA, and microtubules composed of
tubulin.

Classification of Fungi
 The classification of fungi, like that of bacteria, is designed mainly for practical application but it also bears some relation to
phylogenetic considerations.
 The nomenclature is binomial, with a generic and a specific name (eg: Aspergillus niger).
 Species are collected in genera, genera in families (suffix –aceae), families in orders (suffix-ales), and orders in classes
(suffix-mycetes).
 The division of mycota, or fungi and moulds, includes the true slime moulds (Myxomycetes), the lower fungi (Phycomycetes),
and the higher fungi (Eumycetes).
 Alexopolous and Mims proposed fungal classification in 1979. They place the fungi including the slime molds in the
kingdom mycetae of the super kingdom Eukaryota which, in addition, includes four other kingdoms.
 They divide the kingdom mycetae into three divisions namely:
1. Gymnomycota
2. Mastigomycota and
3. Amastigomycota
 The division is subdivided into subdivision, classes, sub-classes, and orders.

Division I Gymnomycota
 It includes phagotrophic organism devoid of cell walls.
  This division comprises two subdivisions.
 These are Acrasiogymnomycotina and Plasmodiogynomycotina.
Subdivision 1. Acrasiogymnomycotina
It includes a single class Acrasiomycetes.
Class 1. Acrasiomycetes  
 Lacks flagellated cells except for one species. The class comprises two subclasses.
 Acrasiomycetidae and Dictyosteliomycetidae.
Subdivision 2. Plasmodiogymnomycotina
It is divided into two classes:
Class 1 Protosteliomycetes
Class 2 Mycomycetes
It includes the true slime mold and comprises three sub class namely:
Sub class 1. Ceratiomyxomycomycetidae
Order – Ceratiomyxales
Sub Class 2. Mycogasteomycetidae
 It comprise four orders.
Order
1. Liceales
2. Echinosteleales
3. Trichlales
4. Physarales
Sub Class 3. Stemonitomycetidae
Order 1. Stemonitales

Division II Mastigomycota
 Includes fungi with absorptive nutrition, unicellular or filamentous, mycelium coemocytic.
 It comprises two sub divisions:
Sub division 1 Haplomastigomycotina
 Includes fungi with uni-or, bi-flagellate zoospores.
Class 1 Chytridiomycetes– Fungi producing zoospores furnished with a single whiplash flagellum inserted at the posterior end.
Class 2 Hyphochytridiomycetes- Motile cells with a single tinsel flagellum inserted at the anterior end.
Class 3 Plasmodiophoromycetes- Parasitic fungi producing biflagellate motile cells with both the flagella of whiplash type inserted
at the anterior end.
Sub division 2. Diplomastigomycotima Sexual reproduction ooagamous, zoospores biflagellate.
Class 1 Oomycetes
– It comprises four orders:
Order 1 Lagenidiales
Order 2 Saprolegnailes
Order 3. Leptomitales
Order 4. Peronosporales

Division III Amastigomycota

Fungi with absorptive nutrition, motile cells lacking, mycelium aseptate or septate.
This includes four sub divisions:
Sub division 1 Zygomycotina
Class 1 Zygomycetes – it includes six orders.
Class 2 Trichomycetes – it comprises five orders.
Sub division 2 Ascomycotina
Fungi usually with a septate mycelium producing haploid ascospores in sac like cells called asci.
Class 1 Ascomycetes- divided into five sub classes:
Sub class 1. Hemiascomycetidae- comprising three orders.
Sub class 2. Plectomycetidae- Five orders
Sub class 3. Hymenoascomycetidae – Ten orders
Sub class 4 Laboulbeniomycetidae – Two orders
Sub class 5 Lowloascomycetidae – five orders
Sub division 3. Basidiomycotina
Septate mycelium, produces basidiospores, exogenously on various types of basidia.
Class 1 Basidiomycetes: it is split into 3 sub clases:
Sub class 1 Holobasidiomycetidae
Sub class 2 Phragmobasidiomycetidae
Sub class 3 Teliomycetidae
Sub division 4. Deuteromycotina
It includes imperfect fungi in which sexual stage is unknown. It comprises a single form class.
Form Class Deuteromycetes with three form sub classes namely Blastomycetidae, Coelomycetidae and Hyphomycetidae.
On the Basis of Spore Production
On the basis of the organisation of the vegetative thallus, the morphology of reproductive structures, the way of spores production
and particular life cycle involved the kingdom mycota is classified into following divisions.
Phycomycetes
 It includes the simplest type of fungi. It is also called as Algae-Fungi because most of the characteristics of them are similar
to algae like Vaucheria.
 They have simple thallus which is unicellular or coenocytic or aseptate filaments.
 They reproduce asexually by the formation of zoospores or non-motile spores.
 Sexual reproduction is isogamous or heterogamous which takes place by gametangial contact.
 The diploid phase is represented by zygote.
 Phycomycetes has been classified into subclasses: oomycetes and zygomycetes.
Oomycetes
 Oomycetes range from a primitive unicellular thallus to a profusely branched filamentous mycelium.
 Many members of them are terrestrial and obligate parasites.
 Asexually they reproduce by biflagellate zoospores.
 Sexual reproduction is oogamy that involves the fusion of male and female gametes to form oospore.
 Oospore undergoes meioses to produce haploid biflagellate zoospores.
 Example; Phytophthora infestans(causes potato blight)
Zygomycetes
 The group is named zygomycetes because a diploid resting spore called the zygospore is formed during the life cycle.
 They are mostly saprophytic, some others are parasites on plants and animals.
 The vegetative body is mycelium which is well developed, profusely branched and coenocytic.
 The absence of motile sexual or asexual cells.
 The asexual reproduction takes place by sporangiospores, aplanospores or by conidia.
 Sexual reproduction occurs by conjugation of gametangia resulting in the formation of zygospore.
 Examples; Rhizopus, Mucor etc
Ascomycetes
 The species of ascomycetes are called the sac fungi because they produce sexual pores within the sac-like vascus.
 General Characteristics
 Ascomycetes are mostly terrestrial occurring as saprophytes or parasites.
 They have well-developed, branched, septate mycelium except yeast. Yeast is a unicellular fungus.
 Asexually they reproduce by non-motile spores, conidia, oidia or chlamydospores.
 Sexual reproduction takes place by the fusion of gametangia of opposite mating types.
 There is absence of motile cells.
 Examples, Saccharomyces cerevisiae, Penicillium, Aspergillus etc.
Basidiomycetes
 The members of basidiomycetes are saprophytic or parasitic. The group is named basidiomycetes as they produce the
basidiospores at the club-shaped basidium during sexual reproduction.
 Mycelium is highly developed, profusely branched and septate.
 The mycelia are differentiated into two mating types; (+ve) and (-ve).
 There are two kinds of mycelium; primary mycelium and secondary mycelium.
 Asexual reproduction takes place by fragmentation, budding, oidia, conidia or chlamydospore.
 The dikaryotic cell is formed during sexual reproduction.
 The absence of motile cell throughout the life cycle.
 Basidiomycetes are the most advanced fungi as their fructifications are often large and prominent.
 Examples; Mushrooms, Puccinia, Ustilago etc.
Deuteromycetes (The Imperfect Fungi)
 Deuteromycetes compromises more than 17000 species of the diverse habits and habitats. It is considered as an artificial
class of fungi.
 The fungi are saprophytes as well as parasites.Parasitic fungi cause serious diseases to plants, animals including human
beings.
 Some of them are unicellular while others are multicellular.
 They reproduce asexually by conidia along with some other types of spores.
 The sexual reproduction is entirely absent.
  The asexual stage or imperfect stage in Deuteromycetes is well defined. But the sexual or perfect stage is absent
in life cycle, therefore, they are called ‘Fungi Imperfecti’.
 Example; Alternaria, Fusarium, Helminthosporium etc.

Classification of Medically Important Fungi

Classification Based on Site
Mycoses are classified as superficial, cutaneous, subcutaneous, or systemic (deep) infections depending on the type and degree of
tissue involvement and the host response to the pathogen.
Superficial mycoses (or tineas) mostly occur in the tropics and are restricted to the outer surface of the hair and skin. Examples
are:
 Piedraia hortae, a filamentous member of the Ascomycota which causes black piedra, a disease of the hair
shaft characterised by brown/black nodules on the scalp hair (actually the ascostromata of the fungus).
 Trichosporon cutaneum, a yeast belonging to the Basidiomycota that is common in soil, water samples, plants, mammals
and birds, as well as being a member of the normal flora of mouth, skin and nails. It causes white piedra, a
superficial infection of the skin, and scalp and pubic hair (although it is emerging as an opportunistic pathogen of the
immunocompromised).
Cutaneous mycoses. There are three genera of fungi that commonly cause disease in the non-living tissues of skin, hair, or
nails/claws of people and animals, by growing in a zone just above where the protein keratin is deposited.
These three genera are Microsporum, Trichophyton and Epidermophyton (all filamentous Ascomycota) and they are
often labelled ‘dermatophytes’ (with the disease being called ‘dermatophytosis’) although, of course, they are not plants, so they
can’t be any sort of ‘-phyte’ and a better term would be dermatomycosis. These fungi all have the ability to degrade keratin and
grow as non-invasive saprotrophs on skin and its appendages, but their growth causes irritation and inflammation of underlying
epithelial cells, this being an allergic reaction that may result in death of these cells.
Subcutaneous mycoses are generally caused by fungi that are normally saprotrophic inhabitants of soil, particularly in tropical and
subtropical areas of Africa, India and South America, which become infective by being introduced through wounds in the skin. Most
infections involve people who normally walk barefoot.
 Madurella mycetomatis and M. grisea (filamentous, Ascomycota) cause human mycetoma (common name: madura foot),
which is a localised infection causing locally invasive tumour-like abscesses, accompanied by chronic inflammation, resulting
in swelling, distortion and ulceration of the infected body part. The fungus is introduced through mild wounds in the skin and
may grow for several years in the cutaneous and subcutaneous tissues, extending to connective tissues and bones.
Mycetomas are usually resistant to chemotherapy, leaving surgery, even amputation, as the only resolution.
 Sporothrix schenckii (thermally dimorphic, Ascomycota) causes sporotrichosis. Sporothrix is the anamorph
and Ophiostoma stenoceras the teleomorph. The fungus occurs in soil worldwide although the disease is localised, with Peru
having the highest prevalence of Sporothrix schenckii infections. Also called ‘rose handler’s disease’, sporotrichosis starts
by entry of the fungus through minor skin injury and can then spread through the lymphatic system. The fungus is dimorphic,
forming septate vegetative hyphae, conidiophores and conidia at 25°C, while at 37°C oval to cigar-shaped budding yeast
cells are produced. As the yeast form is distributed by the lymphatic system, disseminated sporotrichosis can result in
infections of the lungs and bones and joints, endophthalmitis (inflammation of the internal layers of the eye), meningitis and
invasive sinusitis.
Systemic mycoses are infections that affect the whole body. We divide these into mycoses due to primary (usually dimorphic)
virulent pathogens, and those due to opportunistic pathogens. 

Classification Based on Route of Acquisition

 Infecting fungi may be either exogenous or endogenous.
 When classified according to the route of acquisition, a fungal infection may be designated as exogenous or endogenous in
origin.
 If classified as exogenous, an infecting organism may be transmitted by airborne, cutaneous, or percutaneous routes.
 An endogenously-acquired fungal infection may be acquired from colonization or reactivation of a fungus from latent
infection.

Classification Based on Virulence

Primary pathogens can establish infections in normal hosts. Opportunistic pathogens cause disease in individuals with compromised
host defense mechanisms.
 Deep mycoses are caused by primary pathogenic and opportunistic fungal pathogens.
 The primary pathogenic fungi are able to establish infection in a normal host; whereas, opportunistic pathogens require a
compromised host in order to establish infection (e.g., cancer, organ transplantation, surgery, and AIDS).
 The primary deep pathogens usually gain access to the host via the respiratory tract. Opportunistic fungi causing deep
mycosis invade via the respiratory tract, alimentary tract, or intravascular devices.
 The primary systemic fungal pathogens include Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitidis,
and Paracoccidioides brasiliensis.
 The opportunistic fungal pathogens include Cryptococcus neoformans, Candida, Aspergillusspp., Penicillium marneffei, the
Zygomycetes, Trichosporon beigelii, and Fusarium spp.
Pour Plate Technique- Procedure, Advantages, Limitations
Pour Plate Technique- Procedure, Advantages, Limitations
 In nature, microbial populations do not segregate themselves by species but exist with a mixture of many other cell types.
 In the laboratory, these populations can be separated into pure cultures.
 These cultures contain only one type of organism and are suitable for the study of their cultural, morphological, and
biochemical properties.
 At times also the determination of viable cells is very crucial in many microbiological procedures.
 To accomplish this, the serial dilution–agar plate technique is used.
 Briefly, this method involves serial dilution of a bacterial suspension in sterile water blanks, which serve as a diluent of
known volume.
 Once diluted, the suspensions are placed on suitable nutrient media.
 The pour-plate technique is the procedure usually employed.

The pour-plate technique

1. The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette.
2. Molten agar cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample.
3. Following the addition of the molten-then cooled agar, the cover is replaced, and the plates gently rotated in a circular motion
to achieve uniform distribution of microorganisms.
4. This procedure is repeated for all dilutions to be plated.
5. Dilutions should be plated in duplicate for greater accuracy, incubated overnight, and counted on a Quebec colony counter
either by hand or by an electronically modified version of this instrument.
 If the material to be tested is an aqueous liquid, a known volume is simply placed in the base of the Petri dish and 10–25 ml
of molten culture medium (typically tryptone soy agar or plate count agar at 45o C) is poured onto it and quickly mixed by
gentle swirling, then the plate is placed on one side for the agar to set.
 If the sample is a solid that is soluble in water that solid would normally be dissolved, but if it were insoluble then a
suspension would be used. The problem in the last case would be to ensure that the suspension remained uniformly
dispersed during pipetting and any dilution steps.
 Because the sample is dispersed through the medium before the gel sets, the colonies that grow are similarly dispersed
throughout the agar.
 Consequently, the colonies are usually of different sizes because there is less oxygen available within the gel than at its
surface and most organisms are either strict aerobes or facultative anaerobes which will grow best where the oxygen
concentration is highest.

The procedure of Pour Plate Technique

1. Label around the edge of the bottom (not the lid) of a sterile but empty Petri dish with at least your name, the date, the type
of growth medium, and the type of organism to be added to the melted agar medium.
 Include the dilution factor if plating serial dilutions, or a series of repeated dilutions, which results in a systematic reduction in
the concentration of cells in the sample. Preparing serial dilutions is necessary if the number of cells in the sample exceeds
 the capacity of the agar plate, in which the statistically significant range is 30 to 300 CFU. If there are more than 300 CFU on
a plate, then the colonies will be crowded and overlapping.
2. Obtain a tube containing 18 ml of melted agar medium.
 The agar medium should be dispensed into test tubes and pre-sterilized in an autoclave. On the same day, it is needed for
an experiment, the agar should be melted in a steamer for 30 minutes then transferred to a 55 °C water bath. Only as much
agar as is needed for the experiment should be melted as it cannot be re-used.
 Ten minutes before pouring plates, the tubes of melted agar should be transferred from the 55 °C water bath to a heat block
on the laboratory bench set at 48 °C. Once the agar reaches this temperature, it is ready to pour. If the agar is too hot, the
bacteria in the sample may be killed. If the agar is too cool, the medium may be lumpy once solidified.
3. Obtain your sample, which should be either a broth culture or a suspension of cells produced by mixing cells from a colony
into buffer or saline.
 The samples may be derived from a dilution series of a single sample.
 The sample volume to be plated should be between 0.1 and 1.0 ml.
4. Open the lid of the empty Petri dish, and dispense your sample into the middle of the plate. Close the lid.
 Use aseptic technique throughout this procedure.
 Use either a serological pipette or micropipette to transfer your sample to the plate. Control the flow of the sample so it does
not splash out of the plate.
5. Remove the cap from the tube of the melted agar, and pass the rim of the open tube through the flame of the Bunsen burner.
6. Open the lid of the Petri dish containing your sample and pour the agar in carefully. Close the lid then mix the sample with
the agar by gently swirling the plate.
7. Allow the agar to thoroughly solidify before inverting the plate for incubation.

Significance of Pour Plate Technique

 This technique is used to perform viable plate counts, in which the total number of colony-forming units within the agar and
on the surface of the agar on a single plate is enumerated.
 Viable plate counts provide scientists a standardized means to generate growth curves, to calculate the concentration of
cells in the tube from which the sample was plated, and to investigate the effect of various environments or growth conditions
on bacterial cell survival or growth rate.

Advantages of Pour Plate Technique

 Easy to undertake.
 Will detect lower concentrations than surface spread method because of the larger sample volume.
 It requires no pre-drying of the agar surface.
 The most common method for determining the total viable count is the pour-plate method.
 The pour plate technique can be used to determine the number of microbes/ mL in a specimen.
 It has the advantage of not requiring previously prepared plates and is often used to assay bacterial contamination of
foodstuffs.

Disadvantages of Pour Plate Technique

 The use of relatively hot agar carries the risk of killing some sensitive contaminants, so giving a low result.
 Small colonies may be overlooked.
 In the case of solid sample dissolving in water, some species may suffer a degree of viability loss if diluted quickly in cold
water; consequently, isotonic buffer (phosphate-buffered saline for example) or peptone water are used as solvents or
diluents.
 Colonies of different species within the agar appear similar — so it is difficult to detect contaminants.
 The reduced growth rate of obligate aerobes in the depth of the agar.
 Preparation for the pour plate method is time consuming compared with streak plate/and or spread plate technique.

Streak Plate Method- Principle, Methods, Significance, Limitations

Streak Plate Method- Principle, Methods, Significance, Limitations
 In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria.
 The dilution or isolation by streaking method was first developed by Loeffler and Gaffky in Koch’s laboratory, which involves
the dilution of bacteria by systematically streaking them over the exterior of the agar in a Petri dish to obtain isolated colonies
which will then grow into the number of cells or isolated colonies.
 Streaking is rapid and ideally a simple process of isolation dilution.
 The technique is done by diluting a comparatively large concentration of bacteria to a smaller concentration.
 The decrease of bacteria should show that colonies are sufficiently spread apart to effect the separation of the different types
of microbes.
 Streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop.
  Aseptic techniques are used to maintain microbiological cultures and to prevent contamination of the growth medium.

Principle of Streak Plate

 The streak plate method is a rapid qualitative isolation method.
 The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the
inoculums be reduced.
 It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate.
 The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart
on the surface of the agar medium to effect a separation of the different species present.
 In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated microbial culture. The process is
called “picking colonies” when it is done from an agar plate with isolated colonies and is transferred to a new agar or gelatin
plate using a sterile loop or needle.
 The inoculating loop or needle is then streaked over an agar surface.
 On the initial region of the streak, many microorganisms are deposited resulting in confluent growth or the growth of culture
over the entire surface of the streaked area.
 The loop is sterilized by heating the loop in the blue flame of the Bunsen burner, between streaking different sections, or
zones and thus lesser microorganisms are deposited as the streaking progresses.
 The streaking process will dilute out the sample that was placed in the initial region of the agar surface.

Methods of Streak Plate

 There are many different types of methods used to streak a plate. There are two most commonly used streak patterns, a
three sector “T streak” and four-quadrant streak methods.
 Picking a technique is a matter of individual preference and can also depend on how large the number of microbes the
sample contains.

T-Streak
 The three-phase streaking pattern is known as the T-Streak.
 The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop.
 The inoculation loop is first sterilized by passing it through a flame.
 When the loop is cool, it is dipped into an inoculum such as a broth or patient specimen containing many species of bacteria.
 The inoculation loop is then dragged across the surface of the agar back and forth in a zigzag motion until approximately
30% of the plate has been covered.
 The loop then is re-sterilized and the plate is turned 90 degrees.
 Starting in the previously streaked section, the loop is dragged through it two to three times continuing the zigzag pattern.
 The procedure is then repeated once more being cautious to not touch the previously streaked sectors.
 Each time the loop gathers fewer and fewer bacteria until it gathers just single bacterial cells that can grow into a colony.
  The plate should show the heaviest growth in the first section.
 The second section will have less growth and a few isolated colonies, while the final section will have the least amount of
growth and many isolated colonies.

Quadrant method
In the quadrant method, four equally sized sections are streaked. The continuous streaking method typically involves inoculating the
top half of the plate, rotating it 180 degrees, and inoculating the other half of the plate without sterilizing the loop or dragging
bacteria from the previous section.

Discontinuous Streaking Method

1. Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is red hot. Allow it to cool.
2. Pick an isolated colony from the agar plate culture and spread it over the first quadrant (approximately 1/4 of the plate) using
close parallel streaks or insert your loop into the tube/culture bottle and remove some inoculum. You don’t need a huge
chunk.
3. Immediately streak the inoculating loop very gently over a quarter of the plate using a back and forth motion.
4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked, extend the streaks into the
second quarter of the plate.
5. Flame the loop again and allow it to cool. Going back to the area that you just streaked, extend the streaks into the third
quarter of the plate.
6. Flame the loop again and allow it to cool. Going back to the area that you just streaked, extend the streaks into the center
fourth of the plate.
7. Flame your loop once more.

Significance of Streak Plate Method

 The streak plate technique is the most popular method for isolating specific bacteria from a sample containing a mixture of
microorganisms.
 Streak plate technique is used to grow bacteria on a growth media surface so that individual bacterial colonies are isolated
and sampled.
 Samples can then be taken from the resulting isolated colonies and a microbiological culture can be grown on a new plate so
that the organism can be identified, studied, or tested.
 When the bacteria are streaked and isolated, the causative agent of a bacterial disease can be identified.

Limitations of Streak Plate

Streak plating is a microbiology laboratory method that has two major disadvantages.
 Firstly, users will not be able to grow obligate anaerobes using this method.
 Secondly, only organisms that were viable in the original sample are able to be grown.

Bacterial Growth and Factors Affecting Growth of Bacteria

 With respect to humans, the term growth refers to an increase in size; for example, going from a tiny newborn baby to a
large adult.
 Although bacteria do increase in size before cell division, bacterial growth refers to an increase in the number of organisms
rather than an increase in their size.
 Bacterial growth can be defined as an orderly increase of all the chemical components of the cell.
 Cell multiplication is a consequence of growth that leads to an increase in the number of bacteria making up a population or
culture.
 Most bacteria divide by binary fission in which the bacteria undergo cell division to produce two daughter cells identical to the
parent cell.
 Bacterial growth can be equated to cell number: one bacterium divides into two, these two produce four, and then eight, and
so on.
Generation time
 The growth rate of a bacterium is measured by measuring the change in bacterial number per unit time.
 Generation time is the time required for a bacterium to give rise to two daughter cells under optimum conditions.
 The generation time for most of the pathogenic bacteria, such as E. coli, is about 20 minutes.
 The generation time is longer (i.e., 20 hours) for Mycobacterium tuberculosis and longest (i.e., 20 days) for M. leprae.
 A bacterium replicates and multiplies rapidly producing millions of cells within 24 hours.

Factors Affecting Growth of Bacteria

The growth of microorganisms in the body, in nature, or in the laboratory is greatly influenced by temperature pH, moisture content,
available nutrients, and the characteristics of other organisms present.

Oxygen
Bacteria on the basis of their oxygen requirements can be classified broadly into aerobic and anaerobic bacteria.
Aerobic bacteria:
They require oxygen for their growth. They may be:
 Obligate aerobes—which can grow only in the presence of oxygen (e.g., P. aeruginosa).
 Facultative aerobes—which are ordinary aerobes but can also grow without oxygen (e.g., E. coli). Most of the pathogenic
bacteria are facultative aerobes.
 Microaerophilic bacteria—those bacteria that can grow in the presence of low oxygen and in the presence of low (4%)
concentration of carbon dioxide (e.g., Campylobacter jejuni).
 Some fermentative organisms (e.g., Lactobacillus plantarum) are aerotolerant but do not contain the enzyme catalase or
superoxide dismutase. Oxygen is not reduced, and therefore hydrogen peroxide (H2O2) and nascent oxygen (O2) are not
produced.
Anaerobic bacteria:
 Obligate anaerobes are the bacteria that can grow only in the absence of oxygen (e.g., Clostridium botulinum, Clostridium
tetani, etc.).
 These bacteria lack superoxide dismutase and catalase; hence oxygen is lethal to these organisms.

Carbon dioxide
 The organisms that require higher amounts of carbon dioxide (CO2) for their growth are called capnophilic bacteria.
 They grow well in the presence of 5–10% CO2 and 15% O2.
 In candle jar, 3% CO2 can be achieved.
 Examples of such bacteria include H. influenzae, Brucella abortus, etc.

Temperature
The optimum temperature for most of the pathogenic bacteria is 37oC.
The optimal temperature, however, is variable; depending on their temperature range, growth of bacteria is grouped as follows:
 Psychrophiles: These bacteria are cold loving microbes that grow within a temperature range of 0–20o Most of soil and
water saprophytes belong to this group.
  Mesophiles: These are moderate temperature loving microbes that grow between 25oC and 40o Most of pathogenic bacteria
belong to this group.
 Thermophiles: These are heat loving microbes. They can grow at a high temperature range of 55–80o B.
stearothermophilus is an example.

pH
 Most pathogenic bacteria grow between pH 7.2 and 7.6.
 Very few bacteria, such as lactobacilli, can grow at acidic pH below 4.0.
 Many food items, such as pickles and cheese, are prevented from spoilage by acids produced during fermentation.
 V. cholerae is an example of the bacteria that can grow at an alkaline (8.2–8.9) pH.

Light
Depending on the source of energy bacteria make use of, they may be classified as
 Phototrophs (bacteria deriving energy from sunlight)
 Chemotrophs (bacteria deriving energy from chemical sources).

Osmotic pressure
 Microbes obtain almost all their nutrients in solution from surrounding water.
 Hence factors such as osmotic pressure and salt concentration of the solution affect the growth of bacteria.
 Bacteria by virtue of mechanical strength of their cell wall are able to withstand a wide range of external osmotic variations.
 Organisms requiring high osmotic pressures are called osmophilic bacteria.
 Sudden exposure of bacteria to hypertonic solution may cause osmotic withdrawal of water, leading to osmotic shrinkage of
the protoplasm (plasmolysis).
 On the other hand, sudden transfer of bacteria from concentrated solution to distilled water may cause excessive imbibition
of water leading to swelling and bursting of cell ( plasmoptysis).

Bacterial growth curve and its significance

When a broth culture is inoculated with a small bacterial inoculum, the population size of the bacteria increases showing a classical
pattern. When plotted on a graph, a distinct curve is obtained referred to as the bacterial growth curve.

Method of Obtaining Bacterial Growth Curve

 A population growth curve for any particular species of bacterium may be determined by growing a pure culture of the
organism in a liquid medium at a constant temperature.
 Samples of the culture are collected at fixed intervals (e.g., every 30 minutes), and the number of viable organisms in each
sample is determined.
 The data are then plotted on logarithmic graph paper.
 The logarithm of the number of bacteria per milliliter of medium is plotted against time.
The bacterial growth curve shows the following four distinct phases:
1. Lag phase:
 After a liquid culture broth is inoculated, the multiplication of bacteria does not start immediately. It takes some time to
multiply.
 The time between inoculation and beginning of multiplication is known as lag phase.
 In this phase, the inoculated bacteria become acclimatized to the environment, switch on various enzymes, and adjust to the
environmental temperature and atmospheric conditions.
 During this phase, there is an increase in size of bacteria but no appreciable increase in number of bacterial cells. The cells
are active metabolically.
 The duration of the lag phase varies with the bacterial species, nature of culture medium, incubation temperature, etc.
 It may vary from 1 hour to several days.
2. Log phase:
 This phase is characterized by rapid exponential cell growth (i.e., 1 to 2 to 4 to 8 and so on).
 The bacterial population doubles during every generation. They multiply at their maximum rate.
 The bacterial cells are small and uniformly stained.
 The microbes are sensitive to adverse conditions, such as antibiotics and other antimicrobial agents.
 Growth rate is the greatest during the log phase.
 The log phase is always brief, unless the rapidly dividing culture is maintained by constant addition of nutrients and frequent
removal of waste products.
 When plotted on logarithmic graph paper, the log phase appears as a steeply sloped straight line.
3. Stationary phase:
 After log phase, the bacterial growth almost stops completely due to lack of essential nutrients, lack of water oxygen, change
in pH of the medium, etc. and accumulation of their own toxic metabolic wastes.
 It is during this phase that the culture is at its greatest population density.
 However, Death rate of bacteria exceeds the rate of replication of bacteria.
 Endospores start forming during this stage.
 Bacteria become Gram variable and show irregular staining.
 Many bacteria start producing exotoxins.
4. Decline phase:
 During this phase, the bacterial population declines due to death of cells.
 The decline phase starts due to
(a) accumulation of toxic products and autolytic enzymes and
(b) exhaustion of nutrients.
 Involution forms are common in this stage. Some cells assume various shapes, becoming long, filamentous rods or
branching or globular forms that are difficult to identify.
 Some develop without a cell wall and are referred to as protoplasts, spheroplasts, or L-phase variants (L-forms).
 When these involuted forms are inoculated into a fresh nutrient medium, they usually revert to the original shape of the
healthy bacteria.
Significance of the Bacterial Growth Curve
 The study of bacterial growth curves is important when aiming to utilize or inoculate known numbers of the bacterial isolate,
for example to enhance plant growth, increase biodegradation of toxic organics, or produce antibiotics or other natural
products at an industrial scale.
 Knowledge of bacterial growth kinetics and bacterial numbers in a culture medium is important from both a research and
commercial point of view. 
 Growth kinetics is also useful for assessing whether particular strains of bacteria are adapted to metabolize certain
substrates, such as industrial waste or oil pollution.
 Bacteria that are genetically engineered to clean up oil spills, for example, can be grown in the presence of complex
hydrocarbons to ensure that their growth would not be repressed by the toxic effects of oil.
 Similarly, the slope and shape of growth curves produced from bacteria grown with mixtures of industrial waste products can
inform scientists whether the bacteria can metabolize the particular substance, and how many potential energy sources for
the bacteria can be found in the waste mixture. 

Classification of Bacteria
 Bacteria are classified and identified to distinguish one organism from another and to group similar organisms by criteria of
interest to microbiologists or other scientists.
 The classification of bacteria serves a variety of different functions. Because of this variety, bacteria may be grouped using
many different typing schemes.
 The grounds for the classification commonly used may be:

Morphologic Characteristics
 Both wet-mounted and properly stained bacterial cell suspensions can yield a great deal of information.
 These simple tests can indicate the Gram reaction of the organism; whether it is acid-fast; its motility; the arrangement of its
flagella; the presence of spores, capsules, and inclusion bodies; and, of course, its shape.
 This information often can allow identification of an organism to the genus level, or can minimize the possibility that it
belongs to one or another group.

Growth Characteristics
 A primary distinguishing characteristic is whether an organism grows aerobically, anaerobically, facultatively (i.e., in either
the presence or absence of oxygen), or microaerobically (i.e., in the presence of a less than atmospheric partial pressure of
oxygen). The proper atmospheric conditions are essential for isolating and identifying bacteria.
 Other important growth assessments include the incubation temperature, pH, nutrients required, and resistance to
antibiotics. For example, one diarrheal disease agent, Campylobacter jejuni, grows well at 42° C in the presence of several
antibiotics; another, Y. enterocolitica, grows better than most other bacteria at 4° C. Legionella, Haemophilus, and some
other pathogens require specific growth factors, whereas E. coli and most other Enterobacteriaceae can grow on minimal
media.

Antigens and Phage Susceptibility

 Cell wall (O), flagellar (H), and capsular (K) antigens are used to aid in classifying certain organisms at the species level, to
serotype strains of medically important species for epidemiologic purposes, or to identify serotypes of public health
importance.
 Serotyping is also sometimes used to distinguish strains of exceptional virulence or public health importance, for example
with V. cholerae (O1 is the pandemic strain) and E. coli (enterotoxigenic, enteroinvasive, enterohemorrhagic, and
enteropathogenic serotypes).
 Phage typing (determining the susceptibility pattern of an isolate to a set of specific bacteriophages) has been used primarily
as an aid in epidemiologic surveillance of diseases caused by Staphylococcus aureus, mycobacteria, P. aeruginosa, V.
cholerae, and S. Typhi. Susceptibility to bacteriocins has also been used as an epidemiologic strain marker. In most cases
recently, phage and bacteriocin typing have been supplanted by molecular methods.

Biochemical Characteristics
 Most bacteria are identified and classified largely on the basis of their reactions in a series of biochemical tests.
  Some tests are used routinely for many groups of bacteria (oxidase, nitrate reduction, amino acid degrading enzymes,
fermentation or utilization of carbohydrates); others are restricted to a single family, genus, or species (coagulase test for
staphylococci, pyrrolidonyl arylamidase test for Gram-positive cocci).

Classification on the basis of Gram Stain and Bacterial Cell Wall

 Of all the different classification systems, the Gram stain has withstood the test of time. Discovered by H.C. Gram in 1884 it
remains an important and useful technique to this day.
 It allows a large proportion of clinically important bacteria to be classified as either Gram positive or negative based on
their morphology and differential staining properties.
 Slides are sequentially stained with crystal violet, iodine, then destained with alcohol and counter-stained with safranin.
Gram positive bacteria stain blue-purple and Gram negative bacteria stain red.
 The difference between the two groups is believed to be due to a much larger peptidoglycan (cell wall) in Gram positives. As
a result the iodine and crystal violet precipitate in the thickened cell wall and are not eluted by alcohol in contrast with the
Gram negatives where the crystal violet is readily eluted from the bacteria.
 As a result bacteria can be distinguished based on their morphology and staining properties.
 Some bacteria such as mycobacteria are not reliably stained due to the large lipid content of the peptidoglycan. Alternative
staining techniques (Kinyoun or acid fast stain) are therefore used that take advantage of the resistance to destaining after
lengthier initial staining.

Classification of Bacteria on the Basis of Shape

In the year 1872 scientist Cohn classified bacteria to 4 major types depending on their shapes are as follows:
A) Cocci:These types of bacteria are unicellular, spherical or elliptical shape. Either they may remain as a single cell or may
aggregate together for various configurations. They are as follows:
 Monococcus:– they are also called micrococcus and represented by single, discrete round      Example: Micrococcus
flavus.
 Diplococcus:– the cell of the Diplococcus divides ones in a particular plane and after division, the cells remain attached to
each other. Example: Diplococcus pneumonia.
 Streptococcus: – here the cells divide repeatedly in one plane to form chain of cells. Example: – Streptococcus pyogenes.
 Tetracoccus: – this consists of four round cells, which defied in two planes at a right angles to one another. Example:
– Gaffkya tetragena. Staphylococcus: – here the cells divided into three planes forming a structured like bunches of grapes
giving and irregular configuration. Example: – Staphylococcus aureus.
 Sarcina: -in this case the cells divide in three planes but they form a cube like configuration consisting of eight or sixteen
cells but they have a regular shape. Example: –Sarcina lutea.
B) Bacilli: – These are rod shaped or cylindrical bacteria which either remain singly or in pairs. Example: –Bacillus cereus.
C) Vibro: – The vibro are the curved, comma shaped bacteria and represented by a single genus. Example: – Vibro cholerae.
D) Spirilla: – These type of bacteria are spiral or spring like with multiple curvature and terminal flagella. Example: –Spirillum
volutans.
Others
Actinomycetes are branching filamentous bacteria, so called because of a fancied resemblance to the radiating rays of the sun
when seen in tissue lesions (from actis meaning ray and mykes meaning fungus).
Mycoplasmas are bacteria that are cell wall deficient and hence do not possess a stable morphology. They occur as round or oval
bodies and as interlacing filaments.

Classification of Bacteria on the Basis of Mode of Nutrition

1. Phototrophs:
 Those bacteria which gain energy from light.
 Phototrops are further divided into two groups on the basis of source of electron.
 Photolithotrophs: these bacteria gain energy from light and uses reduced inorganic compounds such as H2S as electron
source. Eg. Chromatium okenii.
 Photoorganotrophs: these bacteria gain energy from light and uses organic compounds such as succinate as electron
source.
2. Chemotrophs:
 Those bacteria gain energy from chemical compounds.
 They cannot carry out photosynthesis.
 Chemotrops are further divided into two groups on the basis of source of electron.
 Chemolithotrophs: they gain energy from oxidation of chemical compound and reduces inorganic compounds such as NH3
as electron source. Eg. Nitrosomonas.
 Chemoorganotrophs: they gain energy from chemical compounds and uses organic compound such as glucose and amino
acids as source of electron. eg. Pseudomonas pseudoflava.
3. Autotrophs:
 Those bacteria which uses carbondioxide as sole source of carbon to prepare its own food.
 Autotrophs are divided into two types on the basis of energy utilized to assimilate carbondioxide. ie. Photoautotrophs and
chemoautotrophs.
 Photoautotrophs: they utilized light to assimilate CO2. They are further divided into two group on the basis of electron
sources. Ie. Photolithotropic autotrophs and Photoorganotropic autotrophs
 Chemoautotrophs: They utilize chemical energy for assimilation of CO2.
4. Heterotrophs:
 Those bacteria which uses organic compound as carbon source.
 They lack the ability to fix CO2.
 Most of the human pathogenic bacteria are heterotropic in nature.
 Some heterotrops are simple, because they have simple nutritional requirement. However there are some bacteria that
require special nutrients for their growth; known as fastidious heterotrophs.

Classification of Bacteria on the Basis of Temperature Requirement

Bacteria can be classified into the following major types on the basis of their temperatures response as indicated below:
1.Psychrophiles:
 Bacteria that can grow at 0°C or below but the optimum temperature of growth is 15 °C or below and maximum temperature
is 20°C are called psychrophiles
 Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid nature to the cell membrane even at
lower temperature.
 Examples: Vibrio psychroerythrus, vibrio marinus, Polaromonas vaculata, Psychroflexus.
2. Psychrotrops (facultative psychrophiles):
 Those bacteria that can grow even at 0°C but optimum temperature for growth is (20-30)°C
3. Mesophiles:
 Those bacteria that can grow best between (25-40)o C but optimum temperature for growth is 37C
 Most of the human pathogens are mesophilic in nature.
 Examples: E. coli, Salmonella, Klebsiella, Staphylococci.
4. Thermophiles:
 Those bacteria that can best grow above 45C.
 Thermophiles capable of growing in mesophilic range are called facultative thermophiles.
 True thermophiles are called as Stenothermophiles, they are obligate thermophiles,
 Thermophils contains saturated fattyacids in their cell membrane so their cell membrane does not become too fluid even at
higher temperature.
 Examples: Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus.
5. Hypethermophiles:
  Those bacteria that have optimum temperature of growth above 80C.
 Mostly Archeobacteria are hyperthermophiles.
 Monolayer cell membrane of Archeobacteria is more resistant to heat and they adopt to grow in higher remperature.
 Examples: Thermodesulfobacterium, Aquifex, Pyrolobus fumari, Thermotoga.

Classification of Bacteria on the Basis of Oxygen Requirement

Obligate Aerobes:
 Require oxygen to live.
 Example: Pseudomonas, common nosocomial pathogen.
Facultative Anaerobes:
 Can use oxygen, but can grow in its absence.
 They have complex set of enzymes.
 Examples: E. coli, Staphylococcus, yeasts, and many intestinal bacteria.
Obligate Anaerobes:
 Cannot use oxygen and are harmed by the presence of toxic forms of oxygen.
 Examples: Clostridium bacteria that cause tetanus and botulism.
Aerotolerant Anaerobes:
 Cannot use oxygen, but tolerate its presence.
 Can break down toxic forms of oxygen.
 Example: Lactobacillus carries out fermentation regardless of oxygen presence.
Microaerophiles:
 Require oxygen, but at low concentrations.
 Sensitive to toxic forms of oxygen.
 Example: Campylobacter.

Classification of Bacteria on the Basis of pH of Growth

1. Acidophiles:
 These bacteria grow best at an acidic pH.
 The cytoplasm of these bacteria are acidic in nature.
 Some acidopiles are thermophilic in nature, such bacteria are called Thermoacidophiles.
 Examples: Thiobacillus thioxidans, Thiobacillus, ferroxidans, Thermoplasma, Sulfolobus
2. Alkaliphiles:
 These bacteria grow best at an alkaline pH.
 Example: Vibrio cholerae optimum ph of growth is 8.2.
3. Neutrophiles:
 These bacteria grow best at neutral pH (6.5-7.5).
 Most of the bacteria grow at neutral pH.
 Example: E. coli

Classification of Bacteria on the Basis of Osmotic Pressure Requirement

Halophiles:
 Require moderate to large salt concentrations.
 Cell membrane of halophilic bacteria is made up of glycoprotein with high content of negatively charged glutamic acid and
aspartic acids. So high concentration of Na+ ion concentration is required to shield the –ve charge.
 Ocean water contains 3.5% salt. Most such bacteria are present in the oceans.
 Archeobacteria, Halobacterium, Halococcus.
Extreme or Obligate Halophiles:
 Require a very high salt concentrations (20 to 30%).
 Bacteria in Dead Sea, brine vats.
Facultative Halophiles:
 Do not require high salt concentrations for growth, but tolerate upto 2% salt or more.
Classification of Bacteria on the Basis of Number of Flagella
On the basis of flagella the bacteria can be classified as:
1. Atrichos: – These bacteria has no flagella. Example: Corynebacterium diptherae.
2. Monotrichous: – One flagellum is attached to one end of the bacteria cell. Example: – Vibro cholerae.
3. Lophotrichous: – Bunch of flagella is attached to one end of the bacteria cell. Example: Pseudomonas.
4. Amphitrichous: – Bunch of flagella arising from both end of the bacteria cell. Example: Rhodospirillum rubrum.
 5. Peritrichous : – The flagella are evenly distributed surrounding the entire bacterial cell. Example: Bacillus.

Classification of Bacteria on the basis of Spore Formation

1. Spore forming bacteria:
 Those bacteria that produce spore during unfavorable condition.
 These are further divided into two groups:
i) Endospore forming bacteria: Spore is produced within the bacterial cell.
Examples. Bacillus, Clostridium, Sporosarcina etc
ii) Exospore forming bacteria: Spore is produced outside the cell.
Example.  Methylosinus
2. Non sporing bacteria:
 Those bacteria which do not produce spores.
Eg. E. coli, Salmonella.

Water Quality Analysis by Most Probable Number (MPN)

The most probable number (MPN) analysis is a statistical method based on the random dispersion of microorganisms per volume in
a given sample.
 In this method, measured volumes of water are added to a series of tubes containing a liquid indicator growth medium.
 The media receiving one or more indicator bacteria show growth and a characteristic color change. The color change is
absent in those receiving only an inoculum of water without indicator bacteria.
 From the number and distribution of positive and negative reactions, the MPN of indicator organisms in the sample may be
estimated by reference to statistical tables.
 MPN test is completed in three steps:
1. Presumptive test
2. Confirmed test
3. Completed test

Image Source: Microbe Online and Scharlab.

Presumptive Test
This test, a specific enrichment procedure for coliform bacteria, is conducted in fermentation tubes filled with a selective growth
medium (MacConkey lactose broth), which contains inverted Durham tubes for the detection of fermentation gas. A series of lactose
broth tubes are inoculated with measured amounts of the water sample to be tested. The series of tubes may consist of three or four
groups of three, five, or more tubes.
The main selective factors found in the medium are lactose, sometimes a surfactant such as Na-lauryl sulfate or Na-taurocholate
(bile salt), and often a pH indicator dye for facilitating detection of acid production, such as bromcresol purple or brilliant green. The
selective action of lactose occurs because many bacteria cannot ferment this sugar, whereas coliform bacteria and several other
bacterial types can ferment it. The surfactant and dye do not inhibit coliform bacteria, whereas many other bacteria, such as the
spore formers, are inhibited.

Confirmed Test
This test serves to confirm the presence of coliform bacteria when either a positive or doubtful presumptive test is obtained.
1. A loopful of growth from such a presumptive tube is transferred into a tube of brilliant green lactose bile (BGLB) 2% broth (or
other lactose broth) and incubated at 35°C for 48 hours. This is a selective medium for detecting coliform bacteria in water,
dairy, and other food products. A selective agent in the medium is lactose. The broth tube also contains a Durham tube to
detect gas production.
2. A plate of LES Endo agar (or EMB agar) is streaked with a loopful of growth from a positive tube and incubated at 35°C for
18–24 hours. Typical coliform bacteria (E. coli and Enterobacter aerogenes) exhibit good growth on this medium and form
red to black colonies with dark centers or a sheen. Salmonella typhi exhibits good growth but the colonies are colorless. S.
aureus growth is inhibited altogether.

Completed Test
This test helps to further confirm doubtful and, if desired, positive confirmed test results. A typical coliform colony from an LES Endo
agar plate is inoculated into a tube of brilliant green bile broth and on the surface of a nutrient agar slant. They are then incubated at
35°C for 24 hours. After 24 hours, the broth is checked for the production of gas, and a Gram stain is made from organisms on the
nutrient agar slant. If the organism is a Gram-negative, non-spore-forming rod and produces gas in the lactose tube, then it is
positive that coliforms are present in the water sample.

Objectives of Most Probable Number (MPN)

 To enumerate the number of bacteria present in the drinking water by the MPN method.
 To identify the bacteria present in the drinking water sample.

Procedure of Most Probable Number (MPN)

I. Presumptive Test

Image Source: Microbe Online

1. Prepare MacConkey purple media of single and double strength in test tubes with Durham’s tube and autoclave it.
2. Take three sets of test tubes containing five tubes in each set; one set with 10 ml of double strength (DS) other two
containing 10 ml of single strength (SS) .
3. Using sterile pipettes, transfer 10 ml of water to each of the DS broth tubes. Transfer 1 ml of water sample to each of 5 tubes
of one set of SS broth and transfer 0.1 ml water to five tubes of remaining last set of SS broth tubes.
4. Incubate the tubes at 37°C for 24 hours.
5. After incubation, observe the gas production in Durham’s tube and the color change of the media.
6. Record the number of positive results from each set and compare with the standard chart to give presumptive coliform count
per 100 ml water sample.

II. Confirmed Test

Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to confirm the presence of
coliform, a confirmatory test is done. For this, a loopful of suspension from a positive tube is inoculated into a 3 ml lactose-broth or
brilliant green lactose fermentation tube and to an agar plate (EMB agar or Endo Agar) or slant.
A. Inoculation of the lactose-broth
1. Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2 hours.
2. If no gas production is seen, further incubate up to a maximum of 48 ±3 hours to check gas production.
B. Inoculation in media slants
1. Take a loopful of suspension from a positive tube and inoculate it on the agar surface.
2. The agar slants should be incubated at 37°C for 24± 2 hours.
3. Colonies must be examined macroscopically.

III. Completed Test

1. Transform a typical coliform colony from the agar plate into a tube of brilliant green bile broth with placed Durham’s tube and
on the surface of a nutrient agar slant.
2. Incubate at 35°C for 24 hours.
3. After 24 hours, check the broth for the production of gas, and perform Gram staining for organisms on the nutrient agar slant.

Results of Most Probable Number (MPN)

A. Presumptive Test
Positive: The formation of 10% gas or more in the Durham tube within 24 to 48 hours, together with turbidity in the growth medium
and the color change in the medium constitutes a positive presumptive test for coliform bacteria, and hence for the possibility of
fecal pollution.
 The test is presumptive only because under these conditions several other types of bacteria can produce similar results.
Negative: No growth or formation of gas in Durham’s tube.

B. Confirmed Test
Positive: Formation of gas in lactose broth and the demonstration of a coliform-like colony on the EMB agar indicate the presence
of a member of the coliform group in the sample examined.
Coliforms produce colonies with a greenish metallic sheen which differentiates them from non-coliform colonies (show no sheen).
The presence of typical colonies at high temperatures (44.5 ±0.2) indicates the presence of thermotolerant E.coli.
Negative: The absence of gas formation in lactose broth or the failure to demonstrate coliform-like colonies on the EMB agar.

Completed Test
Positive: The presence of gas in the brilliant green bile broth tube and Gram-negative, non-spore-forming rods on NA slant
constitutes a positive completed test for the presence of coliform bacteria, which, in turn, infers possible contamination of the water
sample with fecal matter.
Negative: Absence of growth and gas formation in the broth. Absence of gram-negative, non-sporing rods on Gram staining.

Uses of Most Probable Number (MPN)

 It is commonly used in estimating microbial populations in soils, waters, agricultural products.
 The technique is particularly useful with samples that contain particulate material that interferes with plate count enumeration
methods.
  It has also been suggested as a consideration for an alternate method to trend environmental monitoring studies.
 It is also useful for counting bacteria that reluctantly form colonies on agar plates or membrane filters but grow readily in
liquid media.

Advantages of Most Probable Number (MPN)

 Ease of interpretation, either by observation or gas emission
 Sample toxins are diluted
 Effective method of analyzing highly turbid samples such as sediments, sludge, mud, etc.
 Permits samples that cannot be analyzed by membrane filtration.

Limitations of Most Probable Number (MPN)

 Poor accuracy and precision associated with MPN count usually mean that the method is one of last resort — to be
considered only when other counting methods are inappropriate.
 Laborious and expensive in terms of materials, glassware, and incubator space.
 It has relatively a large margin of error.

Water Quality Analysis by Membrane Filter (MF) Technique

 The Membrane Filter (MF) Technique was introduced in the late 1950s as an alternative to the Most Probable Number
(MPN) procedure for microbiological analysis of water samples.
 It involves the use of membrane filters, which are thin porous sheet structures composed of cellulose esters or similar
polymeric materials.
 They act essentially as two-dimensional screens and as such, all particles, both biological and non-biological, which exceed
the pore size, are retained upon the surface of the filter from fluids passing through.
 Bacteria-tight membrane filters capable of retaining microorganisms larger than 0.45 micrometer (μm) are frequently used for
analysis of water.

Objective of Membrane Filter (MF) Technique

To determine the quality of water samples using the membrane filter method.

Principle of Membrane Filter (MF) Technique

Membrane filters have a known uniform porosity of predetermined size (generally 0.45 µm) sufficiently small to trap microorganisms.
A water sample is passed through a sterile membrane filter that is housed in a special filter apparatus contained in a suction flask.
Following filtration, the filter disc that contains the trapped microorganisms is aseptically transferred to a sterile Petri dish containing
a pad saturated with the appropriate medium. The passage of nutrients through the filter during incubation facilitates the growth of
organisms in the form of colonies, on the upper surface of the membrane. Membrane filtration and colony count techniques assume
that each bacterium, clump of bacteria, or particle with bacteria attached, will give rise to a single visible colony. Each of these
clumps or particles is, therefore, a colony forming unit (cfu) and the results are expressed as colony forming units per unit volume.
Discrete colonies thus formed can be easily transferred to confirmation media.  Following incubation, the colonies present on the
filter are counted with the aid of a microscope.

Procedure of Membrane Filter (MF) Technique

1. Collect the sample and make any necessary dilutions.
2. Select the appropriate nutrient or culture medium. Dispense the broth into a sterile Petri dish, evenly saturating the
absorbent pad.
3. Flame the forceps, and remove the membrane from the sterile package.
4. Place the membrane filter into the funnel assembly.
5. Flame the pouring lip of the sample container and pour the sample into the funnel.
6. Turn on the vacuum and allow the sample to draw completely through the filter.
7. Rinse funnel with sterile buffered water. Turn on vacuum and allow the liquid to draw completely through the filter.
8. Flame the forceps and remove the membrane filter from the funnel.
9. Place the membrane filter into the prepared Petri dish.
10. Incubate at the proper temperature and for the appropriate time period.
11. Count the colonies under 10 – 15 X magnification.
12. Confirm the colonies and report the results.

Uses of Membrane Filter (MF) Technique

1. It is used to analyze a series of dilutions of water samples collected upstream and downstream from an outlet of a sewage
treatment plant.
2. EPA-approved guidelines for the determination of fecal contaminating organisms (EPA Method 1103.1) are routinely utilized
worldwide to examine water samples before treated water is released into a nation’s waterways.
3. A total count of coliform bacteria determines the potability of the water sources.
4. Delicate media components that cannot withstand steam sterilization by autoclaving (e.g., serum, certain carbohydrate
solutions, certain antibiotics, and other heat-labile substances) can be sterilized by membrane filtration.
5. The pharmaceutical and cosmetics industries typically focus on monitoring their process water for Pseudomonas species.

Merits of Membrane Filter (MF) Technique

1. Results are available in a shorter period of time
2. Larger volumes of sample can be processed
3. Because of the high accuracy of this method, the results are readily reproducible.
4. Allows isolation and enumeration of discrete colonies of bacteria
5. Allows for removal of bacteriostatic or cidal agents that would not be removed in Pour Plate, Spread Plate, or MPN
techniques.
6. It involves less preparation than many traditional methods, and is one of a few methods that will allow the isolation and
enumeration of microorganisms.  

Limitations of Membrane Filter (MF) Technique

1. During the processing of turbid specimens that contain large quantities of suspended materials; particulate matter clogs the
pores and inhibits passage of the specific volume of water.
2. When small quantities of sample (for example, of sewage effluent or of grossly polluted surface water) are to be tested, it is
necessary to dilute a portion of the sample in sterile diluent to ensure that there is sufficient volume to filter across the entire
surface of the membrane.

Isolation of Actinomycetes from Soil sample

 Actinomycetes are classified as a group of gram-positive bacteria that are unique for their spore forming abilities and
formation of mycelia structures.
 They show marked chemical and morphological diversity but form a distinct evolutionary line of organisms that range from
coccoid and pleomorphic forms to branched filaments.
 These bacteria have been noted to serve as rich reservoirs of medicinal antibiotics and are therefore extremely relevant to
scientists, pharmaceutical industries and agricultural industries.
Figure: Free living actinomycetes are ubiquitous in soil environments as well as in marine and fresh water ecosystems. In addition,
they have an important ecological role in the turnover of organic material. Many actinomycetes have evolved to live in symbiosis
with plants, fungi, insects and animals. Most such actinomycete–host interactions are beneficial, whereby actinomycetes produce
NPs that allow their host to protect itself against pathogens or pests, or enzymes to degrade resilient natural polymers like
lignocellulose.
Image Source: Gilles P. van Wezel, Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE
Leiden, The Netherlands.

Principle of Isolation of Actinomycetes

Actinomycetes is a non-taxonomic term for a group of common soil microorganisms sometimes called “thread or ray bacteria.” They
are known for decomposing more resistant organic materials such as chitin, a complex sugar found in the outer skeleton of insects
and elsewhere. The richness and diversity of actinomycetes present in any specific soil, however, is greatly influenced by the soil
type, geographical location, cultivation and organic matter amongst other factors.
Different agar media can be used for the isolation of Actinomycetes from soil sample after dilution of the soil slurry. Actinomycetes
Isolation Agar is a selective medium commonly employed. It contains sodium caseinate as nitrogen source. Asparagine in addition
to being an amino acid is also a source of nitrogen. Sodium propionate is used as a substrate in anaerobic fermentation.
Dipotassium phosphate provides the buffering system. The sulphates serve as source of sulphur and metallic ions. Glycerol serves
as an additional source of carbon. Cycloheximide and nystatin used in the agar, further prevents fungi interference by inhibiting
mRNA translation, hence causing cell growth arrest and cell death of opportunistic fungi.
The chitin agar, on the other hand, contains chitin, a complex sugar which is generally hydrolysed by Actinomycetes. Thus it is able
to support the growth of only the actinomycetes and also in addition prevent the growth of other soil bacteria and fungi.
Procedure of Isolation of Actinomycetes

1. First, soil slurry is made by suspending 1 gm of the collected dry soil in 10 ml distilled water. The slurry is mixed by vortexing
for 2 minutes.
2. Four 1 in 10 fold serial dilutions is made from the slurry in duplicates.
3. 1 ml portions of each dilution, are then plated by spreading on the set agar plates (Actinomycete Isolation Agar
supplemented with cyclohexamide (50 μg/ml) and nystatin (50 μg/ml) and Chitin agar/ Starch Casein Agar).
4. All spread plates are labelled and incubated at 25 ̊C for a period of 7 to 14 days.
5. On day 14, the number of colonies formed in each dilution of both groups of, are counted and recorded.
6. All plates are carefully observed under the microscope to detect diversity in colonies formed. The richness, evenness and
diversity index, are also calculated and recorded.
7. Different colony types are then picked out with sterile forceps and streaked out on glucose yeast extract agar plates, to
obtain pure single colonies.
8. The streaked plates are then incubated and subsequently tested for antibiotic production.

Expected Results of Isolation of Actinomycetes

 On Actinomycete Isolation Agar supplemented with cyclohexamide (50 μg/ml) and nystatin (50 μg/ml), after 7 days
incubation, whitish pin-point colonies, characteristic of actinomycetes, with a clear zone of inhibition around them can be
observed.
 Colonies appearing as large bright white filaments with net-like mycelia, pale white branching filaments with powdery
appearance, dark brown uniformity and crumb-like appearance, light brown appearance with cilia-like mycelia on its
boundaries, a dark brown appearance with embedded with concentric circular patterns, yellow colonies with beautiful
mycelia and transparent boundaries can be expected.
 Some of the actinomycete colonies on chitin agar are surrounded by a clear zone of hydrolysis which facilitates macroscopic
recognition.

Isolation of Bacillus thuringiensis (Bt) from Soil sample

 Bacillus thuringiensis (Bt) is an ubiquitous, Gram-positive and sporulating bacterium that synthesizes insecticidal proteins
with specificity against a wide range of insects during sporulation (Cry and Cyt) and vegetative growth (Vip and Sip).
 These proteins have portrayed Bt as an environment-friendly alternative to chemical insecticides.
Principle of Isolation of Bacillus thuringiensis
Widely used methodologies to isolate Bt from soil consist of a thermal shock treatment followed by selective germination of spores.
Thermal shock is employed to eliminate all bacteria incapable of producing endospores from the sample soil. The samples are then
diluted many folds to eliminate the amount of humic material within the soil and to reduce the overall colony forming units within the
sample. The diluted samples are then cultured on nutrient agar in order to give the spores chance to germinate on media with
adequate nutrients and at optimal temperature. The media offers favourable growth conditions for a wide range of bacteria
including Bacillus thuringiensis. A series of selection tests are thus further employed to identify Bacillus thuringiensis alone from the
range of bacteria present in the crude soil sample population. Common important tests include Sodium acetate selection test,
Gram’s staining, Amino black and Ziehl’s carbol fuchsin staining, Endospore staining, Catalase test, checking for growth above 45 oC
and looking for presence of parasporal bodies among others.

Procedure of Isolation of Bacillus thuringiensis

1. Obtain about 20 g of cultivated or non-cultivated soil sample with a tubular soil sampler after removing the 2-3 cm of the top
layer.
2. Place samples at 4 ºC in 50 ml (sterile) centrifuge tubes or zip-lock bags until isolation.
3. Suspend the soil samples of 1 g in 10ml 0.85% NaCl.
4. Heat with shaking at 70°C for 10 min.
5. Plate aliquots of 100μl of suspension onto nutrient agar (0.5% Peptone, 0.3% beef extract, 0.5% NaCl and 1.5% agar).
6. Incubate plates at 30±2°C for 48h.
7. Sub-culture bacterial colonies exhibiting Bt-like phenotype for single-colony isolation again on fresh plates and incubate.
8. Stain the culture with amino black and Ziehl’s carbol fuchsin and examine under a standard light microscope for preliminary
identification.

Expected Results of Isolation of Bacillus thuringiensis

 Colonies mostly appear matte white colour, flat, dry and with uneven borders.
 Cultures that show parasporal crystals dyed black on microscopic observation may be of importance and require storing.
47 Differences between Prokaryotes and Eukaryotes
Differences between Prokaryotes and Eukaryotes

Here are some of the differences:

S.N. Character Prokaryotes Eukaryotes

Greek for “primitive Greek

1. Term Origin

nucleus” for “true nucleus”

Organisms made up of cells

Organisms made up of cell(s)
that possess a membrane-
2. Definition that lack a cell nucleus or any
bound nucleus as well as
membrane-encased organelles.
membrane-bound organelles.

Bacteria, Archae, and Algae, fungi, protozoa, plants,

3. Major groups
Bluegreen algae animals

4. Origin Around 3.5 billion years ago. Around 2 billion years ago.

5. Size (approximate) 0.5-3.0 μm >5 μm

Usually unicellular (some

6. Cell Type cyanobacteria may be Usually multicellular
multicellular)

7. Complexity Simple Complex organization.

8. Nucleus Location Free in the cytoplasm, attached Contained in membrane bound

 to mesosomes structure

9. Nucleur membrane No nuclear membrane. Classic membrane present.

10. Nucleolus Absent Present

Chromosome
11. One More than one
number

12. Chromosome shape Circular Linear

Expressed in groups called

13. Genes Expressed individually
operons.

14. Genome DNA haploid genome DNA diploid genome

DNA base ratio

15. 28-73 About 40
(G+C %)

Multiple proteins act together to

fold and condense prokaryotic
DNA. Folded DNA is then
DNA wrapping on Eukaryotes wrap their DNA
16. organized into a variety of
proteins around proteins called histones.
conformations that are
supercoiled and wound around
tetramers of the HU protein.

Efficient and compact with little With large amounts of non-

17. Genome nature
repetitive DNA. coding repetitive DNA.

Membrane-bound
18. Absent Present
organelles

Ribosomes
19. (sedimentation 70S (50S + 30S).Smaller. 80S (60S + 40S). Larger.
coefficient)

Free in cytoplasm or bound Attached to rough endoplasmic

20. Ribosome’s location
to cell membrane reticulum

21. Mitochondria Absent Present

22. Golgi bodies Absent Present

Endoplasmic
23. Absent Present
reticulum

24. Mesosomes Present. Performs the function Absent

 of Golgi bodies and
mitochondria and also help in
the separation of chromosome
during cell division.

25. Lysosomes Absent Present

26. Peroxisomes Absent Present

Chloroplasts Absent; chlorophyll scattered in

27. Present (in plants)
the cytoplasm
 

Prokaryotes may have pili

and fimbriae (appendage that
28. Fimbriae can be found on many Gram- Absent
negative and some Gram-
positive bacteria).

29. Microtubules Absent or rare Present

Present except in flowering

30. Centrosome Absent
plants.

31. Cytoskeleton May be absent Present

32. Glycocalyx Present Only in some

Cytoplasmic
33. Absent Present
streaming

Cytoplasmic Does not contain sterols

34. Contains sterols
membrane (except Mycoplasma)

Complex structure containing

Present for plant cells and
35. Cell wall protein, lipids, and
fungi; otherwise absent
peptidoglycans

36. Muramic acid Present Absent

37. Movement Simple flagellum, if present Complex flagellum, if present

38. Respiration Via cytoplasmic membrane Via mitochondria

Electron transport chain located

Energy production Within membrane bound
39. in the cell
site
mitochondria
membrane

40. Metabolic rate Higher due to larger Comparatively slow

 surface area to volume ratio

Sexual and asexual/ Mitotic

41. Reproduction Asexual (binary fission)
division

42. Generation time Shorter Comparatively longer

Genetic
43. Partial, unidirectional transfer Meiosis and fusion of gametes
Recombination

44. Zygote Merozygotic (partially diploid) Diploid

Extrachromosomal Inside the

45. Plasmid
DNA mitochondria

46. DNA replication Occurs in cytoplasm. Occurs in the nucleus.

Transcription occurs in nucleus

Transcription and
47. Occurs simultaneously. and then translation occurs in
translation
cytoplasm.

31 Differences Between Gram Positive and Gram Negative Bacteria

Differences Between Gram-Positive and Gram-Negative Bacteria
Some of the differences are:

Gram-Positive Gram-Negative
 S.N. Character
Bacteria Bacteria
Retain crystal violet dye and Accept safranin after
1.    Gram Reaction stain blue or purple on
Gram’s staining. decolorization and stain pink
or red on Gram’s staining.

2.    Cell wall thickness Thick (20-80 nm) Thin (8-10 nm)

3.    Peptidoglycan Layer Thick (multilayered) Thin (single-layered)

4.    Rigidity and Elasticity Rigid and less elastic Less rigid and more elastic

5.    Outer Membrane Absent Present

Variety of amino acid

6.  Few Several
in cell wall

Aromatic and Sulfur-

7.  containing amino acid Absent Present
in cell wall

8.    Periplasmic Space Absent Present

9.    Teichoic Acids Mostly present Absent

10. Porins Absent Present

Lipopolysaccharide
11. Virtually None High
(LPS) Content

Lipid and Lipoprotein Low (acid-fast bacteria have High (because of presence
12.
Content lipids linked to peptidoglycan) of outer membrane

13. Ratio of RNA:DNA 8:1 Almost 1

14. Mesosomes Quite Prominent Less Prominent

15. Flagellar Structure 2 rings in basal body 4 rings in basal body

16. Magnetosomes Usually absent. Sometimes present.

17. Morphology Usually cocci or spore Usually non-spore forming

forming rods (exception : rods (Exception : Neisseria)
Lactobacillus and
 Corynebacterium)

Some produce endospores

Usually not found to produce
18. Endospore formation during unfavorable
endospores.
conditions.

19. Toxin Produced Exotoxins Endotoxins or Exotoxins

Few pathogenic bacteria

Most pathogens are Gram
20. Pathogens belong to Gram positive
negative.
group.

Nutritional
21. Relatively Complex Relatively Simple
Requirements

Resistance to
22. High Low
Physical Disruption

Low (requires pretreatment

Cell Wall Disruption
23. High to destabilize outer
by Lysozyme
membrane)

Susceptibility to
24. Penicillin and High Low
Sulfonamide

Susceptibility to
Streptomycin,
25. Low High
Chloramphenicol and
Tetracycline

Inhibition by Basic
26. High Low
Dyes

Susceptibility to
27. High Low
Anionic Detergents

Resistance to Sodium
28.    High Low
Azide

29. Resistance to Drying High Low

They can rendered Gram -ve They can rendered Gram

30. Rendering
by increasing acidity +ve by increasing alkalinity

31. Examples Staphylococcus Escherichia

Streptococcus Salmonella
Bacillus Klebsiella
Clostridium Proteus
 Helicobacter
Enterococcus
Pseudomonas

28 Differences Between Bacteria and Virus (Bacteria vs Virus)

Differences Between Bacteria and Virus

Some of the differences between bacteria and virus are as follows:

S.N. Character Bacteria Virus

1 Cell type Prokaryotic cells Acellular

2 Number of cells Single-celled No cell

3 Size Larger than viruses (0.3-2 μ) Minute (0.02-0.3 μ)

Visible under Light Visible only under an Electron

4 Microscopy
Microscope. Microscope.

Viruses typically have

 Common bacterial cell
spherical (polyhedral), rod-
shapes include cocci
shaped, or helically shaped
5 Shape (spherical), bacilli (rod-
capsids while some viruses,
shaped), spiral, and vibrio
such as bacteriophages, have
(comma-shaped).
complex shapes.

Possesses a cellular
6 Cellular Machinery Lack of cellular machinery
machinery 
 Mostly intercellular organisms Intracellular organisms (they
7 Type of organism (i.e. they live in-between infiltrate the host cell and live
cells); some intracellular. inside the cell). 

Genetic material within a

Organelles and genetic
8 Structure capsid, some have an
material within a cell wall
envelope membrane.

Cell wall made of

No cell wall. Protein coat
9 Cell wall peptidoglycan and
presents instead.
lipopolysaccharide.

Cell membranes present. No

sterol except Some are enveloped, but no
10 Cellular membrane
in Mycoplasma cells which membrane function.
have cholesterol.

DNA and RNA DNA or RNA

11 Genome
1 chromosome 1 nucleocapsid except in
No histones segmented or diploid viruses

DNA and RNA floating freely DNA or RNA is enclosed

12 Nucleic acid
in the cytoplasm. inside a coat of protein.

Some have poly-cistronic

Mono- and poly-cistronic
13 mRNA mRNA and post-translational
mRNA
cleavage.

Presence of non-membrane Absent. Uses host organelles;

14 Cell organelles
bound cell organelles. obligate intracellular parasites

15 Ribosomes 70s ribosomes (30s+50s) No ribosomes

Between living and non-living

16 Living attributes Living organisms.
things.

It invades a host cell and

takes over the cell causing it
Binary fission (asexual). DNA
17 Replication to make copies of the viral
replicates cells continuously.
DNA/RNA. Destroys the host
cell releasing new viruses.

The need for host Need a living cell to

18 Able to reproduce by itself.
cell reproduce

In some spore-forming Besides viruses, two other

19 Other forms bacteria, sporulating forms acellular forms exist Viroids
can be seen. and Prions.

20 Cells Infected Animal, Plant, Fungi Animal, Plant, Protozoa,

 Fungi, Bacteria, Archaea

21 Infection Localized Systemic 

A bacterial illness notoriously A viral infection may or may

22 Induction of Fever
causes a fever not cause a fever.

A bacterial illness commonly Most viral illnesses last 2 to

23 Duration of illness
will last longer than 10 days. 10 days.

Food poisoning, gastritis, and

AIDS, common cold,
24 Diseases/Infections ulcers, meningitis,
influenza, chickenpox, etc
pneumonia, etc

Susceptibility to Most bacteria are susceptible The virus does not respond to
25
Antibiotics to antibiotics. antibiotics.

26 Treatment Antibiotics Antiviral drugs

Viruses are not beneficial.

Some bacteria are beneficial However, a particular virus
27 Beneficial use (as normal flora, probiotics, may be able to destroy brain
fermenters, etc.) tumors. Viruses can be useful
in genetic engineering.

E.coli, Salmonella spp.,
Listeria spp., Mycobacteria HIV, Hepatitis A virus, Rhino
28 Examples
spp., Staphylococcus Virus, Ebola virus, etc.
spp., Bacillus anthracis, etc.