INTRODUCTION

TO

MICROBIOLOGY

STM 211

THE HISTORY AND SCOPE OF MICROBIOLOGY

Microbiology is the study of microorganisms usually less than 1mm in diameter which requires some form
of magnification to be seen clearly. In other words, Microbiology is the study of organisms and agents that
are generally too small to be seen clearly by the unaided eye, but by the use of microscope. These
organisms include viruses, bacteria, algae, fungi, and protozoa.

Historical Perspectives

Scientists that contributed to the history of microbiology and their contributions

• Robert Hooke (1665) – He reported that life’s smallest structural units were ‘little boxes’ or ‘cells’. This
marked the beginning of cell theory – that all living things are composed of cells.
• Van Leuwenhoek (1673) – He discovered the ‘invisible’ world of microorganisms ‘animalcules’.

• Francesco Redi (1668) – He was a strong opponent of spontaneous generation. He demonstrated that
maggots appear on decaying meat only when flies are able to lay eggs on the meat.

• John Needham (1745) – He claimed that microorganisms could arise spontaneously from heated nutrient
broth.

• Lazzaro Spallanzani (1765) – He repeated Needhams experiments and suggested that Needham’s results
were due to microorganisms in the air entering the broth.

• Rudolf Virchow (1858) – He brought about the concept of biogenesis that is, living cells can arise only
from preexisting cells.

• Louis Pasteur (1822-1895) – His discoveries led to the development of aseptic techniques used in the
laboratory and medical procedure to prevent contamination by microorganisms that are in the air.

Fermentation and Pasteurization:

• Pasteur found that yeast ferments sugars to alcohols and that bacterium can oxidize the alcohol to acetic
acid.

• Heating processes called pasteurization is used to kill bacteria in some alcoholic beverages and milk.

The Germ theory of disease: Previously, people thought that disease was punishment for an individual's
crimes, due to poisonous vapors etc. Before it was experimentally proved that microbes are the etiological
agent of diseases, some scientists had expressed a strong support for the theory.

Paul Ehrlich (1845) developed modern concept of chemotherapy and chemotherapeutic agents.

Elie Metchnikoff (1845) discovered phagocytosis.

Hans Christian Gram (1853) developed Gram’s staining techniques.

• Joseph Lister (1860s) – He introduced the use of disinfectant (Phenol, a carbolic acid) to clean surgical
dressings in order to control infection in humans.

• Agostino Bassi (1934) and Pasteur (1865) – showed a casual relationship between microorganisms and
disease.

• Robert Koch (1876) – He demonstrated that anthrax disease of sheep and cattle is caused by Bacillus
anthracis. He proved that microorganisms transmit disease. Koch’s postulates which are used today to
prove that a particular microorganism causes a particular disease.

• Koch introduced pure cultures. He also developed techniques for isolating organisms he developed
tuberculin and studied various diseases in Africa and Asia. His studies on Tuberculosis won him Nobel
Prize for philosophy and medicine in 1905.

Vaccination:

• Immunity is conferred by inoculation with a vaccine.

• Edward Jenner (1798) – demonstrated that inoculations with cowpox material provides humans with
immunity from small pox.

Spontaneous Generation or Abiogenesis: This is the belief that life could originate from non-
living/abiotic source or decomposing matter.

Supported by:

Aristotle (384-322 BC) – He believed that simple invertebrates could arise by spontaneous Generation.

John Needham (1748) – supported spontaneous generation of microbes by showing that even after boiling
mutton broth and pouring into sealed containers, growth of microbes occurred after a period of time

Felix Pouchet (1859) – He proved growth without contamination from air.

Disproved by:

Francesco Redi (1626-1697) – first to challenge theory of spontaneous generation by showing that if raw
meat was protected from flies, by covering with gauze the formation of maggots was prevented.

Schwann, Friedrich Schroder and von Dusch (1830s) – Air allowed to enter flask but only after passing
through a heated tube or sterile wool

John Tyndall (1820-1893) – Omission of dust, no growth. He demonstrated heat resistant forms of
bacteria (endospores)

Louis Pasteur (1822 - 1895) trapped airborne organisms in cotton; he also heated the necks of flasks,
drawing them out into long curves, sterilized the media, and left the flasks open to the air; no growth was
observed because dust particles carrying organisms did not reach the medium, instead they were trapped
in the neck of the flask; if the necks were broken, dust would settle and the organisms would grow; in this
way Pasteur disproved the theory of spontaneous generation.

1776: L. Spallanzani- He challenged spontaneous generation as it pertained to microbes by showing that

sealed containers do not produced microbes.

Scope of microbiology

1.Medical microbiology: It deals with the identification of plans and measures to prevent and control/
cure diseases of human and animals which are infectious to them. Some of these aspects include
antibiosis, chemotherapy, immunization and diagnostic measures. Other areas of medical microbiology
are

i. Public health and epidemiology: Studies and controls transmission, frequency, and distribution of
disease.

ii. Immunology: This deals with the study of immune system which protect the body from pathogens.

2. Agricultural/ soil microbiology: This has to do with the impact of microbes on agriculture, use of
biofertlizers and biopesticides and also the use of microbes in reclamation of sodic (alkaline) soil. Also,
useful in the prevention of the diseases that mainly damage the useful crops. Microbes are found in
association with plants in symbiotic relationships, they are used to maintain soil fertility, used for
biodegradation or bioremediation while some are devastating plant pathogens, but others act as biological
control agents against these diseases.

3. Microbial ecology: This deals with relationships between microbes and their habitats. It also involves
the use of microbes to study the occurrence of microbes in the solar system, for maintenance of
carbon/oxygen balance in space, for prospecting for oil, the use of biological weapons in warfare, for
water and sewage treatments.

4. Food microbiology: It deals with prevention of food borne disease. It involves the prevention of
spoilage of food and food borne diseases and the uses of microbes to produce cheese, yoghurt, pickles and
beer. Food infections are caused by microorganisms such as Salmonella while food intoxications are
caused by certain microbes like Staphylococcus and Clostridia. Many microbes are beneficia to humans in
the area of fermentation.

5. Industrial microbiology: It deals with commercial use of microbes to produce finished products such
as medicines, food supplements, alcoholic beverages, enzymes, antibiotics, steroids, alcohol, vitamins and
amino acids.

6. Biotechnology: This deals with the manipulation of organisms to form useful products. Other areas
are:

i. Microbial genetics and Molecular biology– It deals with the nature of genetic information and how it
regulated the development and function of cells and organisms. It is also useful for the development of
new microbial strains that is more efficient in synthesizing useful products.

ii. Genetic engineering – This arose from work of microbial genetics and molecular biology. Engineered
microorganisms are used to make hormones, antibiotics, vaccines and other products useful for human
beings. New genes can also be inserted into plants and animals. Bacteria can produce important
therapeutic substances such as insulin, human growth hormone, and interferon. Because microbes
reproduce very quickly, they are useful for studies involving the transfer of genetic information.

7. Microbial physiology and Biochemistry – study the synthesis of antibiotics and toxins, microbial
energy production, microbial nitrogen fixation, effects of chemical and physical agents on microbial
growth and survival etc.

8. Environmental microbiology: This deals with the study ofmicrobes that are responsible for the
cycling of carbon, nitrogen phosphorus (geochemical cycles). Microbes are used to maintain ecological
balance on earth and may also be used to clean up the environment of toxic compounds.
 THE MICROSCOPE

Microscope is a powerful and crucial basic tool in the field of microbiology (the science that deals with
the organismstoo small to be seen with unaided eye). The existence of microbial life to the world
wasintroduced first by Antony Van Leeuwenhoek in 1673, with the help of simple, crude, self-made,
single-lens microscope having a magnification of about 300. Over the years,microscopes have evolved to
increase the magnification several thousand fold. Modernday microscopes are either light microscopes or
electron microscopes.

Early Microscopes

Early microscopes were of two kinds. The first was a simple microscope with a single lens of short focal
length, which does not differ in principle from ordinary hand lens. The second type is the compound
microscope with a double lens system consisting of eye piece lens and the objective lens. It has a greater
magnification and eventually displaced the use of simple microscope.

Types of Microscopes

Various types of microscopes are available for use in the microbiology laboratory. The microscopes have
varied applications and modifications that contribute to their usefulness. There are 2 major types which
are:

1. The light or optical microscope

2. The electron microscope

Light Microscope

Modern light microscopes are compound microscopes because they contain two types of lenses that
function to magnify an object. The ocular lens which is closer to the eye and the objective lens closer to
the object. Here the magnifiedimage formed by the objective lens is further enlarged by one or more
additional. A variety of light microscopes are used now-a-days by microbiologistsfor different purposes.
These include:

(a) Bright Field Microscopes –This is the most commonly used microscope in biologyand microbiology
courses. It is called so because it forms a dark imageagainst a brighter background. It contains 2 lenses
systems for magnification:

The ocular lens in eyepiece and the objective lens located in nosepiece. The specimen is illuminated by a
light focusedon it by a sub-stage lens called a condenser. With this microscope,specimens are made
visible because of the differences in contrast thatexist between them and the surrounding medium.
Contrast differencesarise because cells absorb or scatter light in varying degrees. Live cells,however, are
difficult to observe through this microscope due to absenceof contrast between specimen and the
surrounding medium. It is therefore,used to observe nonviable and stained preparations where contrast
isincreased and variations in colour between cell structures are evident.

(b)Dark Field Microscope --- This microscope contains a special condenser that scatters light and causes
it to reflect off the specimen at an angle. A light object is seen on a dark background. In this microscope,
condenser system is modified, so that only reflected and refracted light by specimen forms animage. Here,
the object is brightly illuminated while the field surrounding the object appears black. This microscope
can be used to observe live unstained cells andorganisms for example, live spirochetes especially such that
cause syphilis. The internal cell parts such as mitochondria, lysosomes, and the Golgi body can also be
seen with this instrument.

(c) Phase Contrast Microscope – Unpigmented and unstained living cells can be easilyobserved by
phase contrast microscope. It has a special objective and a condenserthat converts slight differences in
refractive index and cell density into easily detectedvariations in light intensity and produce visible image
of the structure under study. The special condensers throw light “out of phase” and cause it to pass
through the object at different speedsThe image appears dark against a light background. Phase contrast
microscopy isused widely to study microbial motility, determining the shape of living cells anddetecting
microbial structures like inclusion bodies and endospores etc.

(d)Fluorescent Microscope – In above said microscopes, i.e. (a)-(c), image is produced fromlight that
passes through a specimen but in fluorescent microscope, specimenimage is formed because of the light
emitted by the object itself. This microscope isused more frequently to visualize specimens that are tagged
with a fluorescent dye.The source of illumination here is an ultraviolet light obtained from a high-pressure
mercury vapour arc lamp or hydrogen quartz lamp. Fluorescent dye absorbs theultraviolet light of desired
wavelength and re-emits the energy in visible range.Fluorescent portion of the dye becomes visible against
a black background. Thefluorescent microscope has become an essential tool in medical microbiology
andmicrobial ecology for identifying the pathogens and other microbes.The forms of light microscopy just
considered, forms two-dimensional images. Besides,certain new forms of light microscopy have been
developed which gives three-dimensionalimages. These are differential interference contrast microscopy,
atomic force microscopyand confocal scanning laser microscopy.

Electron Microscope

The energy source used in the electron microscope is a beam of electrons. Since the beam has an
exceptionally short wavelength, it strikes most objects in its path and increases the resolution of the
microscope significantly. The electrons travel in a vacuum to avoid contact with deflecting air molecules,
and magnets focus the beam on the object to be viewed. An image is created on a monitor and viewed by
the technologist.

Electron microscopes have higher magnification and resolving power than lightmicroscopes. it is
important to note that in electronmicroscope, the limit of resolution is 0.2 nm and magnification can be up
to more than onemillion. These microscopes can be used to visualize submicroscopic cellular
particles,viruses etc. Electron microscopes are of two types:

(a) Transmission Electron Microscope –The more traditional form of electron microscope is
the transmission electron microscope (TEM). To use this instrument, one places ultrathin and dehydrated
slices of microorganisms or viruses on a wire grid and then stains them with gold or palladium before
viewing. Electrons pass through the specimen and form image on to photographic film. Theseare used to
observe internal cell structure.

(b) Scanning Electron Microscope –The scanning electron microscope (SEM) is the more contemporary
form electron microscope. Although this microscope gives lower magnifications than the TEM, the SEM
permits three‐dimensional views of microorganisms and other objects.
 It can be used to observe intact cells or cellcomponents directly, whole objects are used, and gold or
palladium staining is employed. Then sections are not necessary. It is used for visualizingsurface
characteristics rather than intracellular structures. A narrow beam of electronsscan back and forth,
producing a three-dimensional image as electrons are reflectedoff the specimen’s surface.Though a variety
of optical instruments are available for routine laboratory work, compoundbright field microscope is the
one commonly found in all biological laboratories.

Principle of Microscopy

When the light passes from one medium to another, refraction occurs, i.e., the ray is bent at the interface.
The direction and the magnitude of bending are determined by the refractive indices of the two media
forming the interface. The refractive index is a measure of how greatly a substance slows the velocity of
light when light passes from air to glass or vice versa. When the light rays strike the lens, a convex lens
will focus these rays at specific point called focal point. The distance between the center of the lens and
the focal point is called the focal length. Convex lens act as a magnifier. It provides a clear magnifying
image at a much closer range. Lens strength is related to focal length. A lens with a short focal length has
a more magnification power than a lens having a longer focal length.

Magnification means enlargement. Though magnification is important, it has limits. Microscope should
not only produce the enlarged image but also the clear image. There is a degree of magnification beyond
which any further enlargement from the objective does not bring into view any more detail but only a
more highly magnified image which is increasingly blurred, this is known as empty or useless
magnification

To magnify an object, light is projected through an opening in the stage, where it hits the object and then
enters the objective. An image is created, and this image becomes an object for the ocular lens, which
remagnifies the image. Thus, the total magnification possible with the microscope is the magnification
achieved by the objective multiplied by the magnification achieved by the ocular lens.

A compound light microscope often contains four objective lenses: the scanning lens (4X), the low-power
lens (10X), the high-power lens (40 X), and the oil-immersion lens (100 X). Most microscopes
are Parfocal meaning that the microscope remains in focus when one switches from one objective to the
next objective.The ability to see clearly two items as separate objects under the microscope is called
the resolution of the microscope. The resolution is determined in part by the wavelength of the light used
for observing. Visible light has a wavelength of about 550 nm, while ultraviolet light has a wavelength of
about 400 nm or less. The resolution of a microscope increases as the wavelength decreases, so ultraviolet
light allows one to detect objects not seen with visible light. The resolving power of a lens refers to the
size of the smallest object that can be seen with that lens. The resolving power is based on the wavelength
of the light used and the numerical aperture of the lens. The numerical aperture (NA) refers to the
widest cone of light that can enter the lens; the NA is engraved on the side of the objective lens.

However, the oil-immersion lens is exceedingly narrow, and most light misses it. Therefore, the object is
seen poorly and without resolution. To increase the resolution with the oil-immersion lens, a drop
of immersion oil is placed between the lens and the glass slide. Immersion oil has the same light-bending
ability (index of refraction) as the glass slide, so it keeps light in a straight line.
 (a) The important parts of a common light microscope. (b) How immersion oil gathers more light for use
in the microscope.

Parts of Microscope

Microscope is a metal body composed of a base and an arm to which remaining parts are attached. The
components of the microscope are;

i) Light Source – It is either mirror or electric illuminator present at the base. Some microscopes have
reversible mirror with one side flatand other concave. Concave side of the mirror is used for sunlight and
its flat side isused for artificial light, e.g., tungsten lamp. Other microscopes have built in lightsource.

ii) Condenser – It is present beneath the stage. It collects and focuses a cone of light on the slide. Its
position can be adjusted verticallyto regulate the light passing on to the specimen. It also has iris
diaphragm- a shutterthat is also used to adjust the amount of light entering into the lens system.

iii) Stage – It is a fixed platform positioned about halfway up the arm and providesurface for keeping the
slide. Slide is held in position either by simple slide clip or by a mechanical stage clip. Latter helps the
viewer inmoving the slide around during viewing by use of stage control knobs. Stage has anopening in
the center through which light can pass from an illuminating source.

iv) Body Tube – Above the stage and attached to the arm is the body tube, to whicheye piece(s) or ocular
lens and revolving nosepiece is attached. Microscopes havingeyepieces for both eyes are called binocular
microscopes. Nosepiece containsthree to five objective lenses of different magnifying power, which can
be rotated atwill. Microscope should be parfocal, i.e., the image should remain in focus whenobjectives
are changed. The objectives are nearer the specimen and it produces thereal magnified image of the
specimen at focal plane. This image is further magnifiedby ocular lens to produce the final image.

v) Objective Lenses

The objective lenses combine with the eyepiece lens to increase magnification levels. Microscopes
generally feature three or four objective lenses, with magnification levels ranging 4x to 100x.

vi)The Rack Stop

The rack stop prevents users from moving the objective lenses too close to the slide, which could damage
or destroy the slide and specimen.

Use and care of Microscope

Proper care and maintenance of microscope is needed. Following points should be kept in mind while
handling the microscope:

 Instrument should be kept in special cabinets while not in use.

 Microscope should be held firmly by holding the arm with right hand and base withleft arm e.g.
Hold the plug (not the cable) when unplugging the illuminator.

 All the lens systems should be cleaned with lens tissue to remove dust, oil, etc.,which may
decrease the efficiency of the microscope. Blotting paper, cloth or towelshould not be used for
cleaning. NEVER use a paper towel, a kimwipe, your shirt, or any material other than good quality
lens tissue or a cotton swab (must be 100% natural cotton) to clean an optical surface. Be gentle!
You may use an appropriate lens cleaner or distilled water to help remove dried material. Organic
solvents may separate or damage the lens elements or coatings.

 If the lens is sticky or oily, the lens can be cleaned with xylol followed by 95%alcohol. The lens is
wiped dry with lens paper. This should be performed only ifnecessary as consistent use of xylol
may loosen the lens. EVERYTHING on a good quality microscope is unbelievably expensive, so
be careful

 Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.

 Always make sure the stage and lenses are clean before putting away the microscope.

 Cover the instrument with a dust jacket when not in use.

 Focus smoothly; don't try to speed through the focusing process or force anything. For example if
you encounter increased resistance when focusing then you've probably reached a limit and you
are going in the wrong direction.
Microbial staining techniques
Microbial staining is a technique that can be used to better visualize cells and cell components under a
microscope. Microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before
they can be viewed with the light microscope. In some cases, staining is unnecessary, for example when
microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be
placed directly on the slide and observed. A preparation such as this is called a wet mount. A wet mount
can also be prepared by placing a drop of culture on a cover‐slip (a glass cover for a slide) and then
inverting it over a hollowed‐out slide. This procedure is called the hanging drop.
Cells may be stained to highlight metabolic processes or to differentiate between live and dead cells in a
sample. Cells may also be enumerated by staining cells to determine biomass in an environment of
interest.
By using different stains, one can preferentially stain certain cell components, such as a nucleus or a cell
wall, or the entire cell. Most stains can be used on fixed, or non-living cells, while only some can be used
on living cells; some stains can be used on either living or non-living cells.
Staining and preparation of slides
Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the
following procedures may be required to prepare a sample:
• Permeabilization - treatment of cells, generally with a mild surfactant, which dissolves cell
membranes in order to allow larger dye molecules to enter inside the cell.
• Fixation - serves to "fix" or preserve cell or tissue morphology through the preparation process.
This process may involve several steps, but most fixation procedures involve adding a chemical fixative
that creates chemical bonds between proteins to increase their rigidity. Common fixatives include
formaldehyde, ethanol, methanol, and/or picric acid.
• Mounting - involves attaching samples to a glass microscope slide for observation and analysis.
Cells may either be grown directly to the slide or loose cells can be applied to a slide using a sterile
technique. Thin sections (slices) of material such as tissue may also be applied to a microscope slide for
observation.
• Staining - application of stain to a sample to color cells, tissues, components, or metabolic
processes. This process may involve immersing the sample (before or after fixation or mounting) in a dye
solution and then rinsing and observing the sample under a microscope. Some dyes require the use of a
mordant, which is a chemical compound that reacts with the stain to form an insoluble, colored precipitate.
The mordanted stain will remain on/in the sample when excess dye solution is washed away.

In preparation for staining, a small sample of microorganisms is placed on a slide and permitted to air dry.
The smear is heat fixed by quickly passing it over a flame. Heat fixing kills the organisms, makes them
adhere to the slide, and permits them to accept the stain.
Simple staining techniques: This can be performed with basic dyes such as crystal violet or methylene
blue, positively charged dyes that are attracted to the negatively charged materials of the microbial
cytoplasm. Such a procedure is the simple stain procedure. An alternative is to use a dye such as nigrosin
or Congo red, acidic, negatively charged dyes. They are repelled by the negatively charged cytoplasm and
gather around the cells, leaving the cells clear and unstained. This technique is called the negative stain
technique.
Differential staining techniques
i. Gram staining - This differential technique separates bacteria into two groups, Gram‐positive bacteria
and Gram‐negative bacteria. Crystal violet is first applied, followed by the mordant iodine, which fixes the
stain. Then the slide is washed with alcohol, and the Gram‐positive bacteria retain the crystal‐violet iodine
stain; however, the Gram‐negative bacteria lose the stain. The Gram‐negative bacteria subsequently stain
with the safranin dye, the counterstain, used next. These bacteria appear red or pink under the oil‐
immersion lens, while Gram‐positive bacteria appear blue or purple, reflecting the crystal violet retained
during the washing step.
ii. Acid‐fast staining - This technique differentiates species of Mycobacterium from other bacteria. Heat
or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed
with a dilute acid‐alcohol solution. Mycobacterium species resist the effect of the acid‐alcohol and retain
the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene
blue stain (blue). Thus, the acid‐fast bacteria appear bright red, while the non acid‐fast bacteria appear
blue when observed under oil‐immersion microscopy.
iii. Endospore staining - The endospore stain is a differential stain used to visualize bacterial endospores.
The main purpose of endospore staining is to differentiate bacteria spores from other vegetative cells and
to differentate spore formers from non- spore formers.
Endospores are formed by a few genera of bacteria, such as Bacillus. By forming spores, bacteria can
survive in hostile conditions. Spores are resistant to heat, dessication, chemicals and radiation. Bacteria
can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions. The
normally-growing cell that forms the endospore is called a vegetative cell. Spores are metabolically
inactive and dehydrated. They can remain viable for thousands of years. When spores are exposed to
favorable conditions, they can germinate into a vegetative cell within 90 minutes.
Endospores can form within different areas of the vegetative cell. They can be central, sub terminal, or
terminal. Central endospores are located within the middle of the vegetative cell. Terminal endospores are
located at the end of the vegetative cell. Sub terminal endospores are located between the middle and the
end of the cell. Endospores can also be larger or smaller in diameter than the vegetative cell. Those that
are larger in diameter will produce an area of "swelling" in the vegetative cell. These endospore
characteristics are consistent within the spore-forming species and can be used to identify the organism.
Because of their tough protein coats made of keratin, spores are highly resistant to normal staining
procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells
with heat. Since malachite green is water-soluble and does not adhere well to the cell, and since the
vegetative cells have been disrupted by heat, the malachite green rinses easily from the vegetative cells,
allowing them to readily take up the counterstain.
The staining procedure:
1. Perform a bacterial smear of Bacillus or the organism you want to stain,
2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green.
3. Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper
saturated with malachite green during heating. If the slide is steaming, you’re okay; if it stops
steaming, add more malachite green!
4. Remove the bibulous paper from the slide, and rinse the slide gently with water. Dispose of the
used bibulous paper in the trash. DO NOT leave the bibulous paper in the sink or drain!
5. Counterstain with safranin for 2 minutes.
6. Rinse the slide gently with water.
7. Carefully blot the slide dry with bibulous paper.
8. Observe the slide under the microscope, using proper microscope technique.
Endospores will stain green. Parent cells will stain red/ pink.
iv. Flagella stain - The flagella stain allows observation of bacterial flagella under the light microscope.
Bacterial flagella are normally too thin to be seen under such conditions. The flagella stains employ a
mordant to coat the flagella with stain until they are thick enough to be seen. Staining bacterial flagella
differs from staining other bacterial structures because it usually requires extraordinary care for the slides,
stain, and cells. The number and arrangements of flagella are critical in identifying species of motile
bacteria.
Two techniques for staining flagella are in use:
1. A wet-mount procedure (Ryu method)
2. Dried-smear preparation (Leifson staining technique)
A wet-mount technique for staining bacterial flagella is highly successful when a stable stain and regular
slides and cover slips are used. This technique is simple for routine use when the number and arrangement
of flagella are critical in identifying species of motile bacteria.
The preparations are not permanent because the stain precipitates as the wet mount dries.
Method:
1. Grow the organism to be stained at room temperature on blood agar for 16-24 hours.
2. Add a small drop of water to a microscope slide.
3. Dip a sterile inoculating loop into the sterile water
4. Touch the loopful of water to the colony margin briefly (this allows motile cells to swim into the
droplet of water)
5. Touch the loopful of motile cells to the drop of water on the slide. NOTE: Agitating the loop in the
droplet of water on the slide causes the flagella to shear off the cell.
6. Cover the faintly turbid drop of water on the slide with a cover slip. A proper wet mount has barely
enough liquid to fill the space under a cover slip. Small air spaces around the edge are preferable.
7. Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not seen, do
not proceed with the stain.
8. If motile cells are seen, leave the slide at room temperature for 5 to10 minutes. This allows time
for the bacterial cells to adhere to either the glass slide or the cover slip.
9. Apply 2 drops of RYU flagella stain gently to the edge of the cover slip. The stain will flow by
capillary action and mix with the cell suspension. Small air pockets around the edge of the wet mount are
useful in aiding the capillary action.
(Note: The Ryu stain has two components. Solution I, the mordant, contains 10 ml of 5% aqueous solution
of phenol, 2 g of tannic acid, and 10 ml of saturated aqueous solution of aluminum potassium sulfate-12
hydrate. Solution II, the stain, is a saturated ethanolic solution of crystal violet (12 g in 100 ml of 95%
ethanol). The final stain was prepared by mixing 1 part solution Il with 10 parts solution I and then
filtering the mixture through filter paper to remove coarse precipitate)
10. After 5 to 10 minutes at room temperature, examine the cells for flagella.
11. Cells with flagella may be observed at 100× (oil) in the zone of optimum stain concentration, about
half way from the edge of the coverslip to the center of the mount.
12. Focusing the microscope on the cells attached to the coverslip rather than the cells attached to the
slide facilitates visualization of the flagella. The precipitate from the stain is primarily on the slide rather
than the cover slip.
 CHARACTERISTICS OF MICROORGANISMS

Major characteristics of microbes fall into the following categories:

1. Morphological characteristics which include cell shape, size, cel arrangement, staining reactions,
motility etc.
2. Chemical compositions i.e the various chemical constituents of the cells.
3. Cultural characteristics like nutritional requirements, physical conditions required for growth and
manners of growth.
4. Metabolic characteristics such as the way cells obtain and use their energy to carryout chemical
reactions and also how the reactions are regulated.
5. Antigenic characteristics-this incudes special large chemical components of the cell
(antigens),distinctive for certain kinds of microbes.
6. Genetic characteristics: This involves the characteristics of the hereditary materials of the cell
(DNA), occurrence and functions.
7. Pathogenicity: This is the ability to cause diseases in various plants and animals or microbes.
8. Ecological characteristics: This is the habitat and the distribution of the organism in nature and the
interactions between and among species in natural environments.

Prokaryotics and Eucaryotic cells

Microorganisms are a heterogeneous group of several distinct classes of living things. Based on the
difference in cellular organization and biochemistry, the kingdom protista has been divided into two
groups namely prokaryotes andeukaryotes. Bacteria and blue-green algae(cyanobacteria) are prokaryotes,
while fungi, other algae, slime moulds and protozoa are eukaryotes.

The prokaryotic cells have no membrane bound nucleus or any other well developed membrane bound
compartments in the cytoplasm. Eukaryotic cells on the other hand have a well-defined nucleus and
specialized membrane bound organelles.

Assignment: In a tabular form, give at least 10 differences between prokaryotic cells and Eukaryotic cells
in terms of the composition of their organelles.

Morphological characteristics of Microrganisms

A. Bacteria

Bacteria are unicellular organisms. The cells are described as prokaryotic because they lack a nucleus.
They exist in four major shapes: bacillus (rod shape), coccus (spherical shape), spirilla (spiral shape), and
vibrio (curved shape). Most bacteria have a peptidoglycan cell wall; they divide by binary fission; and
they may possess flagella for motility. The difference in their cell wall structure is a major feature used in
classifying these organisms.

According to the way their cell wall structure stains, bacteria can be classified as either Gram-positive or
Gram-negative when using the Gram staining. Bacteria can be further divided based on their response to
gaseous oxygen into the following groups: aerobic (living in the presence of oxygen), anaerobic (living
without oxygen), and facultative anaerobes (can live in both environments).
According to the way they obtain energy, bacteria are classified as heterotrophs or autotrophs. Autotrophs
make their own food by using the energy of sunlight or chemical reactions, in which case they are called
chemoautotrophs. Heterotrophs obtain their energy by consuming other organisms. Bacteria that use
decaying life forms as a source of energy are called saprophytes.

Shapes of Bacteria

Depending on their shape, bacteria are classified into 3

1. Cocci (from kokkos meaning berry) are spherical or oval cell

2. Bacilli (from baculus meaning rod) are rod shaped cells

3. Vibrios are comma shaped curved rods and derive their name from theircharacteristics vibratory
motility. This includes Spirilla which are rigid spiral forms; Spirochetes (from speira meaning coil and
chaite meaning hair) are flexuous spiral forms

Shapes of bacteria.

Bacteria sometime show characteristic cellular arrangement or grouping.According to the plane of cellular
division, cocci may be arranged in pairs(diplococci), chains (streptococci), groups of four (tetrads) or eight
(sarcina),or grape like clusters (staphylococci).

Arrangement of Cocci.
Bacterial Structure

The outer layer or cell envelope consists of two components, a rigid cell wall and beneath it a cytoplasmic
or plasma membrane. The cell envelope enclosesthe protoplasm, comprising the cytoplasm, cytoplasmic
inclusions such as ribosomes and mesosomes, granules, vacuoles and the nuclear body.

Cell wall

Beneath the external structures is the cell wall. It is very rigid & gives shape to the cell. Its main function
is to prevent the cell from expanding & eventually bursting due to water uptake. Cell Wall constitutes a
significant portion of the dry weight of the cell and it is essential for bacterial growth and division. The
cell wall cannot be seen by direct light microscopy and does not stain with simple stains. It may be
demonstrated by micro dissection, reaction with specific antibodies, mechanical rupture of the cell,
differential staining procedures or by electron microscopy. Chemically the cell wall is composed of
peptidoglycan. Mucopeptide (peptidoglycan or murien) formed by N acetyl glucosamine and N acetyl
muramic acid alternating in chains, cross linked by peptide chains. Embedded in it are polyalcohol called
Teichoic acids. Some are linked to Lipids & called Lipoteichoic acid. Lipotechoic acid link peptidoglycan
to cytoplasmic membrane and the peptidoglycan gives rigidity. The functions of Teichoic acid are: It gives
negative charge, major antigenic determinant, transport ions, anchoring and external permeability barrier.

Outer Membrane

Outer membrane is found only in Gram-negative bacteria, it functions as an initial barrier to the
environment and is composed of lipopolysaccharide (LPS) and phospholipids

Lipopolysaccharide (LPS)

The LPS present on the cell walls of Gram-negative bacteria account for their endotoxic activity and
antigen specificity. A bacterium is referred as a protoplast when it is without cell wall. This cell is easily
lysed and it is metabolically active, not susceptible to infection by bacteriophages but unable to reproduce.
Cell wall may be lost due to the action of lysozyme enzyme, which destroys peptidoglycan..

A bacterium with a damaged cell wall is referred as spheroplasts and they are not clean protoplasts. It is
caused by the action of toxic chemical or an antibiotic, they show a variety of forms and they are able to
change into their normal form when the toxic agent is removed, (i.e. when grown on a culture media),
forming L-forms. Gram negative yields spheroplasts while gram positive yields protoplasts.

Cytoplasmic membrane

Cytoplasmic membrane is present immediately beneath the cell wall, found in both gram positive &
negative bacteria and it is a thin layer lining the inner surface of cell wall and separating it from
cytoplasm. It acts as a semi permeable membrane controlling the flow of metabolites to and from the
protoplasm.

Cytoplasm

The cytoplasm is a colloidal system containing a variety of organic and inorganic solutes containing 80%
Water and 20% Salts, Proteins. They are rich in ribosomes, DNA and fluid. DNA is circular and haploid.
They are highly coiled with intermixed polyamines & support proteins. Plasmids are extra circular DNA.

Ribosomes

They are the centers of protein synthesis. They are slightly smaller than the ribosomes of eukaryotic cells

Mesosomes

They are vesicular, convoluted tubules formed by opening of plasma membrane into the cytoplasm. They
are principal sites of respiratory enzymes and help with cell reproduction.

Cytoplasmic Inclusions

The Inclusion bodies are aggregates of polymers produced when there is excess of nutrients in the
environment and they are the storage reserve for granules, phosphates and other substances. Volutin
granules are polymetaphosphates which are reserves of energy and phosphate for cell metabolism and they
are also known as metachromatic granules.

Nucleus

The Nucleus is not distinct and has no nuclear membrane or nucleolus and the genetic material in it
consists of DNA. The cytoplasmic carriers of genetic information are termed plasmids or episomes.

Capsule

Capsule is the outer most layer of the bacteria (extra cellular). It is a condensed well defined layer closely
surrounding the cell. They are usually polysaccharide and if polysaccharide envelops the whole bacterium
it is the capsule and their production depends on growth conditions. They are secreted by the cell into the
external environment and are highly impermeable. When it forms a loose mesh work of fibrils extending
outward from the cell they are described as glycocalyx and when masses of polymer that formed appear to
be totally detached from the cell and if the cells are seen entrapped in it, they are described as slime layer.
The Capsule protects against complement and is antiphagocytic. The Slime layer and glycocalyx helps in
adherence of bacteria either to themselves forming colonial masses or to surfaces in their environment and
they resists phagocytosis and desiccation of bacteria.
Flagella

Flagella are long hair like helical filaments extending from cytoplasmic membrane to exterior of the cell.
Flagella is highly antigenic and functions in cell motility, sensation, adhesion and moves liquid over the
surfaces of cell. Flagella are generally accepted as being important virulence factors. . The location of the
flagella depends on bacterial species as polar situated at one or both ends which swims in back and forth
fashion and lateral at along the sides. The parts of flagella are the filament, hook and the basal body.
Filament is external to cell wall and is connected to the hook at cell surface, the hook & basal body are
embedded in the cell envelope. Basal body is attached to the cytoplasmic membrane by ring-like
structures. Hook & filament is composed of protein subunits called as flagellin. Flagellin is synthesized
within the cell and passes through the hollow centre of flagella. The arrangement of flagella may be
described as

(i) Monotrichous– single flagella on one side e.g. Vibrio cholerae

(ii) Lophotrichous – tuft of flagella on one side e.g.Bartonella bacillifornis

(iii) Amphitrichous – single or tuft on both sides e.g.Pirillumserpens

(iv) Peritrichous – surrounded by lateral flagellae.g. Escherichia coli

Structures of flagella

Pili / Fimbriae

Hair-like proteinaceous structures that extend from the cell membrane to external environment are pili
which are otherwise known as fimbriae. They are thinner, shorter and more numerous than flagella and
they do not function in motility. The fimbriae is composed of a subunit called Pilin.There are two types
pili namely Non-sex pili (Common pili) e.g. fimbriae or type IV and the sex pili. The fimbriae are
antigenic and mediate their adhesion which inhibits phagocytosis. The sex pili help in conjugation.

Spore

Some bacteria have the ability to form highly resistant resting stage called spores, which helps them to
overcome adverse environmental conditions that are unfavorable for vegetative growth of cell. They are
not a reproductive form and not a storage granule. These spores are resistant to bactericidal agents and
adverse physical conditions. Each spore can give rise to only one endospore which play a role in heat
resistance. Spores consists of three layers namely core, cortex and spore coat
 B. Archaea

Archaea or Archaebacteria differ from true bacteria in their cell wall structure and lack peptidoglycans.
They are prokaryotic cells with avidity to extreme environmental conditions. Based on their habitat, all
Archaeans can be divided into the following groups: methanogens (methane-producing organisms),
halophiles (archaeans that live in salty environments), thermophiles (archaeans that live at extremely hot
temperatures), and psychrophiles (cold-temperature Archaeans). Archaeans use different energy sources
like hydrogen gas, carbon dioxide, and sulphur. Some of them use sunlight to make energy, but not the
same way plants do. They absorb sunlight using their membrane pigment, bacteriorhodopsin. This reacts
with light, leading to the formation of the energy molecule adenosine triphosphate (ATP).

C. Fungi

Fungi (mushroom, molds, and yeasts) are eukaryotic cells (with a true nucleus). Most fungi are
multicellular and their cell wall is composed of chitin. They obtain nutrients by absorbing organic material
from their environment (decomposers), through symbiotic relationships with plants (symbionts), or
harmful relationships with a host (parasites). They form characteristic filamentous tubes called hyphae
that help absorb material. The collection of hyphae is called mycelium. Fungi reproduce by releasing
spores. Fungi like yeasts are non-motile,round or oval organisms that reproduce by budding.

D. Protozoa

Protozoa are unicellular aerobic eukaryotes. They have a nucleus, complex organelles, and obtain
nourishment by absorption or ingestion through specialized structures. They make up the largest group of
organisms in the world in terms of numbers, biomass, and diversity. Their cell walls are made up of
cellulose. Protozoa have been traditionally divided based on their mode of locomotion: flagellates produce
their own food and use their whip-like structure to propel forward, ciliates have tiny hair that beat to
produce movement, amoeboids have false feet or pseudopodia used for feeding and locomotion, and
sporozoans are non-motile. They also have different means of nutrition, which groups them as autotrophs
or heterotrophs.

E. Algae

Algae, also called cyanobacteria or blue-green algae, are unicellular or multicellular eukaryotes that obtain
nourishment by photosynthesis. They live in water, damp soil, and rocks and produce oxygen and
carbohydrates used by other organisms. It is believed that cyanobacteria are the origins of green land
plants. The blue green algae have chromatin bodies functioning as nucleus while other groups have well
defined nuclei that is similar to those of higher plants. Some are able to move by gliding over the surfaces
and motility is not due to the use of flagella in some.
 F. Viruses

Viruses are non-cellular entities that consist of a nucleic acid core (DNA or RNA) surrounded by a protein
coat. Although viruses are classified as microorganisms, they are not considered as living organisms.
Viruses cannot reproduce outside a host cell and cannot metabolize on their own but reproduce by
replication in ahost capable of mutation. Viruses often infest prokaryotic and eukaryotic cells causing
diseases. Generally, the viruses of humans and animals are spherical, those of plants are rod shaped or
many sided and those of bacteria (bacteriophages) are tadpole shaped. Viruses are mainly classified by
phenotypic characteristics, such as: Morphology, nucleic acid type, mode of replication, host organisms
and type of disease they cause. Viruses of all shapes and sizes consist of a nucleic acid core, an outer
protein coating or capsid, and sometimes an outer envelope.  Filamentous viruses are long and cylindrical.
Many plant viruses are filamentous, including TMV (tobacco mosaic virus). Isometric viruses have shapes
that are roughly spherical, such as poliovirus or herpesviruses. Enveloped viruses have membranes
surrounding capsids. Animal viruses, such as HIV, are frequently enveloped. Head and tail viruses infect
bacteria. They have a head that is similar to icosahedral viruses and a tail shape like filamentous
viruses. In general, there are five main morphological virus types:

Assignment: Make an outline of the structures of some common viruses.

1. Helical – These viruses are composed of a single type of capsomer stacked around a central axis to
form a helical structure, which may have a central cavity, or hollow tube.

2. Icosahedral – Most animal viruses are icosahedral or near-spherical with icosahedral symmetry.

3. Prolate – This is an icosahedron elongated along one axis and is a common arrangement of the
heads of bacteriophages.

4. Envelope – Some species of virus envelop themselves in a modified form of one of the cell
membranes, either the outer membrane surrounding an infected host cell or internal membranes
such as nuclear membrane or endoplasmic reticulum, thus gaining an outer lipid bilayer known as
a viral envelope.

5. Complex – These viruses possess a capsid that is neither purely helical nor purely icosahedral, and
that may possess extra structures such as protein tails or a complex outer wall.

A complete virus particle, known as a virion, consists of nucleic acid surrounded by a protective coat of
protein called a capsid. These are formed from identical protein subunits called capsomeres. Viruses can
have a lipid “envelope” derived from the host cell membrane. The capsid is made from proteins encoded
by the viral genome and its shape serves as the basis for morphological distinction. Virally coded protein
subunits will self-assemble to form a capsid, in general requiring the presence of the virus genome.
Complex viruses code for proteins that assist in the construction of their capsid. Proteins associated with
nucleic acid are known as nucleoproteins, and the association of viral capsid proteins with viral nucleic
acid is called a nucleocapsid.
 G. Rickettsia

Rickettsia is a genus of non-motile, Gram-negative, non-spore-forming, highly pleomorphic bacteria that

may occur in the forms of cocci 0.1 μm in diameter, rods 1–4 μm long, or threads of up to about 10 μm
long. The bacterial genus Rickettsia was named after Howard Taylor Ricketts, in honour of his pioneering
work on tick-borne spotted fever. Being obligate intracellular parasites, rickettsias depend on entry,
growth, and replication within the cytoplasm of living eukaryotic host cells
(typically endothelial cells). Accordingly Rickettsia species cannot grow in artificial nutrient culture; they
must be grown either in tissue or embryo cultures; typically, chicken embryos are used. Their organization
is basically that of prokaryotic cell with fastidious nutritive requirement. Rickettsiae possess cell wall,
RNA ,DNA, certain enzymes and are able to carry out a number of metabolic activities. They multiply by
binary fission. Rickettssia, Rochalimaee and Coxiella are the 3 genera in the family Rickettsiaceae that
contain important pathogen for man.

H. Mycoplasma

These do not have true cellwall ,hence they have unusual features.They are ubiquitous,saprophytes,
parasites and pathogens of many hosts.They are non-motile, gram negative,smaller than ordinary bacteria
and about the size of some larger viruses. They are closer to bacteria in theirchemical structure and
contain both DNA and RNA. They are of various forms such as coccoid,granular,filamentous, cluster-like,
ring shaped and filterable forms.They can be non-pathogenic and pathogenic causingserious diseases in
cattle, birds and man e.g. pneumonia,arthritis and infections of the kidney. They are the smallest
organisms which can be cultivated on cell-free media.

Classification of microoganisms

Various groups of microorganisms are generally classified depending on their morphological

characteristics and biochemical characteristics.The Morphological characteristics include shape, size,
possession of flagella, capsule, vacuoles, chloroplasts, gram stain, endospores etc. The biochemical tests
include tests such as catalase, oxidase, coagulase, methyl red, urease production etc.

Morphological classification of:

Bacteria

I. Direct microscopic examination

A i. Staining reactions which could be gram positive or negative

Ii. Acid fast stain reaction which could be positive or negative

B.Size

C. Shape e.g. cocci, bacillus and vibro (spirillum and spirochetes)

E. Capsule formation which could either be encapsulated or non- capsulated

II.Culture

A. Food requirements e.g. autotrophs, saprophytes and parasites

 B. Suitable media for growth e.g chemically simple media, special media or no growth on media.
C. Appearance of growth on different media
D. Oxygen requirement e.g Obligate aerobes, Microaerophiles. Anaerobes etc.
E. Optimum temperatures e.g. Psycrophiles, mesophiles, thermophiles etc.
F. Characteristics of pure culture
G. Production of hemolysins on blood agar e.g. Alpha, beta and gamma hemolysis

III. Biochemical reaction

A. Catalase test: To differentiate Staphylococci (catalase positive) from Streptococci (catalase test
negative)

B. Citrate utilization test: To differentiate members of Enterobacteriaceae family.

C. Coagulase test: Coagualse test is used to identify Staphylococcus aureus. Coagulase test

differentiates Staphylococcus aureus (positive) from coagulase negative staphylococci, such as S.
epidermidis, S. saprophyticus.

D. Indole test: This test is used to determine the ability of an organism to split tryptophan to form the
compound indole. It is used differentiate gram negative rods particularly E. coli in microbiology
laboratory.

E. Oxidase test: To help identify Neisseria, Pasteurella, Vibrio, Pseudomonas. This test is used to
determine the presence of bacterial cytochrome oxidase.

F. Urease test: Urease test is used to determine the ability of an organism to produce urease (an
enzyme) which hydrolyzes urea. This test is done to help identify Proteus, Morganella, Yersinia
enterocolitica, Helicobacter Pylori.

IV. Animal inoculation

A. Disease production(virulence test) e.g pathogenic or non-pathogenic

B. Toxin production e.g exotoxin and endotoxin producers

Fungi

Fungi belongs to Kingdom Mycota. It has two divisions :Myxocota and Eumycota

The division Eumycota are the true fungi which lack chlorophyll. These include yeasts,molds and some
related forms. Four major subdivisions are known based on the characteristics of sexual spores and
fruiting bodies present during the fruiting stage of their life cycles. These are :

I. Class Zygomycetes: These are water molds which reproduce either sexually or asexually. Examples
are: Rhizopus and Mucor.

II. Class Ascomytes: They reproduce by means of sexual spores formed within spacially developed sac
or ascus.Examples are yeasts(Genus Saccharomycetes).
III.Class Basidiomycetes: These are known as clubfungi.They form Basidiospores o speciallised
structure called Basidium. Examples are mushrooms, toadstools, puffballs and the plant smuts and rusts.
Certain members produce poisons.

IV. Class Deuteromycetes: These are known as imperfect fungi as they do not produce sexual spores.
Examples areAspergillus and Penicillium.

Protozoa

The protozoa are classified into four divisions. These are:

Class mastigophora:They are flagellate protozoa. The group include the phytoflagellates and the
zooflagellates e.g. Euglena.

Class Rhizopoda /sacormastigophora: These are the amoeboid protozoaand aremotile by means of
pseudopodia.

Class Sporozoa : These are parasitic protozoa, immotile or show gliding movement without cilia and
flagella, Possess one or many sporozoites.

Class Ciliata: They are called the ciliates and are motile by means of cilia. They have two types of
nucleus which differ in structure and function e.g. Paramecium

Algae

The major divisions are :

I. Cyanophycophyta which are the blue-green algae and are prokaryotic .

II Euglenophyta: These are unicellular organisms which are motile by means of flagella and reproduce
by cell division. E.g.Euglena

II Chlorophycophyta: They are motile by flagella action. Their cells have well defined nucleus, a cell-
wall, chloroplasts etc. Examples are Chlamydomonas, and Volvox.

IV. Chrosophycophyta: These are golden-brown algae. Some are flagellates while some are amoeboid.
Example is Chysamoeba with flagella.

V. Pyrrophycophyta: These includes the motile dinoflagellates and non-motile phytodinads which
produce spores with flagella. They have characteristic arrangement of flagella. Some possess cell
wall while others do not.

VI. Phaeophycophyta: These are multicellular and contain brown pigment. They live in water. Many
have hold fasts and air bladders which gives them buoyancy.

VII. Rhodophycophyta: These are the red-algae, smaller than most phaeophycophyta, they do not have
flagella.
Viruses

Classification of viruses are based on biological properties like Nucleic acid((DNA or RNA)) present,
size, Structure(geometric pattern of capsid and number of capsomeres), presence or absence of an
envelope, sensitivity to physical and chemical agents, immunological aspects and epidemiological
features.
NUTRITIONAL REQUIREMENTS OF MICROORGANISMS

A characteristic of microorganisms is their ability to grow and form a population of organisms. One of the
results of microbial metabolism is an increase in the size of the cell. The many requirements for successful
growth include those both chemical and physical. While all microorganisms share the characteristic of
being tiny, their specific environmental requirements are quite different. They have the same type of
requirements, but there are huge differences in what is needed to meet them.
Chemical requirements.

 In order to grow successfully, microorganisms must have a supply of water as well as numerous other
substances including mineral elements, growth factors, and gas, such as oxygen.

1.Carbon: Virtually all chemical substances in microorganisms contain carbon in some form, whether
they be proteins, fats, carbohydrates, or lipids. 50 percent of a bacterium's dry weight is carbon. Carbon
can be obtained from organic materials in the environment, or it may be derived from carbon dioxide.
Carbon is normally added to the growth medium by adding carbon dioxide if the microbe is an autotroph,
meaning they make their own food. If the microbe is a heterotroph, they cannot make their own food.
Both chemoautotrophic and photoautotrophic microorganisms obtain their energy and produce their
nutrients from simple inorganic compounds such as carbon dioxide. Chemoautotrophs do so through
chemical reactions, while photoautotrophs use photosynthesis. Organic compounds such as
carbohydrates are added for them to break down and extract the carbon. Fungi require a higher
concentration of carbon than bacteria and other microbes

2.Nitrogen: is used for the synthesis of proteins, amino acids, DNA, and RNA. Bacteria that obtain
nitrogen directly from the atmosphere are called nitrogen-fixing bacteria. They include species
of Rhizobium and Azotobacter, both found in the soil. DNA have to be duplicated for cellular replication
in multicellular microbes and reproduction in bacteria and multicellular microbes. Nitrogen is usually
added to a medium by adding ammonium.
3.Phosphorus: is an essential element for nucleic acid synthesis and for the construction of phospholipids.

4.Oxygen: is used by aerobic bacteria during the process of cellular respiration as a final electron
acceptor. For aerobic organisms, oxygen is an absolute requirement for their energy-yielding properties.
Certain microorganisms grow in oxygen-free environments and are described as anaerobic. Organisms
such as these produce odoriferous gases in their metabolism, including hydrogen sulfide gas and methane.
Certain pathogenic species, such as Clostridium species, are anaerobic. Certain species of microorganisms
are said to be facultative. These species grow in either the presence or absence of oxygen. Some bacteria
species are microaerophilic, meaning that they grow in low concentrations of oxygen.
Other chemical requirements for microbial growth include such trace elements as iron, copper, and zinc.
These elements often are used for the synthesis of enzymes. Organic growth factors such as vitamins may
also be required by certain bacteria. Amino acids, purines, and pyrimidines should also be available.

Physical requirements.
 Certain physical conditions affect the type and amount of microbial growth.
1. Temperature of the environment, and microorganisms are classified in three groups according to their
temperature preferences: psychrophilic organisms (psychrophiles) prefer cold temperatures of about 0°C
to 20°C; mesophilic organisms (mesophiles) prefer temperatures at 20°C to
40°C; thermophilic organisms (thermophiles) prefer temperatures higher than 40°C .
A minimum and a maximum growth temperature range exist for each species. The temperature at which
best growth occurs is the optimum growth temperature. 
The correct temperature for growing microorganisms is maintained using incubators, cold rooms,
and refrigerators.
2.pHof a solution. For most bacteria, the optimum pH is between 6.5 and 7.5. Since the pH of most human
tissue is 7.0 to 7.2, these neutrophilic bacteria usually grow well in the body. Certain bacteria, such as
those in sauerkraut and yogurt, prefer acidic environments of 6.0 or below. These bacteria are said to
be acidophilic. Molds and yeasts are among other common acidophilic microorganisms. Most fungi grow
best at a lower pH, somewhere between pH 4 to 6, while bacteria thrive in a more neutral pH around 6.5 to
7. Alkalophilic microorganisms grow at pH starting from 8.

3. Osmotic Pressure: Microbial growth proceeds best when the osmotic pressure is ideal. Normally, the
salt concentration of microbial cytoplasm is about 1 percent. Microorganisms that live in marine
environments can tolerate high salt concentrations. These organisms are said to be halophilic. They
include diatoms and dinoflagellates, two types of unicellular algae that lie at the base of oceanic food
chains. There are many other species of halophilic bacteria, fungi, protozoa, and algae.

MICROBIAL GROWTH CURVE

When a fresh medium is inoculated with a given number of cells, and the population growth is monitored
over a period of time, plotting the data will yield a typical bacterial growth curve as shown below:

Four characteristic phases of the growth cycle

1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains
temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in
volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.The
length of the lag phase is apparently dependent on a wide variety of factors including the size of the
inoculum; time necessary to recover from physical damage or shock in the transfer; time required for
synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible)
enzymes that are necessary to metabolize the substrates present in the medium.

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all
the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells
divide at a constant rate depending upon the composition of the growth medium and the conditions of
incubation.

3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed
system such as a test tube or flask). Population growth is limited by one of three factors:

 exhaustion of available nutrients

 accumulation of inhibitory metabolites or end products
 exhaustion of space, in this case called a lack of "biological space".

During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells
are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing
and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria
that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth
cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is
during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of
genes that may be involved in sporulation process.

4. Death Phase. If incubation continues after the population reaches stationary phase, a death phase
follows, in which the viable cell population declines. During the death phase, the number of viable cells
decreases geometrically (exponentially), essentially the reverse of growth during the log phase.

Growth Rate and Generation Time

As mentioned above, bacterial growth rates during the phase of exponential growth, under standard
nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium's generation time.
Generation times for bacteria vary from about 12 minutes to 24 hours or more. The generation time for E.
coli in the laboratory is 15-20 minutes, but in the intestinal tract, the coliform's generation time is
estimated to be 12-24 hours. For most known bacteria that can be cultured, generation times range from
about 15 minutes to 1 hour. Symbionts such as Rhizobium  tend to have longer generation times. Many
lithotrophs, such as the nitrifying bacteria, also have long generation times. Some bacteria that are
pathogens, such as Mycobacterium tuberculosis and Treponema pallidum, have especially long generation
times, and this is thought to be an advantage in their virulence
Calculation of Generation Time

When growing exponentially by binary fission, the increase in a bacterial population is by geometric
progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the
second generation, 8 cells in the third generation, and so on. The generation time is the time interval
required for the cells (or population) to divide.

G (generation time) = (time, in minutes or hours)/n(number of generations)

G = t/n

t = time interval in hours or minutes

B = number of bacteria at the beginning of a time interval

b = number of bacteria at the end of the time interval

n = number of generations (number of times the cell population doubles during the time interval)

b = B x 2n (This equation is an expression of growth by binary fission)

Solve for n:

logb = logB + nlog2

n = logb - logB 
           log2

n = logb - logB 
           .301

n = 3.3 logb/B

G = t/n

Solve for G

G =        t 
       3.3 log b/B
CULTURE MEDIA
A culture media is a special medium used in microbiological laboratories to grow different kinds of
microorganisms. A growth or a culture medium is composed of different nutrients that are essential for
microbial growth. Since there are many types of microorganisms, each having unique properties and
requiring specific nutrients for growth, there are many types based on what nutrients they contain and
what function they play in the growth of microorganisms.

A culture may be solid or liquid. The solid culture media is composed of a brown jelly like substance
known as agar. Different nutrients and chemicals are added to it to allow the growth of different
microorganisms.

Culture media contains nutrients and physical growth parameters necessary for microbial growth. All
microorganisms cannot grow in a single culture medium and in fact many can’t grow in any known
culture medium.Organisms that cannot grow in artificial culture medium are known as obligate
parasites. Mycobacterium leprae, Rickettsias, Chlamydias, and Treponema pallidum are obligate
parasites. Microbial culture media can be distinguished on the basis of composition, consistency and
purpose.

Classification of culture media used in Microbiology laboratory on the basis of consistency

1. Solid medium
Solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying
agent. Solid medium has physical structure and allows bacteria to grow in physically informative or
useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for
determining the colony characteristics of the isolate.
2. Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have soft custard like
consistency and are useful for the cultivation of microaerophilic bacteria or for determination of
bacterial motility.
3. Liquid (Broth) medium
These media contain specific amounts of nutrients but don’t have trace of gelling agents such as
gelatin or agar. Broth medium serves various purposes such as propagation of large number of
organisms, fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VR
broth.

Classification of culture media based on the basis of composition

1.Synthetic or chemically defined medium: A chemically defined medium is one prepared from purified
ingredients and therefore whose exact composition is known.Synthetic medium may be simple or complex
depending up on the supplement incorporated in it. A simple non-synthetic medium is capable of meeting
the nutrient requirements of organisms requiring relatively few growth factors whereas complex non-
synthetic medium support the growth of more fastidious microorganisms.

2. Non synthetic or chemically undefined medium: Non-synthetic medium contains at least one
component that is neither purified nor completely characterized nor even completely consistent from batch
to batch. Often these are partially digested proteins from various organism sources. Nutrient broth, for
example, is derived from cultures of yeasts.

Classification of Bacterial Culture Media based on the basis of purpose/ functional use/ application

Many special purpose media are needed to facilitate recognition, enumeration, and isolation of certain
types of bacteria. To meet these needs, numerous media are available.

1. General purpose media/ Basic media

Basal media are basically simple media that supports most non-fastidious bacteria. Peptone
water, nutrient broth and nutrient agar are considered as basal medium. These media are generally used
for the primary isolation of microorganisms.
2. Enriched medium (Added growth factors):
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them
enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar,
chocolate agar, Loeffler’s serum slope etc are few of the enriched media. Blood agar is prepared by adding
5-10% (by volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or
lysed blood agar.Unlike selective media, enrichment culture is typically used as broth medium.
Enrichment media are liquid media that also serves to inhibit commensals in the clinical
specimen. Selenite F broth, tetrathionate broth and alkaline peptone water (APW) are used to recover
pathogens from fecal specimens.
3.Selective media: This is designed to suppress the growth of some microorganisms while allowing the
growth of others. Selective media are agar based (solid) medium so that individual colonies may be
isolated.Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the
pathogen of interest. Various approaches to make a medium selective include addition of antibiotics,
dyes, chemicals, alteration of pH or a combination of these.
Examples of selective media include:
 Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics; vancomycin,
colistin and nystatin.
 Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.Potassium
 tellurite medium used to recover C.diphtheriae contains 0.04% potassium tellurite.
 MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits most
gram positive bacteria.
 Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide (antiseptic
agent)
 .Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
 Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating
malachite green.
 Wilson and  Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye
brilliant green.
 Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens have
elevated pH (8.5-8.6), which inhibits most other bacteria
4.Differential/indicator media:
Certain media are designed in such a way that different bacteria can be recognized on the basis of their
 colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that
those bacteria that utilize them appear as differently coloured colonies. Such media are called
differential media or indicator media. Differential media allow the growth of more than one
microorganism of interest but with morphologically distinguishable colonies.
Examples of differential media include:
 Mannitol salts agar (mannitol fermentation = yellow)
 Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
 Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter produces pale
or colorless colonies.
 TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)
5. Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent
overgrowth of contaminating organisms or commensals. This can be achieved by using transport media.
Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and
inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in
consistency. Addition of charcoal serves to neutralize inhibitory factors.
 Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to
transport feaces from suspected cholera patients.
 Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering
from bacillary dysentery.
 Pike’s medium is used to transport streptococci from throat specimens.
6. Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced
oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media
may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any
dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red
hot iron filings can render a medium reduced. Before use the medium must be boiled in water bath to
expel any dissolved oxygen and then sealed with sterile liquid paraffin.
Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium spps contains a 2.5
cm column of bullock heart meat and 15 ml of nutrient broth. Thioglycollate broth contains sodium
thioglycollate, glucose, cystine, yeast extract and casein hydrolysate.
Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the
medium. Under reduced condition, methylene blue is colorless.
7. Assay media
These media are used for the assay of vitamins, amino acids and antibiotics. E.g. antibiotic assay media
are used for determining antibiotic potency by the microbiological assay technique.
Other types of medium includes;
 Media for enumeration of Bacteria,
 Media for characterization of Bacteria,
 Maintenance media etc.
VARIOUS CULTURE CHARACTERISTICS ON AGAR

Colony appearance on agar plates

Margin

Elevation
Growth on agar slants

PURE AND MIXED CULTURE

Microorganisms exist in nature as mixed populations. However, to study microorganisms in the laboratory
we must have them in the form of a pure culture, that is, one in which all organisms are descendants of the
same organism. Pure culture, in microbiology, a laboratory culture containing a single species of
organism. A pure culture is usually derived from a mixed culture (one containing many species) by
transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual
cells across the medium surface or by thinning the sample manyfold before inoculating the new medium.
Both methods separate the individual cells so that, when they multiply, each will form a discrete colony,
which may then be used to inoculate more medium, with the assurance that only one type of organism will
be present. Isolation of a pure culture may be enhanced by providing a mixed inoculum with a medium
favouring the growth of one organism to the exclusion of others.
Two major steps are involved in obtaining pure cultures from a mixed population:

1. First, the mixture must be diluted until the various individual microorganisms become separated
far enough apart on an agar surface that after incubation, they form visible colonies isolated from
the colonies of other microorganisms. This plate is called an isolation plate.
2. Then, an isolated colony can be aseptically "picked off" the isolation plate and transferred to new
sterile medium. After incubation, all organisms in the new culture will be descendants of the same
organism, that is, a pure culture.

METHODS OF OBTAINING PURE CULTURE FROM MIXED CULTURE

A. STREAK PLATE METHOD OF ISOLATION

The most common way of separating bacterial cells on the agar surface to obtain isolated colonies is the
streak plate method. It provides a simple and rapid method of diluting the sample by mechanical means.
As the loop is streaked across the agar surface, more and more bacteria are rubbed off until individual
separated organisms are deposited on the agar. After incubation, the area at the beginning of the streak
pattern will show confluent growth while the area near the end of the pattern should show discrete
colonies.

B. THE POUR PLATE AND SPIN PLATE METHODS OF ISOLATION

With the pour plate method, the bacteria are mixed with melted agar until evenly distributed and
separated throughout the liquid. The melted agar is then poured into an empty plate and allowed to
solidify. After incubation, discrete bacterial colonies can then be found growing both on the agar and in
the agar.

The spin plate method involves diluting the bacterial sample in tubes of sterile water, saline, or broth.
Small samples of the diluted bacteria are then pipetted onto the surface of agar plates. A sterile, bent-glass
rod is then used to spread the bacteria evenly over the entire agar surface. 

C. USE OF SPECIALIZED MEDIA

To supplement mechanical techniques of isolation such as the streak plate method, many special-purpose
media are available to the microbiologist to aid in the isolation and identification of specific
microorganisms.

VARIOUS METHODS OF CONTROL OF MICRO-ORGANISMS

Control of microbial growth; means to inhibit or prevent growth of microorganisms. This control is
affected in two basic ways: (1) by killing microorganisms or (2) by inhibiting the growth of
microorganisms.
Microbial control is necessary to maintain a balance between man and microbes and for the following
reasons :
 prevention of disease transmission and infection
  prevention of decomposition and spoilage
 prevention of contamination (communication of diseaseorganisms through contact, dispersal,
fomites)

Mode of Action - How Control Agents Work

Damage usually occurs at the level of:

 the cell wall
 the cell membrane
 the internal proteins and cytoplasm

a) Damage to the cell wall - maintains the integrity of thecell and is the site of various enzymatic
reactions; porous but protective; some Gram + bacteria are attacked by LYSOZYME (enzyme in tears,
mucous secretions, white blood cells) which helps control skin Staphylococcus; penicillins and
cephalosporinsinhibit cell wall synthesis in rapidly growing cultures

b) Alteration of Cell Membrane Permeability - responsible for selective transport; "leakage" is term used
when cell membrane is destroyed; phenolic compounds, detergents, quaternary ammonium compounds,
all destroy selective permeability and cause leakage; alcohol (dissolve lipids and denature proteins)and
heat have similar effects

c) Alteration of Colloidal Nature of Cytoplasm - death is rapid when proteins are denatured; alcohol and
high temperatures, organometallics; denature proteins and coagulate proteins

d) Inhibition of Enzyme Action - cytochrome oxidase is inhibited by cyanide, trivalent arsenic interferes
with the Krebs cycle' glycolysis is inhibited by halogens (chlorine, fluorine, etc.)
mercury, hydrogen peroxide, sulfanilamide, some antibiotics, usually stop protein synthesis; oxidative
phosphorylation is stopped by the nitrophenols
e) Interference with Synthetic Processes - antimetabolites; blockage of folic acid by sulfonamides needed
for PABA;common TB treatment

DEFINITION OF SOME TERMS

Basic terms used in discussing the control of microorganisms include:

1. Sterilization 
Sterilization is the process of destroying all living organisms and viruses. A sterile object is one free of all
life forms, including bacterial endospores, as well as viruses.

2. Disinfection 
Disinfection is the elimination of microorganisms, but not necessarily endospores, from inanimate objects
or surfaces.
3. Decontamination
Decontamination is the treatment of an object or inanimate surface to make it safe to handle.

4. Disinfectant 
Disinfectant usually chemicals which kill growing forms (vegetative) of pathogens but not necessarily
spore forms or encapsulated forms or viruses; they are commonly applied to
inanimate objects and can be used as sprays or fogs; dependent on concentration they can be static or
cidal.
5. Antiseptic 
Antiseptic a chemical agent that arrests or prevents the growth or action of microbes; it controls by
destruction or inhibition dependent upon concentration; works on cuts, scrapes, abrasions, wounds on or in
the body; ERGO chemical disinfection of living tissue.
6. Sanitizer
These are chemical agents that reduce microbial populations to safe levels (as determined by the PHS);
usually destroys 80 -90 percent; normally used on inanimate objects such as in dairy industry; more
importantly to the public sanitizers imply cleanliness as well as disinfection
A sanitizer is an agent that reduces microbial numbers to a safe level.

7. Antibiotic 
An antibiotic is a metabolic product produced by one microorganism that inhibits or kills other
microorganisms.

8. Chemotherapeutic synthetic drugs 
Synthetic chemicals that can be used therapeutically.

9. Cidal 
An agent that is cidal in action will kill microorganisms and viruses.Thus, the term bactericidal refers to
killing bacteria: thusbactericide kills bacteria, a fungicide kills fungi, and so on.

10. Static 
An agent that is static in action will inhibit the growth of microorganisms. A bacteriostaticagent  refers
to inhibiting the growth of bacterial cells.

PHYSICAL METHODS OF MICROBIAL CONTROL

HEAT

This is most important and widely used method of sterilization. For sterilization one must consider the
type of heat, and most importantly, the time of application and temperature to ensure destruction of all
microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their
destruction guarantees sterility. It must take into account what is being subjectedto high or low
temperature. Thermal Death Point (TDP) lowest temperature required tokill ALL microorganisms in a
liquid suspension within 10minutes (time is held constant, temperature varies). Thermal Death Time
(TDT) minimal length of time in which all bacteria in a liquid culture will be killed at a given temperature
(temperature is held constant, time varies) useful in situations such as pasteurization where
certainproducts will be rendered unusable if temperature becomestoo high

Dry heat

 Flame or incineration:is used for inoculating loops and needles and for contaminated paper cups,
bags, dressings.Incinerationburns organisms and physically destroys them. Used for needles,
inoculating wires, glassware, etc. and objects not destroyed in the incineration process.
 Dry heat (hot air oven): basically, the cooking oven. The rules of relating time and temperature
apply, but dry heat is not as effective as moist heat (i.e., higher temperatures are needed for longer
periods of time). For example, 160o/2hours or 170o/1hour is necessary for sterilization. The dry
heat oven is used for glassware, metal, and objects that won't melt.

Moist Heat
Moist heat is thought to kill microorganisms by causing denaturation of essential proteins. Death rate is
directly proportional to the concentration of microorganisms at any given time.
 Boiling need a minimum of 10 continuous minutes of boiling to destroy spores of fungi, many
viruses, and mostbacterial pathogens; endospores and some viruses mayrequire up to 25 hours for
destruction.
 Autoclaving (steam under pressure or pressure cooker). Autoclaving is the most effective and
most efficient means of sterilization. All autoclaves operate on a time/temperature relationship.
These two variables are extremely important. Higher temperatures ensure more rapid killing. The
usual standard temperature/pressure employed is 121ºC/15 psi for 15 minutes. Longer times are
needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal
for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic
media, liquids, and many other things.
Low Heat
Low temperature normally used to restrict reproduction;may slow down enzymatic action but does not
stop it;freezing or cold normally does not kill unless done slowlyso that ice crystals may develop within
the cells.
 Pasteurization is the use of mild heat to reduce the number of microorganisms in a product or
food. In the case of pasteurization of milk, the time and temperature depend on killing potential
pathogens that are transmitted in milk, i.e., staphylococci, streptococci, Brucella
abortus and Mycobacterium tuberculosis.  Milk is usually pasteurized by heating, typically at 63°C
for 30 minutes (batch method) or at 71°C for 15 seconds (flash method), to kill bacteria and extend
the milk's usable life. The process kills pathogens but leaves relatively benign microorganisms that
can sour improperly stored milk. During the process of ultrapasteurization, also known as ultra
high-temperature (UHT) pasteurization, milk
is heated to temperatures of 140°C for one or two seconds.

B. Filtration
This involves the physical removal (exclusion) of all cells in a liquid or gas. It is especially important for
sterilization of solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids,
vitamins, etc.). Portable units can be used in the field for water purification and industrial units can be
used to "pasteurize" beverages. Essentially, solutions or gases are passed through a filter of sufficient pore
diameter (generally 0.22 micron) to remove the smallest known bacterial cells.

C. Dessication reproduction affected but viability of many organisms is unchanged; ineffective against
endospores and some viruses; used in preservation certain foods

D. Osmotic Pressure increase in sugar or salt concentration will lead to desiccation and is common
method of food preservation; may not be effective against all fungi, endospores,
or some viruses

G.Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is commonly used to sterilize
the surfaces of objects, although x-rays, gamma radiation and electron beam radiation are also used. 
Ultraviolet lamps are used to sterilize workspaces and tools used in microbiology laboratories and health
care facilities. UV light at germicidal wavelengths (two peaks, 185 nm and 265 nm) causes adjacent
thymine molecules on DNA to dimerize, thereby inhibiting DNA replication
Gamma radiation and electron beam radiation are forms of ionizing radiation used primarily in the health
care industry. Gamma rays, emitted from cobalt-60, are similar in many ways to microwaves and x-rays.
Gamma rays delivered during sterilization break chemical bonds by interacting with the electrons of
atomic constituents. Gamma rays are highly effective in killing microorganisms and do not leave residues
or have sufficient energy to impart radioactivity.
Electron beam (e-beam) radiation, a form of ionizing energy, is generally characterized by low
penetration and high-dose rates. E-beam irradiation is similar to gamma radiation in that it alters various
chemical and molecular bonds on contact. Beams produced for e-beam sterilization are concentrated,
highly-charged streams of electrons generated by the acceleration and conversion of electricity. 

Recommended use of heat to control bacterial growth

Treatment Temperature Effectiveness
Vaporizes organic material on
nonflammable surfaces but
Incineration >500o
may destroy many substances
in the process
30 minutes of boiling kills
microbial pathogens and
o
Boiling 100 vegetative forms of bacteria
but may not kill bacterial
endospores
Three 30-minute intervals of
boiling, followed by periods of
Intermittent boiling 100o
cooling kills bacterial
endospores
o
Autoclave and 121 /15 minutes kills all forms of life including
pressure cooker (steam at 15# pressure bacterial endospores. The
under pressure) substance being sterilized must
be maintained at the effective
 T for the full time
For materials that must remain
dry and which are not
destroyed at T between
Dry heat (hot air oven) 160o/2 hours
121o and 170o Good for
glassware, metal, not plastic or
rubber items
Same as above. Note
Dry heat (hot air increasing T by 10 degrees
170o/1 hour
oven)  shortens the sterilizing time by
50 percent
kills most vegetative bacterial
cells including pathogens such
Pasteurization (batch o
63 /30 minutes as streptococci, staphylococci
method)
and Mycobacterium
tuberculosis
Effect on bacterial cells
similar to batch method; for
Pasteurization (flash o milk, this method is more
72 /15 seconds
method) conducive to industry and has
fewer undesirable effects on
quality or taste
o
Ultrapasteurization 140 /2 seconds Effect on most bacterial cells
(direct method) is lethal. For milk, this method
creates a product with
relatively long shelf life at
refrigeration temperatu
res.

CHEMICAL METHODS OF MICROBIAL CONTROL

Characteristics of an ideal chemical agent

1. Toxicity to microbes broad spectrum, effective against spores, waxy coats; lowest concentration still
effective
2. Solubility in water, in tissue fluids; aesthetic qualities clear or cloudy
3. Stability shelf life water soluble products tend to last long; alcohol-based products have reduced shelf
life
4. Toxicity to man, other animals and plants skin tests,kidneys will usually show first; conjunctiva test
and respiratory tests; if substance goes in or on the body it is under the control of FDA and the National
Institutes ofHealth studies the protocol aspects
5. Homogeneity of mixture will it separate on standing
6. Combining power with organic substances want low affinity or will cause dilution test with organisms
coated with mucin; does it combine with lipids in the body, toxic to man
 7. Temperature toxicity at room temperature or body temperature; materials must not change at very high
or very low temperature on the shelf
8. Penetrating power - few compounds penetrate skin (ex. DMSO); usually refers to cut, wounds,
abrasions
9. Noncorroding and non-staining
10. Deodorizing quality organometallics are especially good;usually the result of washing out of the
atmosphere
11. Detergent action removal of dead tissue from wounded area
12. Availability in large quantities
13. Affordable

Common antiseptics and disinfectants

Chemical Action Uses
Denatures proteins
Ethanol (50-70%) and solubilizes Antiseptic used on skin
lipids
Denatures proteins
Isopropanol (50-70%) and solubilizes Antiseptic used on skin
lipids
Reacts with NH2,
Disinfectant, kills
Formaldehyde (8%) SH and COOH
endospores
groups
Antiseptic used on skin
Tincture of Iodine (2% I2
Inactivates proteins Disinfection of drinking
in 70% alcohol)
water
Forms
Disinfect drinking
hypochlorous acid
Chlorine (Cl2) gas water; general
(HClO), a strong
disinfectant
oxidizing agent
General antiseptic and
Precipitates
Silver nitrate (AgNO3)  used in the eyes of
proteins
newborns
Inactivates proteins Disinfectant, although
Mercuric chloride by reacting with occasionally used as an
sulfide groups antiseptic on skin
Detergents (e.g.
Disrupts cell Skin antiseptics and
quaternary ammonium
membranes disinfectants
compounds)
Phenolic compounds (e.g. Antiseptics at low
Denature proteins
carbolic acid, lysol, concentrations;
and disrupt cell
hexylresorcinol, disinfectants at high
membranes
hexachlorophene) concentrations
Ethylene oxide gas  Alkylating agent Disinfectant used to
sterilize heat-sensitive
objects such as rubber
 and plastics
Ozone Generates lethal Purification of water,
oxygen radicals sewage

 Common food preservatives and their uses

Effective
Preservative Uses
Concentration
Propionic acid and Antifungal agent in breads, cake,
0.32%
propionates Swiss cheeses
Sorbic acid and Antifungal agent in cheeses,
0.2%
sorbates jellies, syrups, cakes
Benzoic acid and Antifungal agent in margarine,
0.1%
benzoates cider, relishes, soft drinks
Sodium diacetate 0.32% Antifungal agent in breads
Antimicrobial agent in cheeses,
Lactic acid unknown buttermilk, yogurt and pickled
foods
Sulfur dioxide, Antimicrobial agent in dried
200-300 ppm
sulfites  fruits, grapes, molasses
Antibacterial agent in cured
Sodium nitrite 200 ppm
meats, fish
Prevents microbial spoilage of
Sodium chloride unknown
meats, fish, etc.
Prevents microbial spoilage of
Sugar unknown preserves, jams, syrups, jellies,
etc.
Prevents microbial spoilage of
Wood smoke unknown
meats, fish, etc

Chemotherapeutic agents (synthetic antibiotics): Antimicrobial agents of synthetic origin useful in the
treatment of microbial or viral disease. Examples are sulfonilamides, isoniazid, ethambutol, AZT,
nalidixic acid and chloramphenicol. Antibiotics are antimicrobial agents produced by microorganisms
that kill or inhibit other microorganisms. Antibiotics are low molecular-weight (non-protein) molecules
produced as secondary metabolites, mainly by microorganisms that live in the soil.

PROCEDURE FOR TRANSPORTING CULTURE SAMPLES FROM ONE LABORATORY TO

THE OTHER

Transport medium may be used to preserve microorganisms during transportation:

 Charcoal medium improves the isolation of bacteria by neutralising toxic substances such as
naturally occurring fatty acids found on the skin. 
  For virology specimen, a viral transport medium should be used as many viruses do not survive
well outside the body. The viral transport medium must not be used after the expiry date. 

 Samples submitted for bacterial polymerase chain reaction (PCR) should not be in transport
medium.

What does a transport media contain?

 Contains only buffers and salt.

 Doesn’t contain any nutritional ingredients such as carbon, nitrogen, and organic growth factors so
as to prevent microbial multiplication.
 Addition of antibiotics and other substances like glycerol may be added for transporting specimens
for tissue culture.

What samples are collected in transport medium?

All types of samples that may contain pathogens but could not be processed immediately requires
transport medium. It may be Stool, urethral swabs, Nasal and throat swabs and specimens for tissue
culture etc.

Equipment used in transporting culture sample

This will vary according to the specimen required but must include:  

 disposable gloves

 additional personal protective equipment (PPE) where applicable (eg apron/gown, respirator,
visor) 

 a plastic tray 

 a sterile container for the specimen 

 appropriate transport medium, if required 

 laboratory specimen form 

 a polythene transportation bag 

 biohazard label, if required