CLINICAL BACTERIOLOGY (LECTURE)

LESSON 2: STERILIZATION AND DISINFECTION

=
nd
2 SEMESTER I S.Y. 2021-2022
TRANCRIBED BY: JEAN HERSHEY REYES

OUTLINE Methods of Sterilization and Disinfection

I. Introduction VI. Chemical Agents That

II. Applications of Denature Proteins Acids A. Physical Agents
Sterilization and and Alkalies 1. Sunlight
Disinfection VII. Chemical Agents 2. Drying
III. Methods of Sterilization commonly used as 3. Heat
and Disinfection disinfectants and a) Dry heat
IV. Chemical Methods Antiseptics b) Moist heat
 Classification VIII. 3 types of Pasteurization 4. Filtration
V. Chemical agents that IX. Control of 5. Radiation
damage the cell Microorganisms using 6. Ultrasonic and Sonic Vibrations
membrane heat methods B. Chemical Agents
 Surface Active X. List and the a) Agents that damage the cell membranes:
Agents recommended 1. Surface-active Disinfectants
 Phenolic concentrations of 2. Phenolic Compounds
Compounds disinfectant commonly 3. Alcohols
 Organic used in the hospitals
Solvents XI. Various procedures of b) Agents that damage protein
sterilization/disinfection of 1. Acids and Alkalies
some important materials 2. Alcohols
XII. Device classification and
Methods of Effective c) Agents that modify functional groups of protein and
Disinfection nucleic acids
1. Heavy metals
Introduction 2. Oxidizing agents
3. Dyes
4. Alkylating agents
 Sterilization: Destruction of all forms of microbial life including
spores; complete removal
Chemical Methods
 Disinfection: Destruction of microbes that cause disease; may
not be effective in killing spores; destroy only a define
 Chemical agents destroy any type of microbes without showing
microorganism
any form of selectivity unlike antibiotics.
 Antisepsis: destruction or inhibition of microorganisms in
living tissue there by limiting or preventing the harmful effect of The efficacy of these agents depends on the following factors;
infection;
o limiting the number of bacteria
o antiseptics: not sporicidal 1. Concentration  There is a relationship between the
of the Agent concentration of the agent and the
Divided into Two Groups time required to kill a given fraction of the
microbial population.
1. Chemical methods (disinfection) of sterilization and disinfection  Highly concentration/ heavily diluted
2. Physical methods (sterilization) of sterilization and disinfection disinfectant may not yield the desired
result
Chemical agent may be also used as sterilant  Concentration must be based on
standard set port by institutions
Mode of reaction
1) Reaction w/ the bacterial plasma membrane 2. Time of  Microbes are killed with a reasonable
2) Denaturation/ coagulation of bacterial proteins Exposure length of time with chemical agents
3) Reaction w/ enzymes  Need an ample time of exposure to
4) Damage to nucleic acids have the desired result

Applications of Sterilization and Disinfection 3. Temperature  Bactericidal potency of the chemical

agent increases with an increase in
 Aseptic techniques: Used in microbiological research, the temperature.
preservation of food and the prevention of the disease.  An increase in 100 C doubles the
 Sterile apparatus and culture media: Laboratory work with pure bacterial death rate.
cultures requires the use of sterile apparatus and culture media.  20-22 C
 The need to avoid infecting the patient.  Increase temperature and Increase
activity
 Decrease temperature and Decrease

Page 1 of 6
 activity Chemical agents that damage the cell membrane

4. Nature of the  Species of the bacteria Surface Active Agents

organism  Growth phase of bacteria in culture
 Presence of capsule, spore and other A. Cationic agents (+ ions)
special structures Lower the surface of tension enabling lipids to dissolve
 Number of bacteria in test system /
material  Quaternary ammonium compounds (Quates)
o Not effective to tubercle bacilli and spores
5. Presence of  Organic materials like serum, blood or o Disinfectant of non-critical areas such as floors, bench,
Extraneous pus makes chemicals inactive that are resto
Materials highly active in their absence. o Inactivated by organic materials
o Pseudomonas aeruginosa is resistant to Quates

 Causes loss of cell membrane semi

permeability leading to loss of nutrients and
essential metabolites.
 Denatures protein

 More active in Gram-positive

bacteria than in Gram negative
bacteria.
 More active at alkaline pH
 Inactivated by organic materials

B. Anionic agents (- ions)

Soaps and fatty acids

o Causes gross disruption of cell membrane lipoprotein

framework.
o More active in Gram-positive bacteria than in Gram-
negative bacteria.
o Active at acidic PH

Phenolic Compounds

 Phenol groups
 Used in hospitals and household
 Germicidal soap
 Active even in the presence of organics
 Disinfectant screening called phenol coefficient

Test organisms of phenol coefficient:

1. Staphylococcus Aureus
2. Salmonella typhi

 Highly effective in Gram positive bacteria.

 Clinically not used because of its neurotoxic effect.
 Currently used as a standard for measuring bactericidal potency
of new chemicals i.e. phenol coefficient.

o Phenol coefficient is the ratio of the concentration of

the new chemical agent being tested to the
 Greater time of exposure greater number of microbes killed
concentration of the reference standard (phenol)
required to kill in a specific time.
Classification of Chemical Methods

1. Chemical agents that damage the cell membrane

o Surface active agents
o Phenols o If phenol coefficient is <1, the new chemical agent is
o Organic solvents less effective than phenol.
2. Chemical agents that denature proteins o If phenol coefficient is =1, the new chemical agent is
o Acids and alkalies equal to phenol in efficacy.
3. Chemical agents that modify functional ,groups of proteins and o If phenol coefficient is >1, the new chemical agent is
nucleic acids more effective than phenol.
o Heavy metals
o Oxidizing agents Organic Solvents
o Dyes
o Alkylating agents
Alcohol: Ethyl or Isopropyl alcohol
 Widely used
 Non-sporicidal
 Effective against mycobacterium tuberculosis
 Deactivated by the presence of organic material
Page 2 of 6
 o Disorganize cell membrane lipid structure. site of blood collection with contact
o Denatures protein. time of 30 secs.
o Active against Gram-positive bacteria, Gram-negative o Iodine tincture- alcohol in iodine solution
bacteria and acid-fast bacilli.
2. Hydrogen Peroxide (3%)
Uses:  Used for cleansing of wound, disinfecting medical-surgical
1. Potent skin disinfectants devices and plastic contact lenses.
2. 2Disinfects clinical thermometer  Agua oxinada is active against vegetative cells and
bacterial spores
Note:
 Ethanol is potent at concentration of 70% and is used in Dyes
preparation of puncture site
Brilliant green / Malachite green / Crystal violet
Chemical agents that denatures Proteins Acids and Alkalies  Highly selective for Gram-positive bacteria.

 Denatured protein Uses:

 Interrupting biochemical processes  For treatment of dermatological lesions.
 Limited due to their corrosiveness  For formulation of selective culture media.
o Causes conformational alteration of proteins (unfolding of
polypeptide chain) resulting in irregular looping and coiling Alkylating Agents
of polypeptide chain.
1. Formaldehyde
o Acids like benzoic acid, citric acid and acetic acid are  37% aqueous
helpful as food preservatives: extending storage life of
food products Uses:
o Preservation of fresh tissues.
Chemical Agents That Modify Functional Groups of Proteins and o Preparation of vaccines from bacterial
Nucleic Acids surfaces, viruses and toxins.
o Bactericidal including spores.
Heavy Metals
Formaldehyde in liquid form:
1. Mercurial:  Tissue preservative
 Mercuric chloride – limited use because of toxicity.  Sterilizer being an irritant and a carcinogen limit its use
 Organic mercurial – less toxic than inorganic mercuric
salts. Formaldehyde in gas form:
 Is used to sterilized biosafety cabinets
Use:
o As antiseptics 2. Glutaraldehyde
Example: Merthiolate / Mercurochrome  10 times more effective than formaldehyde.
 Cold sterling for medical-surgical instruments.
2. Silver Compounds  Sustained even in the presence of organic matter but it is
 e.g. Silver nitrate, Silver sulfasalazine only active in alkaline environment
o 1% of silver nitrate/ creed prophylaxis has been  Does not corrode metal or rubber
used as an eye drop solution against
ophthalmia neonatorum 3. Ethylene oxide
 ophthalmia neonatorum or  Gaseous sterling chemical.
conjunctivitis of the newborn occurs  Most common gas used for sterilization
the birth canal of the mother is
infected with neisseria Uses:
gonorrhoeae – causative agent of o Sterilize medical-surgical devices that would
gonorrhea be damaged by heat.

Use: Antiseptic Agents

 As ophthalmic and wound
antiseptic. (e.g. burn  Disinfectants that are applied on animate bodies (skin or
patients) tissue).
Oxidizing Agents Characteristics:
o Never be toxic to cells
 Converts functional –SH group into non-functional –S-S o Never be corrosive
group. o Should never change nature of skin
 Example: Savlon / Alcohol( 70%) / Iodine tincture /Iodophor
1. Halogens
o Useful chemical agents for the control of
microorganism
o Chlorine: inactivated by organic materials;
used in form of hypochlorite such as sodium
hypochlorite found in bleach
 Recommended time for bleach
solution is about 3 mins or longer.
o Iodine: effective skin disinfectant
 Iodophor- iodine combined with
detergent; used for disinfecting the
Page 3 of 6
 Chemical Agents Commonly Used as Disinfectants and o Moist heat-denatures and coagulates protein; requires
Antiseptics water/moisture

Type Agents Heat

Alcohol (50-70%) Ethanol, isopropanol, benzyl alcohol
DRY HEAT
Actions Applications and Precautions
Denature proteins; Skin antiseptics Dry heat can be used by the following methods:
make lipids soluble 1) Flaming: Scalpels and neck of flasks, bottles and tubes
are exposed for a few seconds, but it is of uncertain
efficacy.
Type Agents
2) Hot Air Sterilizer (Oven): it is essential that hot air
Aldehydes (in Formaldehyde (8%), Glutaraldehyde
should circulate between the objects being sterilized and
solution) (2%)
these must be loosely packed and adequate air space
to ensure optimum heat transfer.
Actions Applications and Precautions o Done by applying 160 - 1800C for 1.5 - 3
React with NH2, SH, Disinfectants; kill endospores; toxic to hours.
and –COOH groups humans o Use: Sterilizes glassware, oils, greases,
lubricants and powders.
Type Agents
Halogens Tincture of iodine (2% in 70% alcohol) MOIST HEAT
 Kills microorganisms through denaturation of proteins and
Chlorine and chlorine compounds enzymes and degradation of nucleic acids

Actions Applications and Precautions Preferred to dry heat due to more rapid killing. Moist heat
Inactivates proteins Skin disinfectants can be used by the following methods.

React with water to Used to disinfect drinking water, surface 1. Boiling: It is not reliable method of sterilization.
form hypochlorous acid disinfectants o It is done by applying 100oC for 15 minutes.
(HClO); oxidizing o Used for sterilizing catheters, dressing and
agents fabrics.

Type Agents 2. Tyndallization: Intermittent steaming (Fractional/Intermittent

Heavy Metals Silver Nitrate (AgNO3) sterilization).
o Done at 100oC for 30mins on three
Mercuric Chloride (HgCl2) consecutive days.
o Spores which survived the heating process
Actions Applications and Precautions would germinate before the next thermal
Precipitates proteins Eye drop (1% solution) exposure and then would be killed.
o Used for sterilizing heat sensitive culture media
Reacts with –SH Disinfectant: Toxic at high concentrations containing materials such as carbohydrates,
groups; lyses cell egg or serum.
membrane o Instruments: Arnold’s Sterilizer
o Called fractional sterilization because it
involved 3 separate heating cycles done
Type Agents
within three consecutive days.
Detergents Quaternary ammonium compounds
3. Pasteurization: application of heat at temperature of 63oC for 30
Actions Applications and Precautions minutes (Holder method) or 72oC for 15 seconds (Flash
Disrupt cell Skin antiseptics; disinfectants method) followed by rapid cooling to discourage bacterial growth.
membranes o Develop by Louis Pasture
o Uses: Pasteurization of milk, Preparation of
Type Agents bacterial vaccines.
Phenolics Phenol, carbolic acid, Lysol, o Used to remove foodborne pathogens
hexachlorophene
3 Types of Pasteurization
Actions Applications and Precautions  Low temperature holding or batch
Denature proteins; Disinfectants at high concentrations; method- 63 C for 30 mins
disrupt cell membranes used in soaps at low concentrations  High temperature/ flash
pasteurization- 72 C for 15 secs.
Type Agents  Ultra-high temperature- 140 C for 3
Gases Ethylene oxide secs

Actions Applications and Precautions 4. Autoclaving: Steam under pressure it is based on the principle
Alkylating agent Sterilization of high-sensitive objects that when water is boiled at increased pressure, hot saturated
steam will be formed which penetrates and gives up its latent
heat when it condenses on cooler objects.
Physical Methods
 Microbiology Laboratory
 Destroy all organisms vegetative cells or spores
HEAT
except prions
 The most reliable and universally applicable method of
sterilization. Autoclaving: Hot saturated steam in autoclaving acts as an excellent
 Mechanism of action agent for sterilization because of:
o Dry heat-denatures protein.  High temperature
Page 4 of 6
  High latent heat Use:
 Ability to form water of condensation  IONIZING: Sterilize surgical sutures, catheters, petri
 Contraction in volume that occurs during condensation dishes, and culture media while dispensing and
pharmaceutical products like hormones, enzymes and
Note: Most culture media are sterilized at a pressure of 15 lb/in, at a antibiotics.
temperature of 121OC for 15 minutes. It destroys bacterial  NON-IONIZING: It also sterilizes biological safety cabinet
endospores and vegetative cells. (Laboratory rooms).
 Uses: Sterilize solid and fluid culture media, gowns, medical
and surgical equipment. Control of Microorganisms using Heat Methods
 Can also be used to decontaminate medical waste but the
conditions must be 132 C, 15 lb/square inch for 30-60
minutes
Methods of Controlling Sterilization (Autoclave)
o Recording of temperature and time of each
sterilizing cycle.
o Heat-sensitive autoclave tape fixed to the outside of
each pack.
 Color change of autoclave tape from blue
to brown-black indicates complete
sterilization.

o Biological indicator : Use of paper strips

impregnated with spores of Bacillus
stearothermophilus

5. Freezing: Inactivation of living bacteria by cold.

 It prevents active multiplication of bacteria by decreasing
the metabolic activity of bacteria.
o Bactericidal- agents that kills bacteria List and the recommended concentrations of disinfectants
o Bacteriostatic- agents inhibits the growth of commonly used in the hospitals
microorganisms
o Treponema pallidum; syphilis; can be killed when Disinfectants Concentration
expose to the temp. of 2-8 C for 72 hours Betadine (Iodophore) 2%
Bleaching powder (calcium hypochlorite) 14g in one liter of water
6. Lyophilization : Freeze-drying
Dettol (chloroxylenol) 4%
 Involves rapid freezing with subsequent drying.
Ethyl Alcohol 70%
Use:
Glutaraldehyde 2%
o Preservation of microbial cultures.
o Preservation of vaccines Lysol 2.5%
Savlon (chlorhexidine and cetrimide) 2%, 5%
FILTRATION Sodium hypochlorite 1%, 0.1%

 Mechanical sieving through membrane filters. Various Procedures of Sterilization/Disinfection of some important
 Vaccines, carbohydrates, antibiotic solutions etc. materials
 Membrane filters with pores
 Used to remove microbes form the air Materials Methods
1. Metallic inoculating wires Red heat
Uses: 2. Infective materials like soiled Burning (incineration)
o Sterilization of thermolabile parental and ophthalmic dressings, bed
solutions, sera and plasma. 3. Glasswares-syringes, Hot air oven
o Microbial evaluation of water purity. petridishes, test tubes, flasks,
o Determination of viral particle size universal containers, oily
fluids (paraffin)
RADIATION 4. Metal instruments Autoclaving, hot air oven, infrared
radiation
 Ion and ultraviolet radiation 5. Serum, body fluids, bacterial Water bath, at 56 C x 1 hour,
 Ion radiation includes χ ray, γ ray and β ray. vaccines vaccine bath at x 1 hour
 These induce break down of single stranded or sometimes 6. Gloves, aprons, dressings, Autoclaving
double stranded DNA. catheters, surgical
 Ultraviolet radiation has less quantum energy with low instruments except sharp
penetrating power than ionic radiation. instruments

2 types: Sharp instruments 5% cresol

o Ionizing- short wavelength gamma rays; sterilized 7. Suture materials except Autoclaving
plastic syringes catheters, gloves and antibiotics catgut

o Non-ionizing- ultraviolet rays; used in exposed Catgut Ionizing radiation

surfaces- lab. rooms and biosafety cabinet 8. Milk Pasteurization
9. Most of the culture media Autoclaving
Radiation: Spore forming bacteria are more resistant to ionic and 10. Culture media containing egg Tyndallization
ultraviolet radiation than vegetative bacteria because of: serum or sugar
 The spore coat confers protection. 11. Toxin, ascitic fluid, serum, Filtration
 DNA is in different state in spores. sugar and antibiotic solutions

Page 5 of 6
12. Rubber, plastic and Gamma radiation, ethylene oxide
polythene tubes including gas
disposable syringes
13. Feces and urine, vomitus Bleaching powder, cresols,
sputum formalin burning, autoclaving
14. Disposable syringes, rubber Ionizing radiation
or plastic disposable goods,
bone and tissue grafts,
adhesive dressings
15. Antitoxic sera, serum, urine Merthiolate (1:10,00)
16. Operation theater, wards and Formaldehyde gas and cresols
laboratory or floor space (Lysol) sodium hypochlorite (1%)
17. Polythene tubing, fabrics, Ethylene oxide
machine
18. Water Chlorine as hypochlorites (0.2%)
19. Skin Tincture iodine, spirit (70%
ethanol), Savlon (phenol
derivative)
20. Woollen blankets, wool and Formaldehyde gas
hides
21. Sterilization of operation Formaldehyde gas (50 cc formalin
theater and 25g KMnO4 per 100 cu ft.
space)

Device Classification and Methods of Effective Disinfection

Page 6 of 6