This document discusses various methods of sterilization. It defines sterilization as the complete killing of microorganisms, while disinfection is the destruction of pathogenic organisms. There are physical and chemical methods of sterilization. Physical methods include heat (dry and moist), filtration, radiation, and ultrasound. Moist heat using an autoclave at 121°C for 15 minutes is the most reliable sterilization method. Chemical sterilization agents include alcohols, aldehydes, halogens, phenols, and metallic salts. The key factors affecting sterilization are temperature, time, and the type of material being sterilized. Proper sterilization is important in fields like bacteriology, surgery

1. By Dr. Rakesh Prasad Sah
Assistant Professor, Microbiology

2. INTRODUCTION
 Microorganism are present everywhere. (Air, Dust,
surfaces, of all articles, Clothes, soil, vegetables, water,
also human skin)
 Cause contamination , infection & decay.
 Removal of these organism by sterilization.

3. EXTREMELY IMPORTANT IN
 Bacteriology - Isolation of bacteria in pure form
from the patient samples.
 Surgery & invasive diagnostic procedures instruments to be
used - free from any bacteria/other microorganism.
 Pharmaceutical industry - free from any bacteria/other
microorganism.
 Drug to be given to patient - free from any bacteria/other
microorganism.
 Food industry - free from any bacteria/other microorganism.

4. DEFINATION
STERILIZATION:-
 Process of 100% killing of Microorganism either
pathogenic or non pathogenic including spores from
any article or surface is called as sterilization
DISINFECTION:- Destruction of all pathogenic organism
or organism capable of giving rise to infection.
ANTISEPSIS:- Prevention of infection by inhibiting
growth of bacteria in wounds or tissues.
NOTE:-
Bactericidal agents – cidal -
Bacteriostatic agents – static -

5. CLASSIFICATION
Physical agents Chemical agents
1- Sunlight 1- Alcohols - Ethyl, isopropyl
2- Drying 2- Aldehydes – Formaldehyde,
Glutaraldehyde
3- Dry heat – Flaming, Incineration, Hot
air oven
3- Dyes
4- Moist heat – Pasteurisation, Boiling,
steam under
• Normal pressure
• High pressure
4- Halogens
5- Filtration 5- Phenols
6- Radiation 6- Surface active agents
7- Ultrasonic & sonic vibration 7- Metallic salts
8- Gases – Ethylene oxide, formaldehyde,
BPL

6. STERILIZING AGENT,
PRINCIPLE & PROCEDURE
ITEMS STERILIZED
1. SUNLIGHT:- Ultraviolet rays &
heat.
2. DRYING:- Removes water from
bacteria (water, constitutes 4/5
wt. of bacteria & is essential for
viability & growth).
3. HEAT:- Most reliable method of
sterilization.
a. Dry heat:- Kills bacteria and
spores by
a. protein denaturation,
b. oxidative damage (oxidizing their
chemical constituents)
c. Toxic effects of elevated levels of
electrolytes.
b. Moist heat:- Steam
denaturation & coagulation of
protein.
 Kills some bacteria in water, air –
exposed to direct sunlight.
 Not reliable.
 Spores – unaffected.
 For destruction of spores.
 Dry 170o c for 1 hrs.
 Moist 121o c 15 lbs for 15-20
minutes.

7. ADVANTAGES OF MOIST HEAT STERILIZATION
1. Sterilization at lower temp. in shorter time.
2. Penetrating power is better.
3. Liquids chemicals – Retain their structure.
Hot air oven (Dry heat) – Charring of media, cotton.
Steam – 1600 ml steam condenses into 1ml water at 100o c
& liberates 518 calories of heat – which raises temp. of
articles.

8. FACTORS AFFECTING STERILIZATION
 DURATION & TEMPATURE:- Inversely proportion.
spore destroyed - 100o c – 20 hrs.
121o c – 15 min.
 SUSCEPTIBILITY TO HEAT:-
• Most vegetative bacteria, yeast, fungi, viruses killed by moist heat in 30 min at
60 - 65o c
• Spores , yeast & fungi – 5 min at 70o c.
• Bacterial spores – 15 min at 121o c.
 TYPE OF MATERIAL:- Presence of organic matter reduses sterilization
process.

9. Method & items sterilization Procedure
A) DRY HEAT:-
a) Red heat:-
 Inoculation loops, wires,
forceps points,
spatulas.
b) Flaming:-
 Scalpel, needles, glass slides,
mouths of culture tube can
be immersed in spirit & then
burnt off.
 Held in Bunsen burner flame till – Red hot.
 Passed a few times thru the flame.
c) INCINERATION:-
 Soiled dressing, animal,
carcass, pathological
material.
 Plastics, PVC polythene.
 Polysterene material,
 Burnt directly in incinerator chamber.
 ”
 Emit dense black smoke, Hence destroyed
by autoclaving.

10. FLAME STERILIZATION

11. HOT AIR OVEN

12. Hot Air Oven
Principle :
 Based on the principle of Dry heat sterilization.
 Microbes killed by
a. Denaturation of protein,
b. oxidative damage (oxidizing their chemical
constituents)
c. Toxic effects of elevated levels of electrode.
Working :
 Oven is electrically heated and fitted with a fan to ensure
adequate & even distribution of hot air in the chamber. It is
also fitted with a thermostat that maintains the chamber air at
choosen temperature.
 Material should be washed & dried.
 Openings of tubes flask should be plugged with aluminum foil.
 Arrange in basket.
 Keep in hot air oven proper distribution/ No over loading start
power supply & check temperature.

13. Temperature and time : 160o c for 2 hr,
1700 for 1 hr,
1800 for 30 mins
Uses
 Glassware – Test tubes, flask, glass syringes, pipettes.
 Liquid paraffin, dusting powder, greases.
Sterilization Control :
• Paper strip impregnated with 106 spores of Cl. tetani (Non toxic keep
in oven with other material).
• Browne’s tube green colour.
Precautions
 Material should be washed & dried.
 No overloading
 Arranged in a manner which allows free circulation of hot air.
 Petridishes and pipettes should be wrapped in paper.
 Test tubes, flasks should be fitted with cotton plugs.
 start power supply & check temperature.
 Put off power supply & Cool to R.T.

14. Method & items sterilization Procedure
B) MOIST HEAT:-
a) Temp. below 100o c:-
i. Pasteurisation of milk (Mycobacter, Brucella,
Salmonella - Destroyed).
ii. Vaccine bath – Vaccine preparation of non
sporeing bacteria by heat inactivation of
bacterial suspension.
iii. Inspissator – Serum & Egg containing media
sterilized (Lowenstein- Jensen & loeffler’s serum
slope).
63o c for 30 min. (Holder method)
 72o c for 15-20 sec. (Flash method) followed by
cooling quickly at 13o c or lower.
 60o c for 1 hrs.
 Heating at 80-85o c for 30 min. on 3 successive
days. Temp. of 80o c for 5-10 min. destroys vegetative
forms of bacteria, yeast & moulds.
• Poliovirus - 60o c for 30 min.
• HBV - 60o c for 10 hrs.
b) Temp. at 100o c:-
i. Boiling – Not recommended for surgical or
invasive procedures.
ii. Steam at atmospheric pressure(100oc) –
Culture media damaged at higher temp. are
sterilized by this method. Koch’s or Arnold’s
steam sterilizer. (Sugar media – Lactose,
Glucose, Mannitol, sucrose).
 Boiling for 10-30 min vegetative form killed almost
immediately at 90-100o c does not destroy spores.
 Expose to steam at 100o c for 20 min. on 3
successive days.
 Tyndallisation/ Fractitional sterilization.
• Vegetative cells killed first group.
• Spores if present germinate.
• Vegetative cells killed in further exposure.

15. INSPISSATOR

16. Method & items sterilization Procedure
c) Temp. above 100o c:-
i. Autoclave:-
Principle:- Water boil when its vapour pressure
equals surrounding atmosphere. When
pressure inside closed vessel is increased
the
(Boiling point) temp. also increased. Steam
condenses on cooler articles gives up
latent heat. 1600 ml steam at 100o c &
atmosphere pressure . Condenses into 1ml
water at 100o c & gives 518 calories of heat.
Moist heat kills the bacteria by coagulation &
denaturation of proteins & kills spore also.
Autoclave:- Different types
• Laboratory autoclaves.
• Hospital autoclaves.
Sterilization controls:-
i. Paper strip impregnated with 106 spores of
Bacillus stearothermophilus kept along with other
material.
ii. Autoclave tapes – become dark coloured.
iii. Thermo couples.
 All surgical item like face masks, rubber gloves,
gowns, caps, drapes.
 Surgical instruments like artery forceps, scissors are
wrapped in cotton cloth & packed properly kept in SS.
Drum.
 Bacteriological culture media in flask or TT. openings
plugged by cotton & wrapped.
 Holding time & temp. 121o c at 15 lbs pressure for 20
minutes.

17. Autoclave
Principle :
 Based on the principle of Moist heat sterilization.
 Microbes killed by
a. Denaturation & coagulation of proteins and enzymes
b. Degradation of nucleic acid
c. Disruption of cell membrane.
 Water is boiled to produce steam  comes into main
chamber. Hot saturated steam continues to enter inside the
chamber until desired temperature is attained.
 According to boyle’s law, vol. of saturated steam kept
constant, temperature α pressure. Thus, treatment of desired
temperature & pressure usually 1210C at 15psi (pound per
square inch) is attained  15-20mins.
 1600 ml of steam (at 1000C at atm pressure)  cooler surface
 condense to 1 ml of water(at 1000C)  releases
518 calories of heat.

18. Procedure :
 All surgical item like face masks, rubber gloves, gowns, caps,
drapes.
 Surgical instruments like artery forceps, scissors are wrapped in
cotton cloth & packed properly kept in Drum.
 Bacteriological culture media in flask. openings plugged by
cotton & wrapped.
 Holding time & temp. 121o c at 15 psi pressure for 20 minutes.
Sterilization controls:-
i. Paper strip impregnated with 106 spores of Bacillus
stearothermophilus kept along with other material.
ii. Autoclave tapes – Red colour become black coloured.
iii. Thermocouple – record tempr by potentiometer.
iv. Browne’s tube – Red solution turns to Green.

19. Uses:
 To sterilize culture media, rubber material, gowns,
dressing, gloves etc
 Useful for materials which cannot withstand the
higher temperature of hot air oven.

20. AUTOCLAVE

22. FILTRATION
Used to sterilization heat labile liquids like :-
 Antibiotic solution, sera , sugars,
 To obtain bacteria free filtrates of toxins & bacteriophages.
 To obtain bacteria scanty in fluids.
1) Candle filters:- Different grades of porosity
• Uses:- Purification of water for industrial & drinking purpose.
• Types:-
i. Unglazed ceramic filters – eg:- Chamberland filter.
ii. Diatomaceous earth filters- eg:- Berfeld filter.
2) Asbestos filters:- Disposable single use discs. Eg:- Seitz filter.
3) Sintered glass filters:- Made of Powered glass particles.
4) Membrane filters:- Made of Cellulose esters (commonly used) .
i. Purification & analysis of water.
ii. Sterilization of parenteral use solution.
Note:- Average pore diameter – 0.22 µm commonly used.

23. SEITZ FILTER

24. 4) Air Filters :-
 filters are used to deliver clean bacteria free air to a
cubicle or a room.
 High efficiency particulate air (HEPA) filters
 Eg. Laminar air flow (0.3µm).

25. STERILIZATION BY RADIATION
NON IONIZING RADIATION IONIZING RADIATION
MOA: - Damage DNA. MOA :- Denaturation of bacterial
protein & interference with DNA
replication.
Electromagnetic rays with wavelength
longer than visible light is used.
1) Infrared radiation:- Rapid mass
sterilization of glass syringes.
2) Ultraviolet radiation:- Disinfection of
closed area.
• Entry ways
• Operation theater
• Wards
• Inoculation hoods
• Virus labs
Ultrasonic & sonic vibrations
X-rays, gamma rays, cosmic rays,
• Short wave length
• High penetrative power
• No increase in temp. (Cold
sterilization)
Gamma radiation used for sterilization:-
• Plastic, syringes, swabs, culture
plates, catheters on large scales
•Cold sterilization.
 Bactericidal effect.