The document discusses various moist heat sterilization techniques including pasteurization, boiling, tyndallization, steam sterilization, and autoclaving. Moist heat sterilization uses hot water vapor to destroy microorganisms through irreversible protein denaturation. Techniques vary based on temperature, with pasteurization occurring below 100°C, boiling and tyndallization at 100°C, and autoclaving above 100°C in pressurized steam. Autoclaving is the most effective method, achieving sterility through 15 minutes at 121°C or 3 minutes at 134°C under pressure. Various controls ensure sterilization conditions are properly met.

1. STERILIZATION
PRIYA DIXIT
DEPARTMENT OF BIOTECHNOLOGY
ERA UNIVERSITY, LUCKNOW

2. MOIST HEAT
Moist heat sterilization describes sterilization techniques that use hot water vapor as a sterilizing agent.
Moist heat in the form of saturated steam under pressure is the most widely used and the most dependable
method. Steam sterilization is nontoxic, inexpensive, rapidly microbicidal, and sporicidal.
Moist heat destroys microorganisms by the irreversible denaturation of enzymes and structural Proteins. The
temperature at which denaturation occurs varies inversely with the amount of water present. Sterilization in
saturated steam thus requires precise control of time, temperature, and pressure.
This method of sterilization may be used at different temperatures as follows:
At temperature below 100 ˚C
Pasteurization
Inspissation
Vaccine Bath
At temperature100˚C
Boiling
Tyndallisation
Steam sterilizer
At temperature above 100 ˚C
Autoclave

3. Pasteurization:
Pasteurization (or pasteurization) is the process of heating liquids for the purpose of destroying viruses and harmful organisms
such as bacteria, protozoa, molds, and yeasts.
The process was named after its inventor, French scientist Louis Pasteur to prevent the spoilage of beverages.. The first
pasteurization test was completed by Pasteur and Claude Bernard on April 20, 1862.
Unlike sterilization, pasteurization is not intended to kill all micro-organisms (pathogenic) in the food. Instead, pasteurization
aims to achieve a "logarithmic reduction" in the number of viable organisms, reducing their number so they are unlikely to
cause disease (assuming the pasteurized product is refrigerated and consumed before its expiration date).
Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc. Generally Pasteurization involves Milk.
Pasteurization of milk
» Batch method
» Flash pasteurization
» Ultrahigh-temperature pasteurization

4. In Holder method is heated for30 min at 60˚C.
In Flash method it is heated at 72˚C for 15-20 sec.
Ultra-high-temperature (UHT) pasteurization involves heating milk or cream to
138°to 150° C (280° to 302° F) for one or two seconds. Packaged in sterile,
hermetically sealed containers, UHT milk may be stored without refrigeration for
months
 Milk is not heated above its boiling point as it may form curdles or aggregates
resulting in spoiling of milk.
By Pasteurization all non sporing bacteria like Mycobacteria, Brucellae and
Salmonellae are destroyed but Coxiella burnetii being heat resistant survives Holder
method.
Method is used for heat sensitive liquid and pharmaceutical products. Commercial-
scale sterilization of food is not common, because it adversely affects the taste and
quality of the product.

5. Extra Points:
All mesophilic non sporing bacteria are killed on exposure to moist heat at 60˚C for ½ hr, except a few which need a different
set of time and temperature of sterilization.
A temperature of 80˚C for 5-10 min destroys all vegetative forms of bacteria, yeasts and moulds.
Staphylococcus aureus and Stretococcus faecalis - 60˚C for 60 min.
Spores of Clostridium botulinum – 120˚C for 4 min or 100˚C for 330min.
Some viruses like poliovirus & hepatitis B at 60˚C for 30min & 10 hrs respectively. (Unlike most viruses rapidly destroyed at
60 ˚C )

6. Inspissation:
Heating at 80-85°C for half an hour daily on three consecutive days
Inspissator is a convenient and effective system designed to produce large batches of
uniform culture medium four to six times per day. Vessels containing culture medium are
incubated on a shallow tray which is in contact with water held at a constant temperature of
85ºC within a tank, so ensuring that the temperature of the vessels is constant. Capacity for
up to 156 test tubes (Ø16mm diameter x 150mm long) or 162 universal containers
Serum or egg media are sterilized
Vaccine bath:
 Heating at 60°C for an hour daily in vaccine bath for several successive days.
Serum or body fluids can be sterilized by heating at 56°C for an hour daily for several
successive days.

7. Boiling:
Boiling is the process of heating the to be sterilized material in a liquid (generally distilled water) at its
boiling point to kill bacteria and other micro-organisms in it including spores.
Boiling for 10 to 30 minutes may kill most of the vegetative forms but many spores withstand boiling for a
considerable time.
Vegetative bacteria are killed immediately at 90-100˚C.
In boiling the to-be-sterilized material should be immersed in water and boiled for 10-30 min.
Hard water should not be used.
During the process of boiling the lid of the container should not be opened.
An inexpensive method of utilizing free steam (i.e., at atmospheric pressure) to sterilize culture media.
 Container and the medium are simultaneously sterilized.
Steamer:
 Tinned copper cabinet with lagged walls.
Conical lid enabling drainage of condensed steam.
 Perforated tray fitted above the water level ensuring steam to surround the material to be sterilized. Usually
takes 90 min.

8. Tyndallization :
Synonym-Intermittent Sterilization
Named after John Tyndall
Used for Media with sugars or gelatin which require 100˚C for 20 min on 3
successive days.
Principle: 1st exposure kills all vegetative bacteria and spores. Since they are in
favouring media will germinate to be killed in subsequent occasions.
May fail to destroy thermophiles and certain anaerobic spores.
Steam Sterilizer:
Koch’s or Arnold’s sterilizer at 100 ˚C for 90 minutes is usually used for material
which may decompose at high temperature of autoclave.

9. Principle: Water boils when vapour pressure equals that of surrounding. Thus increase in pressure inside closed vessel causes
increase in boiling point of the water. Saturated steam has more penetrating power. When steam comes on contact with a
surface it give off its latent heat, thus sterilizing the surface. The large reduction in volume sucks in more steam to the area &
the process continues till the temperature of that surface is raised to that of the steam. Condensed water ensures moist
conditions for killing the microbes present.
When steam comes in contact with a cooler surface, it condenses to water and liberates its latent heat to that surface. E.g.
1600ml of reduction in volume sucks in more steam to the same site and the process continues till the temperature of the article
is raised to that of steam. The condensed water produces moist conditions for killing the microbes present.
Autoclave (Steam Under Pressure):
Vertical cylinder type Autoclave Horizontal cylinder type Autoclave

10. Steam is the effective means of sterilization, because of its High penetrating capacity.
It gives of large amount of heat to surface with which it comes in contact.
Components of Autoclave
Autoclave is a modified pressure cooker or boiler.
A cylindrical or rectangular chamber, with capacities ranging from 400 to 800 liters.
Water heating system or steam generating system.
Steam outlet and inlet valves.
Single or double doors with locking mechanism.
Thermometer or temperature gauge.
It consist of a vertical or horizontal cylinder of gun metal or stainless steel in a supporting iron case.
The lid is fastened by screw clamps and rendered air tight by asbestos washers.
The lid bears a discharge tap for air and steam, a pressure gauge and a safety valve

11. Procedure
Tank is filled with sufficient water and the material to be sterilized is placed on a tray.
The lid is closed with the discharge tap open.
The heater is put on.
Safety valve is adjusted to required pressure.
After boiling of water, the steam and air mixture is allowed to escape till the air has been displaced.
The discharge tap is now closed.
 The steam pressure now rises inside and when it reaches the desired level, safety valve open releases excess steam. From this point, the
holding period counts.
After the holding period, heating is stopped and autoclave is allowed to cool till the pressure gauge indicates normal pressure.
The lid is now opened and material is allowed to remove.
Advantages of autoclaving:-
 Autoclaving is the most rapid and effective method for sterilizing cloth surgical packs and towel packs (other methods are not suitable
for cloth packs)
Automated models are available, although they may be evaluated with a biological spore test monitoring system.
Disadvantages of Autoclaving
 Items sensitive to the elevated temperature cannot be autoclaved.
 Autoclaving tends to rust carbon steel instruments.
Steam seems to corrode the steel neck and shank portions of some diamond instruments and carbide burs.

12. To achieve sterility, a holding time of at least 15 minutes at 121 °C (250 °F) or 3 minutes at 134 °C
(273 °F) at 15 psi (100 kPa) above atmospheric pressure is required.
Types of Autoclave:
Vertical autoclaves are also known as top loading sterilizers that come in cylindrical shape. These are widely used in hospitals,
Operation Theater (OT), microbiology laboratory for sterilization of various medial and cultures.
A Horizontal Autoclave are Double walled unit, heavy M.S. duly painted or Stainless steel tubular stand. Chamber & Boiler
(Steam Generator) are constructed from S.S. Fitted with Automatic Vacuum Breaker to break the vacuum in case of formation of
vacuum due to steam condensation.
Difference:
Horizontal autoclave will have a door which opens outwards towards you -‘Front loading autoclaves’ and A vertical
autoclave can be referred to as a ‘Top loading autoclave', as the chamber door pivots upwards to allow access to the chamber
beneath.

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

14. Sterilization control of the moist heat
Physical Indicator- An alloy designed to melt only after being subjected to relevant holding time.
Chemical indicator- Strips or tapes that change color once the correct conditions have been met.
Biological indicator- Spores of Geobacillus stearothermophilus are used as the test organisms as it is
toughest organism for an autoclave to destroy.
Its spores require an exposure of 15 mins at 1210c to be destroyed.