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Physical agents are usually preferred over chemical agents for performing
sterilization. Heat is the method of choice wherever possible. Both moist as well as
dry heat can be applied.

Moist Heat
This is heating in the presence of water. The best and widely used examples of this
technique are holder method of Pasteurization where 60°C for 30 minutes is
employed for sterilization and the 'flash' modification of the same wherein objects
are subjected to a temperature of 71.1°C for 15 seconds. Tyndallization is another
methodology in which steaming of the object is done for 30 minutes on each of the 3
consecutive days. The principle is that spores which survived the heating process
would germinate before the next thermal exposure and would then be killed. Dry
saturated steam acts as an excellent agent for
sterilization because of:
· High temperature
· Wealth of latent heat
· Ability to form water of condensation
· Instantaneous contraction in volume that occurs during condensation.
Superheated steam is not that effective because it is hotter than dry saturated steam
and the process is akin to dry heat which is not as efficient as moist heat.
Autoclaves are designed upon the principles of moist heat. The ideal time-
temperature relationship in moist heat and dry sterilization processes are as follows:
Process Temperature Holding period
Moist heat 121°C 15 minutes
(Autoclaving) 126°C 10 minutes
134°C 3 minutes
Dry Heat 160°C 120 minutes
170°C 60 minutes
IS0°C 30 minutes

Steps of functioning of an Autoclave are:

Put sufficient quantity of water in the cylinder
Put the materials to be sterilized on the tray
Start the heating
Screw tight the lid, keeping the discharge tap open
Adjust the safety valve to the required pressure
Allow the steam-air mixture to escape freely till all the air has been displaced.
This can be tested by leading the escaping steam into a pail of water through
a rubber tubing close the discharge tap when no more air bubbles come
Count the holding time from the time when the safety valve opens and the
excess steam escapes
Turn off the heater when the holding time is complete
Allow it to cool, tilt the pressure gauge indicates that the pressure inside is
equal to the atmospheric pressure
Open the discharge tap slowly and let the air into autoclone.

Low Temperature Steam with Formaldehyde

Low temperature steam at 80°C was found to be more effective than water at the
same temperature. The addition of formaldehyde to low temperature steam achieves
a sporocidal effect and it has been found to be suitable for thermolabile equipment.

Mechanism of Microbial Inactivation by Moist Heat

Bacterial spores Non-sporulating bacteria
. Denaturation of spore enzyme .Damage to cytoplasmic membrane
· Impairment of germination · Breakdown of RNA
· Damage to membrane · Coagulation of proteins
· Increased sensitivity to inhibitory · Damage to bacterial chromosome
· Structural damage
· Damage to chromosome
Dry Heat
Dry heat is less efficient process than moist heat and bacterial spores are most
resistant to it. Spores may require a temperature of 140°C for three hours to get
Dry heat can be used by following means:
 Incineration
 Red Heat
 Flaming
 Hot Air Sterilizers (Ovens)

Mechanism of Inactivation:
Microbial inactivation by dry heat is primarily an oxidation process. However, the
possibility of DNA damage is also now incriminated as one of the mechanisms. Dry
heat is employed for sterilization of glassware, glass syringes, oil and oily injections
and metal instruments. The thermal death point is the time required to kill all the
bacteria in a particular culture at a specified temperature. The decimal reduction
time, also known as the DRT or D value, is the length of time needed to kill 90 per
cent of the organisms in a given population at a specified temperature.

Ionizing Radiations
Ionizing radiations include X-rays, gamma rays and beta rays. These can induce
single stranded and sometimes double stranded breakdown of DNA.

Mechanism of Microbial Inactivation:

These radiations induce defects in the microbial DNA. If no repair takes place, DNA
synthesis is inhibited and errors in the protein synthesis manifest resulting into cell
death. Spores are more resistant to ionizing radiations than non-sporulating bacteria.
The ionizing radiations are used for the sterilization of single use disposable medical

Ultraviolet Radiations
Bacterial spores are more resistant to UV rays than the vegetative cells. Even viruses
are often more resistant than vegetative bacteria. Whereas Sarcina lutea is highly
sensitive to UV rays, Salmonella typhimurium is moderately sensitive and organisms
such as Escherichia coli, Staphylococcus and Proteus vulgaris are mildly sensitive to
the action of these rays.

Mechanism of Inactivation:
Because of the presence of UV rays, the sunlight has got bactericidal activity.
Sunlight can inactivate even spores provided the exposure is of prolonged duration.
The desert surfaces are found to be sterile not only because of immense dry heat but
also because of prolonged exposure to UV rays present in the sunlight
Sterilization by Filtration

Several types of filters available are:

Unglazed ceramic filters: These are manufactured in different grades of porosity
and used for large scale clarification of water.
Asbestos filters: These filters have high adsorbing capacity but suffer from
disadvantages of alkalinizing the solutions and possible carcinogenesis.
Sintered glass filters: These contain finely powdered glass particles of different
sizes-the size being adjusted according to the required pore size. These are widely
used nowadays and consist of cellulose ester.

Indicators of Sterilization Process

The commonly employed methods are chemical indicators, autoclave tapes and

Process Physical Chemical Biological test

methods methods organism used
Dry heat B. subtilis var niger
-Temperature -Colour change
Moist heat recording chart indicators B.
Ionizing -Temperature -Colour change stearothermophilus
recording chart indicators
Radiations -Dosimeters -Radiochromic B. pumilus
Filtration -Bubble point -None Ser. marcescens, Ps.
pressure test diminuta

Properties of an ideal disinfectant:
 Broad spectrum: Should always have the widest possible antimicrobial
 Fast acting: should have a rapidly lethal action on all vegetative forms and
spores of bacteria and fungi, protozoa and viruses.
 Not affected by physical factors:
 Nontoxic
 Surface compatibility
 Residual effect on treated surfaces
 Easy to use
 Odorless: an inoffensive odor would facilitate its routine use.
 Economical: cost should not be prohibitively high.