Sterilization

8he terms sterilization and disinfection are used toindicate
the treatment of material so as to destroy or otherwise
eliminate any living organisms present. Howeverthe
termsterilization is used where physical methods are
used and disinfection is used where chemical agents are
made use of

METHODS cOMMONLY USED FOR STERILIZATION
The methods used commonly in practice are:
1. Killing organisms by heat: Heat may be dry or moist
2. Destroying organisms by employing chemical antiseptics,
e.g. lysol, phenol, perchloride of mercury, et
3. Removing organisms mechanically by filtration, e.g.
Seitz, unglazed porcelain.

Sterilization by Heat

Adequate heat is the most certain and rapid method fo
sterilization, The time needed for sterilization is inversel
related to the temperature of exposure-the higher the
temperature, theshorter thetimeneeded. Hightemperature
kills bacteria by coagulating their proteins. Differen
types of bacteria show considerable differences in hea
susceptibility. In general, vegetative forms are destroyed
at lower temperatures, whereas high temperatures are
needed for sporing organisms.

Dry Heat

This is the preferred method for sterilizing glassware, e.g.
of glass syringes and of materials such as oils, jellies and
powders which are impervious to steam. Dry heat requires
a much higher temperature or a much longer time at the
same temperature than does moist heat. Dry heat can be
used in the following ways:

Flaming

The articles are passed through the Bunsen flame, without
letting them become red hot. It is used for scalpels, needles,
mouths of culture tubes, glass slides, coverslips and points
of forceps. Only the surfaces actually touched by the flame
are sterilized.

Red Heat

Platinum loops, inoculating wires and needles are heated
in the Bunsen flame until red hot.

Hot Air Oven

hese are electrically heated and thermostatically
controlled. The oven itselfis a double-walled steel chamber
with a stout door. The top or side contains a ventilator
which is left open during sterilization to disperse any
moisture or volatile matter. Air circulates within the oven
by convection currents. Suitable sterilizing times in the
hot air oven are 3 hours at 140°C, l hour at 160°C and
30 minutes at 180°C. All dry glassware, such as test tubes,
petri-dishes, flasks, pipettes and throat swabs, etc. are
made sterile by using hot air oven.
This method is not suitable for sterilizing culture
media, liquids, rubber connections, glass to metal fitting
and fabrics, e.g. masks, towels or gowns.

Moist Heat

Temperature

A temperature of 60 to 65°Ckills most vegetative bacteria
(made use of in pasteurization of milk and preparation of
vaccines).

Boiling

Boiling is frequently used for sterilizing syringes, etc. but is
not adequate as many spores withstand this temperature.

Steam

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Steam is the most effective technique of moist heat
sterilization. Steam may be employed in three ways.
Steam at 100°C
The apparatus used commonly is called Koch’s steamer. It
has a vertical metal cylinder with a conical
lid. It is fitted
with a thermometer and has a small opening for escape
of steam.
Sterilization by free steam can be done in two ways.
Prolonged exposure: For 1½ hours, used for broth or
nutrient agar.
Intermittent heat or tyndallization: It involves exposure for
20 minutes on three successive days and is used to sterilize
sugars and gelatin which decompose on higher temperatures.
Principle: Spores would germinate after first steaming and
destroyed on the next, three steamings would eliminate all
spores and their vegetative forms.
Low Temperature Steam
This method is employed for sterilizing materials (blankets,
polyethene tubing, etc.), which would be damaged at
higher temperatures.
Steam at Temperatures above 100°C (Autoclaving)
Autoclaves are made of strong metal jackets; strong enough
to withstand high pressures required (Figs 2.1A to C). The
autoclave door is hermetically sealed. It has a safety valve
set to blow off at a predetermined pressure. The principle
is that water boils when its vapor pressure is equal to the
pressure of the surrounding atmosphere. If the pressure
is raised inside a closed vessel, the temperature at which
water boils will rise above 100°C. At 15 lbs pressure water
boils at 120°C.
Following are the measures that must be taken care of
during autoclaving:
a. The steam must be saturated.
b. There must be complete discharge of air from the
sterilizing chamber.
c. The autoclave must be loaded in such a way that all the
materials to be sterilized
can be adequately penetrated
by steam.
d. The duration of autoclaving would depend on the
pressure inside and hence on the steam temperature.

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water boils will rise above 100°C. At 15 lbs pressure water
boils at 120°C.
Following are the measures that must be taken care of
during autoclaving:
a. The steam must be saturated.
b. There must be complete discharge of air from the
sterilizing chamber.
c. The autoclave must be loaded in such a way that all the
materials to be sterilized
can be adequately penetrated
by steam.
d. The duration of autoclaving would depend on the
pressure inside and hence on the steam temperature.

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Method
1. Fill boiler with water to a point just below the basket
bottom.
2. Place articles within the basket, bottles should not be
more than 3/4 full and should have loosely screwed on
caps.
3. Close the lid and tighten the screws.
4. Open outlet valve and adjust safety valve to the
required pressure.
5. Turn on the heat source and when steam flows
smoothly, close the vent-cock and let the internal
pressure rise. See that all air has been expelled from
the cylinder.
6. Let pressure rise to the required level and maintain at
that level for the required period of time.
7. Switch off the heat source and let the pressure
meter
register zero. Open the vent-cock and the lid slowly.
(If the autoclave pressure is taken down very quickly—the
fluid-filled bottles may burst).
Timings
10 lbs Pressure for 10 minutes—culture media.
15 lbs Pressure for 20 minutes—infected material.
20 lbs Pressure for 30 minutes—rubber gloves.
Inspissation
Used to sterilize serum containing
media, e.g. Loeffler’s
for diphtheria and Dorset’s, or Lowenstein’s media for
TB. The inspissator consists of a double-walled copper
box, with water flowing between the 2 walls; the temperature
is controlled between
75 to 80°C thermostatically.
Sterilization is done for 2 to 3 hours on each of 3 successive
days. A higher temperature may cause bubbling of the
surface of the media.

Cold
Not used clinically.

Cold Shock
A sudden drop of temperature (e.g. 45 to 15°C) without
actual freezing causes irregular
contraction of cytoplasmic
organelles leading
to disorganization of cellular structures
(95% drop in E. coli viable number is reported by using this
method).
Freezing
This helps by (1) formation of ice crystals outside the cell
by the withdrawal of water from the cell interior, increases
the intracellular
salt concentration—
protein denaturation,
and (2) formation of ice within the cell.
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Ultraviolet Radiation
Ultraviolet (UV) rays of wavelength 2400 to 2800 Angstrom
units are most effective. Low pressure mercury vapor type
lamps can be used to produce UV rays. Take care that the
UV rays do not directly enter the eyes. Gram-negative
bacteria are destroyed
more rapidly than gram-positive
bacteria,
spores are highly resistant and susceptibility
of
viruses is variable.
Ionizing Radiations
Cathode rays and gamma rays are the most effective and
are being increasingly used to sterilize disposable items.
These radiations have considerable disinfectant action.
Filtration
It may be used for the preparation from cultures of cell-free
bacterial products, e.g. toxins and enzymes, to free virus
containing fluids from bacteria and for the sterilization
of media or media ingredients, which would be damaged
by heating. For this purpose, filters with pores sufficiently
small to hold back bacteria must be employed. Filtration is
usually carried out under negative pressure, the fluid being
sucked through the filter into a receiving flask, which is
connected to an exhaust pump. During filtration,
the filter
surface may adsorb material carrying
an opposite charge—
the material adsorbed may be the one desired in the filtrate.
Seitz Filter
This employs filter that consists of a flat disk or asbestos
material of special composition and is inserted into metal
holders, which ensure a tight joint (Fig. 2.2). The disk is
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used only once. The disks are available in three grades, viz.
(1) clarifying (2) normal, and (3) special. This method is
good for obtaining bacteria free filtrates. The large pore
filters are good for filtration of serum for making media.
Collodion or Gradocol Membranes
These are virtually free of any adsorptive effects and have
replaced Seitz filters considerably. These, too, are replaced
after a single use.
Berkefeld and Mandler Filters
These are made from diatomaceous
earth. Grades available
are V—coarse, N—medium and W—fine.
Chamberland and Doulton Filters
These are made of unglazed porcelain. Available in various
grades:
L/a—coarse clarifying.
L/a L2 and L3—medium.
L5 to L15—very fine.
Sintered Glass
It is made of a pad of finely ground glass fused into a glass
cup.
Sand and Paper Pulp Filter
They are used for removing large particles and clearing
emulsions, etc.
Chemical Sterilization
Chemical agents can exert bactericidal or bacteriostatic
effect. The bactericidal agents in lower concentrations exert
bacteriostatic effect. The bactericidal effect is probably
because of enzyme inactivation either by protein denaturation,
oxidation or by a combination of the antibacterial
agent with specific groups of enzyme proteins.
Chemicals Used
Various chemicals used are:
1. Chloroform (volatile antiseptic): Used in preservation
of serum for culture media at 0.25% concentration.
Can be removed by heating to 56oC.
2. Phenol group: Cresol, lysol (strong antiseptics) are mainly
employed for surgical instruments,
discarded routine
cultures, and pipettes, slides, etc. and disinfecting hands.
Phenol 0.5% is used for preserving sera and vaccines.
3. Metallic salts: Perchloride of mercury in 1:1000
strength solution.
4. Glycerol: A 50% solution is used for preservation of
certain viruses. Glycerol also kills contaminating
organisms.
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5. Formalin: It is the only method for sterilizing Perspex
or polythene and for killing cultures on plates. Can also
be used for fumigation purposes.
6. Sulfur: Burning in air forms sulfur dioxide (SO2) for
fumigation.
7. Halogens: Chlorine and iodine. Chlorine for water
disinfection and iodine for skin.
8. Acids and alkalies: Most bacteria grow in pH range of 5
to 9 and many grow at pH 7. Strong acids and alkalies
can be used to disinfect contaminated materials.
9. Alcohols: These are used for disinfecting skin before
injecting and before operations. Alcohols act by
protein denaturation.
10. Two groups of dyes (a) the aniline dyes, and
(b) the acridines have been widely used as skin and
wound disinfectants.
11. Quaternary ammonium compounds are active against
both gram-positive and gram-negative species but are
not effective against spores and mycobacteria. They
are used for sterilizing food utensils in restaurants and
hotels and for disinfecting blankets in hospitals.
12. Glutaraldehyde (Cidex) affects even spores and
mycobacteria. It is employed as 2% solution and is
recommended for sterilizing cystoscopes, etc.
13. Ethylene oxide gas is being widely used to sterilize
disposable plastic syringes, petri dishes, etc.