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
Following are the measures that must be taken care of
a. The steam must be saturated.
b. There must be complete discharge of air from the
c. The autoclave must be loaded in such a way that all the
materials to be sterilized can be adequately penetrated
d. The duration of autoclaving would depend on the
pressure inside and hence on the steam temperature.
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FIG. 2.1C: Precision autoclave
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FIG. 2.1B: Horizontal autoclave
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1. Fill boiler with water to a point just below the basket
2. Place articles within the basket, bottles should not be
more than 3/4 full and should have loosely screwed on
3. Close the lid and tighten the screws.
4. Open outlet valve and adjust safety valve to the
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
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).
10 lbs Pressure for 10 minutes—culture media.
15 lbs Pressure for 20 minutes—infected material.
20 lbs Pressure for 30 minutes—rubber gloves.
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
Sterilization is done for 2 to 3 hours on each of 3 successive
days. A higher temperature may cause bubbling of the
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
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.
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
Cathode rays and gamma rays are the most effective and
are being increasingly used to sterilize disposable items.
These radiations have considerable disinfectant action.
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.
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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(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
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
It is made of a pad of finely ground glass fused into a glass
They are used for removing large particles and clearing
Chemical agents can exert bactericidal or bacteriostatic
effect. The bactericidal agents in lower concentrations exert
bacteriostatic effect. The bactericidal effect is probably
agent with specific groups of enzyme proteins.
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
4. Glycerol: A 50% solution is used for preservation of
certain viruses. Glycerol also kills contaminating
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
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
10. Two groups of dyes (a) the aniline dyes, and
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