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

 

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.

FIG. 2.1A: Vertical autoclave

(Courtesy: Yorco Sales Pvt. Ltd)

FIG. 2.1C: Precision autoclave

(Courtesy: Yorco Sales Pvt. Ltd.)

FIG. 2.1B: Horizontal autoclave

(Courtesy: Yorco Sales Pvt. Ltd)

Sterilization 31

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.

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

FIG. 2.2: Seitz filter

(Courtesy: Yorco Sales Pvt. Ltd)♥

32 Concise Book of Medical Laboratory Technology: Methods and Interpretations 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.

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.