Methods of Sterilisation | Laboratory | Biotechnology

The following points highlight the top eight methods of sterilization. The methods are: 1. Boiling 2. Intermittent Heating 3. Autoclaving 4. Dry Heat Sterilisation Indirect Heat 5. Open Flame Sterilisation 6. Filtration 7. Chemical Sterilisation 8. Pasteurisation.

Method # 1. Boiling:

In the absence of heat resistant spores, boil­ing will free liquids of living cells. However, since it is not sure whether spores are present or not, it is not always safe to boil. Elevation influences boiling point. In an open container at sea level, water boils at 100°C (212°F) but as elevation increases above sea level, the boiling point decreases.

Method # 2. Intermittent Heating (Arnold Sterilisa­tion):

This method is used to sterilise thermolabile (broken down by heat) substances which can withstand a temperature upto 100°C or lower. Normally all vegetative proteins coagu­late at 60°-65°C.

For this, substances like carbohydrates are placed in running steam, after water starts boiling, for 30 minutes. The material is taken out and kept at room temperature for 24 hours. This exposure to temperature will break the dormancy and encourage spores to ger­minate and produce a crop of vegetative cells. The next day the process is repeated at the same time, which will destroy the new crop of cells. Incubate for 24 hours and repeat the process for the third day. This last heating is only as a precaution.

Method # 3. Autoclaving (Steam Under Pressure):

This is the most efficient and reliable method of sterilisation. The instrument, an autoclave, is similar to home pressure cookers. This works on the principle that water boils at 100°C at atmospheric pressure of 760 milli­metre of mercury. But if this steam is built up in a closed chamber, water can be kept boiling until the temperature goes far above 100°C.

The relationship of steam under pres­sure to temperature is:

The most important point to be remem­bered is that it is not the pressure that steri­lises the media/material, but it is the high temperature created by raising the pressure of steam (15 lb) above normal atmospheric pressure that effects sterilisation. It should be made sure that the material in the autoclave is not subject to a mixture of air and steam. For this, after tightening the lid, the valve should be kept open and all the air in the autoclave should be pushed out and replaced by steam.

At this time the pressure gauge will show 5 lb. Then close the valve. As soon as the tem­perature reaches 121°C the pressure gauge will show 15 lb. Note the time and keep it for 15- 20 minutes. All laboratory autoclaves are ad­justed to 15 lb pressure by adjusting the safety valve.

After 15-20 minutes, switch off the auto­clave; allow the pressure to come down to zero. Do not release the pressure quickly. This will make the containers with medium blow up their tops and spill the media. After cooling (0 lbs pr) slowly open the valve allowing air to enter the vacuum in the autoclave, created by the setting of steam.

Culture media in tubes and flasks are plugged with non-absorbent cotton to allow ready access to steam. Do not use absorbent cotton.

All media, glasswares, etc. can be autoclaved. However, the glass-wares should be dried in an oven.

Method # 4. Dry Heat Sterilisation Indirect Heat:

Materials like glass-wares, Petri dishes pipettes, etc. since they should be dry while using, are subject to dry heat. In addition, oils and greases should be heat (dry) sterilised since steam will not penetrate such substances. Protein (of microbes) in the absence of mois­ture will not coagulate at 121°C. It needs temperature well above 160°C and hence the temperature is raised to 170°C (keeping 10°C margin) and maintained for at least 1- 2 hours.

Method # 5. Open Flame Sterilisation:

Needles, loops, forceps, etc. are sterilised by making them red hot on a Bunsen flame, cooled in 95% ethyl alcohol and flame heated by passing it quickly over the flame to burn off the alcohol.

Method # 6. Filtration:

Filtration is used for isolation of toxins and enzymes from parent cells. Besides, some liq­uids like blood serum, sugar solutions, etc. cannot withstand high temperature, since it adversely affects its chemical structure. Such liquids are filtered through filters with pores smaller than that of bacteria (say 0.75 µm).

Liquids are drawn through filters at a rate faster than gravity by applying positive pres­sure. But normally negative pressure (suction) is provided to the receiving flask. A differen­tial of 150-200 millimeters of mercury is rec­ommended.

Ordinary filters will make a liquid free of bacteria but not viral particles. However, fil­ters with sizes much smaller than 0.75 µm can be produced which will retain smaller mi­croorganisms and viral particles. There are dif­ferent types of filters.

A. Earthenware candles – e.g. Berkefeld and Chamberland filter:

Berkefeld filter:

These are made from Kieselguhr, a fossil diatomaceous earth found in deposits in Ger­many and other parts.

This has three grades of porosity:

i. V (viel) the coarsest,

ii. W(wenig) the finest and

iii. N (normal) intermediate.

Out of three, Berkefeld V is the one usually used since it will not allow a small organism like Serratia to pass.

These filters can be autoclaved. Clean them after use with a stiff brush, boil in distilled water and run distilled water through them to make sure that the pores are not clogged and then autoclave. When porcelain or earthenware filters get clogged, they should be made red hot in a muffle furnace and allowed to cool slowly.

Chamberlands filters:

These are made up of unglazed porcelain. They are produced in various porosity grades. These are used to obtain bacterial toxin, free of cells.

B. Seitz filters:

In this an asbestos disk is inserted in a metal frame and fixed by sealing the edges. After use the asbestos disk is discarded. Serum is filtered through this.

C. Sintered glass filters:

These are made of finely ground glass fused to make small particles adhere. After use they must be washed first and cleaned with warm H₂SO₄ to which kno₃ is added and not chromic acid.

D. Cellulose membranes:

Two types of cellulose membranes are avail­able. The older one, gradocol membranes are composed of cellulose nitrate and the mod­ern ones consist of cellulose acetate.

The older ones have average pore size rang­ing from 3 µm, down to 10 nm and are used to determine the size of many viruses.

Modern membrane filter or millipore fil­ters are made of cellulose acetate and are com­posed of two layers, a basal layer with pores of 3-5 µm and an upper layer with pores of 0.5 – 1.0 µm. The upper filter retains bacteria especially Serratia marcescens. They withstand sterilisation for 35—45 minutes at 121°C. These are the most commonly used ones.

Technique of Filtration:

Since fluids do not pass through filters by gravity, a positive or negative pressure is to be applied. Suction is the most convenient method. The fluid drawn from the filter falls into a sterile thick conical flask with a side arm. A similar flask should be included dis­tilled in the vacuum line to prevent back flow­ing and contamination. Positive pressure is generally applied. If negative pressure is ap­plied, start with a small pressure, gradually increase to 100-200 mm of mercury.

Method # 7. Chemical Sterilisation:

Volatile disinfectants like chloroform are used for sterilisation and preservation of serum for culture media.

Disinfectants of the phenol group—lysol and cresol are powerful disinfectants for dis­infecting surgical instruments and discarded cultures.

Formaldehyde kills all bacteria and spores.

Ethylene oxide is a gaseous disinfectant used for sterilising articles labile to damage by heat, e.g. plastic and rubber.

Method # 8. Pasteurisation:

This method was invented by Louis Pasteur to destroy microorganisms in wine and beer. Now it is more popularly used in food and beverage industries. The material is subjected to 62°C for 30 minutes followed by rapid cooling to 10°C or exposed to 71.1°C for 15-17 seconds.