Sterilisation Techniques

Sterilisation is an absolute term meaning the complete destruction or removal of all living organisms. The choice of method depends mainly on the physical nature of the material to be sterilised.

Heat Sterilisation

The temperature and time required for killing are inversely related. The table below shows the minimum times required for effective sterilisation at the temperatures given for both moist and dry heat:

Neurospora contamination of sawdust used in mushroom propagation. Photo: John Leslie 
 

Temperature

Moist heat

Dry heat

100°C

20 hrs

2.5 hrs

50 mins

15 mins

8 hrs

6.5 mins

130°C

2.5 mins

140°C

2.5 hrs

160°C

1 hr

170°C

40 mins

180°C

20 mins

These times do not guarantee sterility however. They are times calculated from experience and are based on normal levels of contamination with heat resistant organisms.

The species, strain and spore forming ability of the microbe greatly affects its susceptibility to heat. In moist heat the vegetative forms of most bacteria, yeasts and fungi and most animal viruses, are killed in 10 minutes by temperatures between 50°C and 60°C. Bacterial spores on the other hand require up to 15 minutes at temperatures ranging from 100°C to 121°C. In dry heat bacterial spores require 1 hour at 160°C.

The nature of the material in which the organisms are heated is also an important factor. A high content of organic substances generally tends to protect spores and vegetative organisms against the lethal action of heat. Proteins, gelatin, sugars, starch, nucleic acids, fats and oils all act in this way. The effect of fats and oils is greatest in moist heat as it prevents access of moisture to the microbes. The addition of an organic or inorganic disinfectant has the opposite effect, facilitating destruction of microbes. The pH is also very important. The heat resistance of bacterial spores is greatest at neutral pH and decreases with increasing acidity or alkalinity. This is put to practical use in the sterilisation of metal instruments. Boiling in water at 2% Na2CO3 is as effective in 10 minutes as boiling in plain water for several hours.

Dry Heat Sterilisation

Dry heat kills microbes by oxidation. The dry heat process is the best method for the sterilisation of dry glassware such as test tubes, Petri dishes, flasks, pipettes, all glass syringes and instruments such as forceps, scalpels, scissors, throat swabs.

The hot air oven is also used for sterilising dry materials in sealed containers and powders, fats, oils and greases that are impermeable to moisture. These materials are penetrated very slowly by heat and must therefore be sterilised in small amounts.

Glassware should be packed so as to allow proper penetration of the hot air throughout the load. This is aided by the fan. The holding period required for sterilisation is 160°C for 1 hour. However most ovens, particularly if packed, will take 2 to 3 hours to reach temperature. Thus 4 hours at 160°C would be the minimum for a big load. Four hours at 170°C allows a safety margin.

Ovens must not be opened during their cycle as one opening for a few seconds may drop the temperature by up to 70°C, which takes the oven perhaps an hour to recover. This leads to the non-sterilisation of that load.

Moist Heat Sterilisation

Moist heat kills microorganisms probably by coagulating and denaturing their enzymes and structural proteins, a process in which water participates. All culture media therefore are sterilised by moist heat.

It is important to realise that killing by moist heat requires contact of the steam and microorganism, and if these are protected from wetting by grease or air pockets they will be subject only to the weaker effects of dry heat sterilisation.

Saturated steam is more effective than dry heat due partly to its greater lethal action and partly to its raising the temperature of the sterilising article very quickly. Pure steam at atmospheric pressure has a temperature of 100°C. A single exposure of 90 minutes seldom fails to sterilise at 100°C as only some thermophiles and very few mesophiles can survive this treatment. A more usual method of sterilisation using steam is 'Tyndallisation', the heating at 100°C for 30-45 minutes on each of three success days.

Autoclaving at temperatures greater than 100°C is the most reliable method and that most widely used for the sterilisation of culture media and surgical supplies. Most autoclaves and pressure cookers operate at 121°C, at which the minimum holding period for sterilisation is 15 minutes.

It is essential that all air is expelled from the autoclave, otherwise it will not reach the correct temperature. Many large autoclaves do this automatically.

If using a pressure cooker or a manual autoclave, allow steam to hiss from the outlet for 2–3 minutes before closing the valve or placing on the cap.

Baskets and not tins should be used for autoclaving and pipettes should not be autoclaved in canisters as localised air pockets will make for inefficient sterilisation. Temperature and NOT pressure is the true criterion for sterilisation procedures.

The autoclave should be adjusted so that the chamber pressure does not fall too rapidly as this results in media boiling over and wetting plugs. Media should be left in the autoclave for about 5 minutes after it has returned to atmospheric pressure, as sometimes solutions remain superheated and when disturbed spray boiling medium or agar over the operator, resulting in nasty burns. If left in the autoclave for longer periods, excessive loss of volume will occur as a vacuum builds up in the autoclave.

An effort should be made to avoid sterilising large and small volumes of media in one load as time must be allowed for large volumes to reach the required holding temperature, and this will result in small volumes receiving too much heat. Here is a rough guide to the extra time that must be added to reach holding temperature:

Volume of liquid

Extra time

Total time at 121°C

Volume of liquid 

Extra time

Total time at 121°C

100 mL bottle

10 mins

25 mins

100 mL conical flask

2 mins

17 mins

250 mL bottle

12 mins

27 mins

250 mL conical flask

4 mins

19 mins

500 mL bottle

18 mins

33 mins

500 mL conical flask

8 mins

23 mins

1000 mL bottle

22 mins

37 mins

1000 mL conical flask

12 mins

27 mins

2000 ml bottle

27 mins

42 mins

2000 mL conical flask

35 mins

Sterilisation by Filtration

Microbes can also be removed from liquids by means of filters with very small pores which trap bacteria. This method is used for sterilising serum, antibiotic solutions, carbohydrate solutions and media that are heat labile.

There are two main types of filter currently in use, the asbestos filter and the membrane filter. The asbestos of Seitz filter has some disadvantages in laboratory work, but due to its cost advantage, is still used extensively in some industries. The membrane filter has however boomed in its use in laboratories over recent years, and discussion will be limited to this.

Membrane filters are thin, porous sheet structures composed of cellulose esters or similar polymeric materials. All particles exceeding the pore size are retained by these filters. Pore sizes range from 14µ to 0.25µ and are very uniform (variation of 0.45µ filter being ±0.02µ) and occupy around 80% of the total filter volume. Thus flow rates are usually far greater than those of other filters having the same size retention capabilities. They are not attacked by water, dilute acids and alkalis, aliphatic or aromatic hydro-carbons or non-polar liquids. In fact, few fluids of interest to microbiologists will attack them. If a compatibility problem does exist, solvent resistant filters are available. They may be autoclaved satisfactorily at 121°C for 15 minutes individually or if assembled, and this is preferable, for a period specified by the holder.

As they are integral structures containing no ionic or particulate material, particulate contamination or alteration of the pH cannot occur.

A 0.22µ membrane filter should normally be used in sterile filtration operations as all reported bacteria are larger than 0.22µ and will therefore be physically retained under all conditions by the filter.

Positive pressure systems are advantageous over negative pressure systems as:

(i) greater pressure can be applied;
(ii) the need for transfer of sterile material from the vacuum flask to point of use is eliminated;
(iii) the possibility of leaks drawing contamination into the flask is eliminated;
(iv) the elimination of bubbling (and therefore denaturation) of protein solutions is achieved.

When sterilising fluid with a high burden of suspended particulate matter a microfibre glass prefilter should be used to prevent unnecessary clogging of the membrane.

Sterilisation of Instruments

Forceps, inoculating needles and other instruments must be sterilised before contact with a culture to avoid cross-contamination.

Inoculating needles are best sterilised by heating to red hot in a flame. The needle must be allowed to cool to room temperature again before being used. Hot needles are the most common cause of failure of subculturing.

Forceps and scalpels are sterilised by standing in a beaker or similar container of alcohol. Before use, the alcohol is burnt off by passing the forceps through a flame to ignite it. Do not hold the instrument in the flame, since this will heat it up too much. Be very careful not to place hot or flaming instruments in or near alcohol, since this is a fire hazard.

Sterilisation of Work Surfaces

Trays, benches and other surfaces may be sterilised with a liquid disinfectant. Alcohol is the most commonly used. Alcohol works best as a sterilant if it contains some water, and a solution of 70% ethanol is suitable. Hypochlorite solutions, such as 1% sodium hypochlorite, are also very effective, but these give off offensive odours, leave a solid residue and are harmful to clothing.