Autoclave, Operating an autoclave, Testing the efficiency of the autoclave

Autoclave:- This is steam above 100°C and it is the most efficient means of sterilisation. It is routinely used for most heat-stable materials. An autoclave is used to achieve the high temperature steam. The boiling point of water is raised by increasing the pressure inside the autoclave. There are different designs of autoclaves in the market. But basically the autoclave is a double-walled or jacketed chamber that can withstand the high pressures required The autoclave has a steam inlet valve and a steam outlet valve. In addition, it is usually equipped with a safety valve (set to open at a pre-determined pressure), a temperature gauge and pressure gauge. The usual sterilization temperature is 121°C at 15 lbs per square inch (psi) pressure for 15 minutes. Greater the pressure, higher the temperature and shorter the time of sterilization.

The cycle however depends on the volume of material to be sterilized and the amount of contamination expected. It is recommended that large volumes of material such as culture media or surgical dressings, be dispensed into small volumes or portions to reduce the time required for sterilization and so decrease the chance of over cooking or damaging the material.

Although autoclaving is a highly recommended method of sterilization, its major drawbacks are that it may cause damage to heat-sensitive materials, and the load is not dried.

The domestic pressure cooker is the simplest form of autoclave. The steam displacement autoclaves which used to be the commonest type, are

becoming obsolete. A mastery of the operation of all types of autoclaves is essential to avoid major accidents due to explosion and to prevent over heating of the material.

 

Operating an autoclave

The load is loosely placed in the chamber and the door or lid securely shut. Both the inlet and outlet valves are open. Steam is introduced into the chamber to force air out from it, leaving only pure steam. (A rubber tubing attached to the outlet valve with the end dipped in a bucket of water will stop bubbling when all the air has been forced out). 

Close the outlet valve, the temperature and pressure will begin to rise. When the desired temperature and pressure are reached, close the inlet valve. Start to time the sterilisation cycle to the desired length of time. 

This period of actual sterilisation is known as the 'holding time'. At the end of sterilisation, open the outlet valve slowly to avoid boiling over of liquid media. The steam begins to escape and at the same time the temperature and pressure begins to fall. Never open the autoclave until the pressure is completely down in order to avoid explosion.

The high vacuum autoclaves are fast and reliable. Before the admission of steam, 98 per cent of the air present in the chamber is rapidly removed by electric pump and sterilisation can proceed without delay. The cycle is short and heat penetration is efficient. Most of the newest models of autoclaves are fully automatic. The operator simply loads the autoclave, presses the start buttons and the autoclave is left unattended to till the cycle is completed.

This table shows the relationship between time of autoclaving, pressure and temperature.

Pressure, holding time and tempreture for autoclaviing
Gauge pressure(PSI*) (pressure above atmospheric)	Holding time (min)	Temperature(*C)
		Pure steam (no air present)	50% air-steam mixture	No air removed
5	40	108	94	72
10	30	115	105	72
15	15	121	112	100
20	10	126	118	109
25	5	130	124	115
30	5	134	128	121

Testing the efficiency of the autoclave

There are several methods of testing whether or not material is adequately sterilised.

The four commonest methods are:

1. Brown's sterilizer control tubes These are tubes containing an indicator liquid and designed for steamer, hot air oven and autoclave. The liquid will change from red to green if the correct temperature time combination has been employed; if not, the liquid turns a reddish-brown color. The tubes are placed in the area of least heat penetration.

2. Biological indicator Filter paper strips impregnated with a sporing organism such as Bacillus stear other mophilus, is enclosed in an envelope and placed at the centre of the load to be sterilised. On removal, the strip is cultured in a suitable medium and incubated at 55°C for one week. No growth indicates proper sterilisation. This method is good when time factor is not important because the organism requires long incubation to grow.

3. Autoclave tapes There are many tapes based on the Bowie-Dick autoclave tape principle of uniform colour change. The tape indicates the degree of heat penetration. The tapes have a water resistant gummed side. When sterilisation is adequate and complete, the tape changes colour uniformly. While the Bowie-Dick tape does not test for adequate exposure to heat in terms of time and temperature, strips of tapes may be useful on individual packages as indicators of exposure to heat.

4. Thermocouples The efficiency of an autoclave is best monitored by means of thermocouples which are placed within the contents and calibrated to show the temperature attained.

Sterilisation failure in the autoclave

Sterilisation failures may be due to:

1. Incomplete removal of air from the autoclave. Temperature of air/steam mixtures at a given pressure is lower than that of pure steam

2. Incorrect methods of packaging.

3. Careless loading into steriliser.

4. Failure to time correctly, the proper period of exposure (holding time).

5. Attempts to sterilise materials impervious to steam in an autoclave, e.g., oils.

6. Faulty equipment.

Inspissation: This is the method of coagulating and further sterilizing culture media that were initially prepared aseptically. The media should contain a coagulable protein such as serum or egg. It is ideal therefore for Loeffler's medium or Lowestein Jensen's medium.

The inspissator is a double walled copper box with water flowing between the two walls.

The inside is made in such a way that culture media bottles lie slopped. The temperature is controlled between 75-80°C thermostatically. Inspissation is carried out for two hours on each of three consecutive days. A higher temperature may denature the protein and cause bubbling of the surface of the media.