All lab equipment and tools may be placed in one of three categories, depending on the nature of their contamination. Equipment that is clean has been washed to remove grease and dirt, and to limit the amount of chemical contaminants that might interfere with the growth of microbes.
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Cleaning means the removal of soil, but never means that the equipment has been sterilized. Dirty equipment is soiled with contaminants that may be chemical or biological in nature.
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In a hospital situation, this equipment has been used often, and has in all likelihood come in contact with patients. Equipment that is sterile contains no living organisms to interfere with the laboratory analysis of a specimen or contaminate others should they come in contact with the equipment after it has been discarded.
Because of the need to successfully grow pure cultures of pathogenic microbes from identification and analysis, it is essential to clean and sterilize all laboratory equipment before it is used.
Several chemical and physical methods have been developed to achieve sterile conditions. Such physical methods include moist-heat sterilization (autoclaving), dry-heat sterilization, and filtration. Chemical sterilization is usually performed by using a gas or chemicals.
An autoclave operates on the same basic principle as a home pressure cooker. Steam under pressure penetrates the materials in the materials in the autoclave more thoroughly and rapidly than dry heat.
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In the operation of an autoclave, the chamber that contains the materials to be sterilized (culture media, glassware, cotton stoppers, and paper materials) is filled with steam as the air is flushed out.
Once this exchange has occurred, additional steam is forced into the chamber and the pressure is increased. Sterilization is accomplished when the materials have been exposed to 121 °C at 15 to 20 pounds per square inch (psi) pressure for about 15 to 20 minutes or in a high-speed vacuum autoclave at 132°C for 3 minutes.
The time, temperature, and pressure can be varied depending on the material to be sterilized and its thickness. However, these conditions are usually sufficient to destroy resistant endospores of bacteria and all vegetative cells.
When the process has been completed, the steam is released from the chamber and the materials removed. Liquid materials such as broth and water must have the steam slowly released from the autoclave to prevent boiling over.
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The heat energy that has been forced into the fluid by the high pressure will cause the fluid to boil if the pressure is reduced too rapidly.
This process is usually done automatically by the autoclave in the slow exhaust cycle. Caution should also be exercised when removing liquid materials from the autoclave since many types of media will boil when shaken, even after slow exhaust cycle.
Non- liquid materials such as empty flasks, test tubes, and bottles can be fast exhausted since there is no danger of boiling over. Containers plugged with cotton or foam caps should be run through a drying cycle following exhausting. Microbes may penetrate the wet caps after the material is taken from the autoclave and contaminate the interior of the tube or flask.
Many materials cannot withstand the high heat of the autoclave, but still must be sterilized. To sidestep this problem, John Tyndall introduced in 1877 an alternative technique now known as Tyndallization.
This process allows heat-resistance endospores to be destroyed at only 100°C by exposing them to a repeated cycle of heating and cooling. The materials to be sterilized are exposed to live steam for a period of about 30 minutes, and then cooled to room temperature for a day. During the cooling process, many of the endospores that might be present germinate into their more heat-sensitive vegetative form.
On the following day, the process is repeated, killing newly germinated cells and stimulating the germination of the remaining endospores. The steam is applied for a last time as a precaution and kills all remaining life forms.
Even though this process takes three days it makes possible the sterilization of culture media containing heat-sensitive sulfur or certain amino acids. For materials that may damaged by the moisture used in autoclaving, dry-heat sterilization may be a good alternative.
Dry-heat sterilization is a very useful procedure for materials that are able to withstand very high temperatures. This process requires that glassware, instruments, or hypodermic needles be heated to between 160°C and 170°C for about 90 minutes.
No special equipment is needed to dry-heat sterilize, and a kitchen even may be used if set above 356°F for at least one hour. Do not sterilize cotton or paper materials in this fashion, since this high temperature will cause them to char and possibly burn.
The problems related to high temperatures and moisture may make it impossible to sterilize many special types of culture media commonly used in a medical laboratory, and an alternative to moist and dry heat must be used.
Media containing such materials as urea, serum, plasma, or other body fluids may be sterilized using the membrane filter method. A special funnel apparatus is used to filter out the microbes found as contaminants in these types of media.
The filter used must have pores so small that the microbes will be captured on its surface as the liquid is pulled through the filter by a vacuum machine. These filters may be made of cellulose, finely granulated glass, asbestos, or diatomaceous earth.
The pore size of many commercially available filters can be specified when the filter is purchased, but most viruses will readily pass through. This is rarely a problem in bacteriological work since both viruses and rickettsias are obligate intracellular parasites and require host cells for growth.
Culture media should be sterilized and not contain cells of any type. The entire apparatus is autoclaved before the fluid is sectioned through and collected in a flask. It is then transferred, using aseptic technique, to another sterile container for use.