Steam Sterilization

Of the many methods or agents that have been developed for sterilizing surgical and dental instruments, microbio- logical media, infectious waste, and other materials not harmed by moisture or heat, steam is still the most effec- tive and most common. The device used most commonly for this purpose is called a steam sterilizer, or autoclave. Autoclaves are relatively safe, easy to operate, and, if used properly, effective at killing all microbial vegetative cells and bacterial endospores.Under atmospheric pressure, water boils at 100°C (212°F). At pressures above atmospheric pressure, water must be heated above 100°C before it will boil. Similar to home pressure cookers, which create pressure and high temperatures to shorten cooking times, autoclaves use super-heated steam under pressure to kill heat-resistant organisms. Examples of heat-resistant organisms include members of the spore-producing genera-Bacillus, Geobacillus, and Clostridium.

In the microbiology laboratory, sterilizing temperature usually is set at between 121°C and 127°C (250°F and 260°F); however, sterilizing time can vary according to the size and consistency of the material being sterilized. At a minimum, to be sure that all vegetative cells and endospores have been killed, items being processed must reach optimum temperature for at least 15 minutes. This includes items deep inside the autoclave container that may be partially insulated from the steam by surrounding items. Understandably, larger loads take longer to process than smaller loads. (Certain sensitive applications, such as microbiological media preparation, in which formula integrity must be maintained and when specific growth inhibiting ingredients are included, lower times and temperatures are acceptable.)

To maintain laboratory safety and comply with laws regarding infectious waste disposal, sterilizers must be checked regularly for operating effectiveness. Special thermometers placed in an autoclave can record the maximum temperature reached inside the chamber but do not measure how low the temperature dips during the normal cycling of the heating elements. Specialized color-coded autoclave tape (Fig. 2.50) can be a fairly good indicator that sterilization is complete, but the only way, with certainty, to determine that sterilization has been achieved is by using a device called a biological indicator.

Biological indicators, as the name suggests, are test systems that contain something living. A typical biological indicator that is particularly useful for testing autoclaves is one that contains bacterial endospores. Bacterial endo- spores, the dormant form of an organism, are highly resistant to both chemical and physical means of control. Therefore, if an autoclave kills the endospores in the test system, it is safe to assume that it will destroy other microbes as well. This is important not only for safety reasons but is of legal importance as well. Public health and safety agencies maintain compliance with hazardous waste disposal regulations by requiring regular testing of autoclaves used to process biohazardous material.

A typical system, and the one selected for today's lab, includes a small, heat-resistant plastic vial containing a glass ampule of sterile fermentation broth. Also inside the vial, but outside of the ampule, is a strip of filter paper containing bacterial endospores. The vial is placed inside the autoclave and heated at 121°C for 15 minutes. After autoclaving, the vial is cooled and crushed with a special device that breaks the inner ampule without damaging the plastic vial. Breaking the ampule allows the fermentation broth to come in contact with the bacterial endospores in the filter paper. The vials are then incubated at 55°C for 48 hours. If the endospores have been killed in the auto- clave, incubation will produce no growth. If they have not been killed, they will germinate and ferment the substrate in the broth. A pH-indicating dye, included in the broth, will reveal any acid produced (during fermentation) with a distinctive color change. No color change during incu- bation is, thus, an indication that sterilization is complete, the endospores have been killed, and the autoclave is operating properly. (For more information on fermentation, refer to Section 5 and Appendix A.)