Autoclaves

Steam sterilization of materials is a dependable procedure for the destruction of all forms of microbial
life. Steam sterilization generally denotes heating in an autoclave utilizing saturated steam under a
pressure of approximately 15 pounds per square inch (psi) to achieve a chamber temperature of at least
121ºC (250ºF) for a minimum of 15 minutes (for small loads). The time is measured after the
temperature of the material being sterilized reaches 121ºC (250ºF).

Mechanical indicators such as pressure gauges and thermometers are widely used to verify proper
autoclave operation. However, these are considered secondary to the use of appropriate biological
indicators placed on or throughout the material being autoclaved. The biological indicator most widely
used for wet heat sterilization is a Bacillus stearothermophilus spore suspension or strip. If sterilization is
associated with patient diagnosis, the biological indicator and associated documentation is required by
law, as these are the best indicator of sterilization.

The FSU Biological Safety Office can test departmental autoclaves on an as-needed basis to verify
sterilization using the B. stearothermophilus spore test. Additional testing may be requested if
sterilization problems have been observed or are suspected. Call the Biological Safety Office for more
information.

FSU researchers should test every sterilization load with autoclave tape or other indicators. The choice
of sterilization test may be made at the discretion of the researcher. Information provided below may
assist researchers in selecting the appropriate autoclave verification method.

Autoclave Procedures

Materials contaminated with infectious materials must be collected in the appropriate containers and
sterilized or disinfected before disposal. Specific requirements for handling, sterilizing, and disposing
infectious waste must be followed.

When using a steam autoclave, consider the following:

1. Never autoclave FLAMMABLE, REACTIVE, CORROSIVE, TOXIC, or RADIOACTIVE MATERIALS.

2. Always wear safety glasses, goggles or face shield, lab coat or apron, and heat-protective non-
asbestos gloves when opening door or removing item(s) from autoclave.
3. Open door slowly; beware of a rush of steam.
4. Open door only after chamber pressure returns to zero. Leave door open for several minutes to
allow pressure to equalize and for materials to cool.
5. Do not mix loads which require different exposure times and exhaust.
6. Materials that will melt (e.g., plastic lab wear) should be placed in a shallow stainless steel
autoclave pan.

Liquids Autoclaving

• Collect liquids in leak-proof containers such as flasks or bottles.

• Liquid waste containers designed to withstand autoclaving temperatures must be used when
steam sterilization is utilized. To allow pressure equalization, they should not be sealed.
• Non-biohazardous and biohazardous liquid waste may also be chemically disinfected with 10%
bleach for 1 hour (final concentration) and then disposed of into the sink with plenty of water.

Storage and Disposal

Following steam sterilization, innocuous liquids may be poured down the sink with plenty of water.
Contact the Biological Safety Office with any questions about disposing waste down the drain.

NOTE: Do not pour melted agar into sink or floor drains. Allow it to cool and solidify for disposal as a
solid waste in black garbage bags.

Solids Autoclaving

• Non-sharp, solid laboratory waste (e.g., empty plastic cell culture flasks, Petri dishes, empty
plastic tubes, gloves, wrappers, absorbent tissues) which may be, or is known to be,
contaminated with infectious material must be collected in autoclavable bags. If this waste is
not autoclaved immediately, it must be stored in autoclavable bags displaying the biohazard-
warning symbol if the material is infectious, and a clear autoclave bag with no biohazard symbol
for materials that are not infectious (see below).
• Autoclavable bags should be used for solid, non-sharp, infectious waste only and disposed of
appropriately. They should not be used for the collection of other solid hazardous or non-
hazardous waste that may require other treatment or disposal methods.
• For laboratories generating large volumes of agar gel in disposable Petri dishes and tubes
requiring sterilization, such waste should be collected in the laboratory in autoclave bags
contained in an autoclavable plastic pail, tray or bin sufficiently deep to contain the
materials. Autoclavable bags filled with plastic ware containing agar gel tend to leak fluids
during and after the sterilization process. The pail or tray will contain the liquids released by the
agar gel.
• Red biohazard autoclave bags vs. clear autoclave bags – Contact the Biological Safety Office for
red autoclave bags to be used for infectious materials, including human blood and tissues and
BSL2 or BSL3 biological material. Biohazardous/infectious waste must be autoclaved in a red
biohazard bag, and the bag sealed and placed in the biohazard box or bin provided. Clear
autoclave bags (no biohazard symbol) should be used for non-pathogenic biological material like
E. coli. Cultures of non-pathogenic organisms must be destroyed through autoclaving or
bleaching. After autoclaving and cooling, the clear bags of autoclaved waste must be placed into
double-lined black plastic garbage bags (available at most stores and through many vendors)
and discarded in the dumpster. No red bags or biohazard symbol bags may go in the trash or
dumpster.

Sterilization and Disinfection

• Inactivate the biological agents by employing steam sterilization procedures. Autoclaving (steam
sterilization) is the preferred (and generally regarded as the most reliable) method of sterilizing
biological waste. Depending on the volume of waste to be sterilized, it may be necessary to
extend the duration of exposure to high temperature steam under pressure.
 • Steam sterilization is generally not recommended for laboratory waste contaminated with or
containing a combination of viable biological agents and significant amounts of hazardous
chemical or radioactive materials.
• Containers of liquid waste must be placed in an autoclavable tray or pan of sufficient capacity to
contain all liquid in the event of vessel failure or breakage inside the autoclave chamber. Use
extreme caution when handling autoclaved liquids since they are hot and may boil over.
• Autoclavable bags of solid waste should be gently closed (but not sealed airtight) to allow steam
penetration before they are placed into the autoclave chamber. Tightly sealed bags that are
autoclaved may burst.

Autoclave Chemical Indicators

Chemical Color Change Indicators

Chemical indicators for steam autoclaving change colors after being exposed for a few minutes to
normal autoclave operating temperatures of 121ºC (250ºF). Hence, chemical indicators can give a quick
visual reference for heat penetration inside the hood. Chemical indicators should be positioned near the
center of each load and toward the bottom front of the autoclave.

Tape Indicators

Tape indicators are adhesive backed paper tape with heat sensitive, chemical indicator markings.
Commonly used heat sensitive markings include diagonal stripes (autoclave tape) and/or the word
“sterile.” These markings only appear when the tape has been exposed for a few minutes to normal
autoclave decontamination temperatures.

Caution: Color change and tape indicators can only be used to verify that your autoclave has reached
normal operating temperatures for decontamination; they have no time factor. Color change or tape
indicators are not designed to prove that organisms are actually killed during a decontamination cycle.

Autoclave Biological Indicators

Biological indicators are designed to demonstrate that an autoclave is capable of killing microorganisms.
Only Bacillus stearothermophilus spores can be used to monitor the effectiveness of steam autoclaves.

Typical biological indicator systems consist of a vial with spore strips or a small glass ampoule of growth
medium with spores and indicator dye. Refer to manufacturer’s instructions for usage. The biological
indicator is removed from a load after it has been autoclaved. Then the biological indicator is incubated
at 56ºC (132.8ºF) for up to three days. A control vial, which is not autoclaved, should remain clear
without evidence of turbidity (no growth). If the autoclaved biological indicator is turbid (cloudy,
indicating growth) the autoclave did not function properly.

Autoclave Maintenance and Testing

To ensure sterility of materials and adequate decontamination of wastes, it is important for all
departments that operate autoclaves to maintain them and train personnel in their proper use. For
departments with large in-house autoclaves, EH&S can conduct, on request, spore testing to make sure
they are operating properly and the procedures are adequate for the decontamination of biohazardous
waste. Autoclave users must use the prescribed conditions for appropriate decontamination. Users
should be aware that large, dense loads require increased decontamination time. Autoclaves requiring
repair should be reported to the department's facilities manager.

Autoclave Inactivation of Certain Toxins

Dry Heat (ºF)

Toxin Autoclaving
200 500 1000 1500

T-2 Mycotoxin No No No No Yes

Brevetoxin No No No No Yes

Microcystin No No Yes Yes Yes

Tetrodotoxin No No Yes Yes Yes

Saxitoxin No No Yes Yes Yes

Palytoxin No No Yes Yes Yes

Ricin Yes No Yes Yes Yes

Botulinum Toxin Yes Yes Yes Yes Yes

Staphylococcal Enterotoxin B Yes (?) Yes (?) Yes (?) Yes (?) Yes (?)

Wannemacher, R.W. 1989. Procedures for Inactivation and Safety Containment of Toxins.
In: Proceedings of the Symposium on Agents of Biological Origin, U.S. Army Research, Development
and Engineering Center, Aberdeen Proving Ground, MD, pp. 115-122.

Autoclave Safety Training