Autoclave Use

Background Information

An autoclave is used to sterilize equipment and media.

Louis Pasteur showed that once sterility of a medium is achieved, that medium will remain sterile (steril = barren) until contaminated by microorganisms (micro = small). Sterilization by heating is one of the most convenient methods, but may not be effective at the temperature of boiling water (100° C) because many spores can survive at this temperature. However, in a pressure cooker, where the pressure is 15 lb. (1 atmosphere) above normal atmospheric pressure, water boils at 121° C, and all life forms are killed in 15 min. or less. Thus, the standard conditions for sterilizing apparatus or small volumes of liquid is to treat them with steam at 15 lb. of pressure for 15 min.

An autoclave (auto = self; clavi = key, in reference to the fact that it is “self-locking” due to the pressure built up) is an automated pressure cooker in which the conditions inside are thermostatically controlled. When the autoclave's timer is set for 15 min., the autoclave first heats until 121° C is achieved, then the light goes on and the timer starts. The pressure and temperature are maintained for the selected time. At the end of the cycle, the pressure is gradually released, and the door cannot be opened until the pressure reaches zero again. Thus, the 15 min. heating time actually takes 30 to 45 min. to accomplish.

Safety Considerations

Make sure that lids on any containers placed in the autoclave are loose enough to allow the pressure inside and outside the container to equalize as the temperature increases. If lids are on tightly and pressure cannot equalize as the autoclave heats up, you risk having those containers implode. Also, make sure that the placement of items in the autoclave will not interfere with operation of the door.

Do not try to force the door open before the pressure has dropped all the way to zero. If the pressure is still high enough, you won’t be able to open the door. If the pressure is still elevated somewhat but low enough to allow the door to be opened, you run the risk of having extremely hot steam being forcibly blown at you as the pressure is released. Also, if you have just autoclaved liquids, the sudden drop in pressure can cause those liquids to start boiling vigorously enough to overflow their containers.

Even if the pressure has dropped all the way to zero, you need to be extremely careful when opening the door of the autoclave. Stand well back from the door, and especially make sure your face is not close enough to get scalded. Wear heat-proof gloves to help protect your hands and arms. First, slowly release just the latch on the door so the initial burst of steam is released slowly and goes straight up rather than out toward you. Once that steam has been released, then carefully open the door.

Use caution when reaching into the hot autoclave to remove articles. Wear heat-resistant gloves because the items you’ll be handling will be extremely hot. Be very careful that your unprotected arms don’t come into contact with the hot doorway or sides of the autoclave as you remove articles from it.

Make sure that lids are on securely-enough to not fall off any containers, but loosly-enough for pressure-equalization before you remove those containers from the autoclave. Note that if taller containers have been placed behind the position of the open door, you may need to carefully tip them slightly to get them past the door. Be careful that you don’t tip containers of liquids so far that the liquid spills out of the container. Heat-resistant gloves rapidly become non-heat-resistant and extremely uncomfortable if/when soaked with hot liquids, not to mention that you could scald your hands.

Regulations and requirements on autoclaving and disposal of contaminated media (“used” petri dishes with cultures growing on them) keep changing (and have changed numerous times since this protocol was written), so check first with the Lab Manager, before attempting to autoclave any cultures, etc. that are no longer needed.

Parts of the Autoclave

Illustrate the autoclave, labeling the following parts:

• drain valve



• water level (internal)



• shelf (internal)




• door
• door latch


• low water light
• reset button



• pressure indicator



• slow/fast exhaust



• temperature indicator



• timer
• on/off light

Use of the Autoclave

1. Close the drain valve.



2. Pour about 4 L of tap water into the bottom of the autoclave. While use of water from the hot water faucet may be preferred because it is deionized, the most convenient water source is the hose attached to the special tap to the left of the autoclave. Note inside the autoclave the “lip” (near the bottom of the opening) with the instructions to “fill to this level” on it.
3. Load the material to be autoclaved, prepared as follows:
   • liquids in bottles (not over ¾ filled) with caps on slightly loosened, placed far enough back so the autoclave door won’t hit them when it’s open,
   • clean glass petri dishes with lids on, right side up, in stainless steel canister if desired,
   • apparatus wrapped in brown paper with the edges folded over and stapled so the apparatus is completely enclosed,
   • various other glassware such as flasks covered with an aluminum foil “cap”
   • if allowed by current regulations, glass or plastic petri dishes with contaminated media (“used” plates), agar side down, in a dishpan — separate glass and plastic, and/or
   • test tubes with or without media in them, with lids on, in a metal or heat-resistant plastic rack. Note that if screw-top vials are being sterilized, the lids should be on loosely during autoclaving (and may be tightened, somewhat, afterwards).
   If you’re getting ready to run the autoclave, it’s always a good idea to check to see if anyone else has something else that needs to be sterilized, and combine loads.


4. Close the door and latch securely by pressing down firmly on the handle.
5. Select the appropriate exhaust: “slow” for water-containing materials or “fast” for apparatus. If you have a mixture of “wet” and “dry” things to autoclave, set the exhaust on “slow.” This controls how quickly the steam is released and the pressure decreased after autoclaving is finished. If you remember PV=nRT-related topics from chemistry, the boiling point of a liquid is dependent on pressure as well as temperature. If you autoclave liquids, you are heating them higher than their “normal” boiling point, but doing so under pressure, so they don’t boil. If the pressure is released too quickly, they will begin to boil, vigorously, and at least partially, boil out of their container. However, if the pressure is slowly released so the liquids have time to “adjust,” boiling will be kept to a minimum.
6. Set the time of treatment (15 min. for small volumes of liquid (this is what we use for almost everything we autoclave), 30 min. for volumes over 1 L, or 60 min. for volumes over a gallon). Note that the timer light doesn’t go on until the autoclave reaches the proper temperature and pressure. Also, if at any time during the process, the pressure/temperature drop, the timer will stop timing until the autoclave has built up proper pressure/temperature, again. Thus, autoclaving something for 15 min. often takes about 45 min.
7. Periodically, check to see how much time is left, and when the timer has finished, watch the pressure gauge until the pressure drops to zero. After the pressure has returned to zero, open up the door with care — the steam is very hot and can burn if your hands are over the door. Remember to stand back, keep your face away from the door, and wear “hot” gloves. Loosen the door latch first and let the steam escape before trying to raise the door. CAUTION: ESCAPING STEAM CAN SCALD!
8. Remove hot items with the aid of heat-resistant gloves. Watch that your arms don’t touch the hot sides of the autoclave. If the lids have popped off any bottles or test tubes, immediately replace them (without touching the inside) while the contents of the bottle or test tube are still hot enough to remain sterile. Typically, due to all the changes in disposal regulations, we have to use plastic, disposable petri dishes, but if “used” glass petri dishes have been autoclaved, immediately pour out any used agar while it is still liquid (ask the Lab Manager where to put it) and rinse the used glass containers, dishpans, etc., in hot water (When glass petri dishes have been cleaned and are dry, they may be returned to a metal canister and sterilized. Regulations on disposal of used plastic petri dishes keep changing, so check with the Lab Manager first (before you run the autoclave) for current regulations.
9. If this is the last load for the day, open the drain valve to empty the water from the bottom of the autoclave.

Use of Autoclave Tape

Autoclave tape looks like masking tape with white lines on it. These white lines are sensitive to conditions in the autoclave and change to black when properly treated. Thus, canisters of pipets or petri dishes, bottles of medium, and/or packages of wrapped equipment (especially if any of these items are to be stored for a period of time before use) can have a SMALL piece of autoclave tape placed across their lids prior to autoclaving. When the container is removed from the autoclave, the tape is dated. By placing the tape across the lid of a container, it “seals” the container (in that it is a sign that the container has been unopened and is sterile inside), and if the seal is broken (if the tape has been torn or removed), that is an indication the container has been opened and the contents may no longer be sterile.

Other Things to Include in Your Notebook

Make sure you have all of the following in your lab notebook:

• all handout pages (in notebook or separate protocol book)
• all notes you take during the introductory mini-lecture
• all notes and data you gather as you use the autoclave
• a drawing of the autoclave with detail of all knobs and dials, with functions of all parts labeled
• a summary/conclusion in your own words, and any suggestions you may have
• any returned, graded pop quiz