Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111

Table of Contents


Practice the procedures, especially the closing and opening of the autoclave door, without introducing any steam into the autoclave chamber until the entire sequence can be performed without any difficulty. The student is authorized to use the autoclave only after he has demonstrated to the instructor his ability without referring back to this write-up. Do not use the autoclave if the student is not absolutely sure of the proper operating procedures. The pressurized steam is more likely to cause burns than boiling water. First, it is hotter than boiling water. The temperature and pressure relationship is shown in the following table. More importantly, the heat of vaporization released upon the condensation of steam causes much severer damage than does the same quantity of boiling water. Always watch out for the hot metal parts on the autoclave. Wear heat insulating (asbestos) gloves when handling hot autoclaved items.
       Temperature and Pressure Relationship
      Steam              Temperature
     Pressure               (ºC)
                --------AIR DISCHARGE-------
                100%   66%    50%   33%   0%
        0       100.0
        1       101.9
        2       103.6
        3       105.3
        4       106.9
        5       108.4  100    94    90    72
        6       109.8
        7       111.3
        8       112.6
        9       113.9
       10       115.2  109   105   100    90
       11       116.4
       12       117.6
       13       118.8
       14       119.9
       15       121.0  115   112   109   100
       16       122.0
       17       123.0
       18       124.1
       19       125.0
       20       126.0  121   118   115   109
       21       126.9
       22       127.8
       23       128.7
       24       129.6
       25       130.4  126   124   121   115
       26       131.3
       27       132.1
       28       132.9
       29       133.7
       30       134.5  130   128   126   121

Around the working range of 15 psi, each psi raises the autoclave temperature by approximately 1ºC.

Operating Procedures

  1. Check the chamber pressure (GAUGE #2 in the Figure) if the autoclave door is already closed and locked, because a locked door usually indicates that autoclaving is in progress. Do not attempt to open the door unless the chamber pressure is at ambient. Opening a chamber filled with pressurized steam can be suicidal. Although some newer models of autoclaves have built-in safety features to guard against this possibility, never subject one's own well being to such a shaky assumption. Check the jacket pressure (GAUGE #1). If the gauge indicates a pressure of between 15-17 psig, proceed to Step 4; otherwise, continue onto Step 2.
  2. Open the condensate valve (VALVE #1 in the Figure).
  3. Slowly open the steam supply valve (VALVE #2). Allow condensate to drain. When the condensate is completely drained, as evidenced by the flow of steam from the outlet of VALVE #1, close the condensate valve (VALVE #1). (If pipes start to "bang" hard during condensate drainage, close VALVE #2 slightly.) Wait for the jacket pressure to build up to the required 15-17 psig.
  4. Open the door on the autoclave slowly and cautiously. Take out all the items from the previous autoclaving runs if they are found inside the chamber. Place items to be autoclaved inside the chamber. Make sure that all the components of each item can withstand the heat of autoclaving; many plastic materials will melt or deform. Leaving a few drops of water inside a closed container where the steam in the chamber cannot easily penetrate will enhance the sterilization effect. If liquid is to be autoclaved, place all such containers in a stainless steel pan so that any spill can be readily caught. Many liquids will boil over in the autoclave. The boiling is especially violent when the pressure is released at the end of the autoclave cycle. The use of a pan will also facilitate handling if more than one container is to be autoclaved. Vent all containers to avoid explosion. If bottles are capped, make sure that the caps are screwed on lightly so that any excess pressure can escape. The caps can be screwed tightly later as they are taken out from the autoclave. At the same time, make sure that everything is closed so that contaminants cannot enter after autoclaving. Note that the need for pressure relief and prevention of contaminant entry are not contradictory. For example, shaker flasks can be vented with cotton seals; fermentor jars, and nutrient jars can be vented with in-line filter units. On the other hand, some items to be autoclaved cannot be conveniently vented; they must be placed in a protective steel casing. The glass pH electrodes are such examples.
  5. Near the center of the door locking wheel, there is a pin that, when in the inserted position, prevents the door locking wheel from turning freely. The pin is released from its hole by pushing the small (approx. 2 inches) lever parallel to the face of the door locking wheel. To engage the pin, set the lever perpendicular to the face of the door locking wheel, then turn the wheel slightly to allow the pin to drop into one of the holes intended for it. Close the autoclave door. Engage the pin, and turn the wheel clockwise by about 1/8 of a turn to insert all the locking bars in the respective slots on the rim of the autoclave. These bars do not always all slide into the slots at the first attempt. If so, wiggle the bars as the wheel is turned. After make sure that all the the locking bars extend securely through the slots, release the pin so that the door locking wheel is now free to spin. Rotate the wheel clockwise until it is hand-tight; this applies pressure to the door to seal it air tight so that steam pressure can be built up inside the chamber. Do not over tighten the wheel. Engage the pin to lock the wheel and prevent it from turning back. The door is now securely locked.
  6. Turn the four-position selection valve (VALVE #3) on top of the autoclave to the sterilize position (STER) by lining up the STER mark with the arrow on the valve. This position allows the steam to enter the autoclave chamber from the jacket.
  7. Wait for the chamber pressure, as indicated by GAUGE #2, to reach the maximum, where the pressure is to remain stationary during the entire autoclave cycle. The pressurization of the chamber will take approximately 5 minutes. This marks the start of the autoclave. The temperature in the chamber can be read with GAUGE #3 at the lower front of the autoclave.
  8. After autoclaving for 20-30 minutes, terminate the autoclaving cycle by shutting off steam to the chamber and venting it slowly to the ambient pressure. This can be accomplished by turning VALVE #3 from the sterilization position (STER) to the exhaust position (EXH). It takes about 3-5 minutes to vent the excess steam. It is safe to open the door only when the needle of the chamber pressure indicator, GAUGE #2, falls within the narrow white area marked around 0 psig. When the chamber reaches ambient pressure, release the pin that locks the door locking wheel. Slowly turn the wheel counterclockwise to relieve pressure on the door. When the door locking wheel is fully released, engage the pin and turn the wheel 1/8 turn further to disengage the locking bars from the slots. Open the door slowly. Watch out for a small puff of residual hot steam being released as the door is first opened. Note that a slight vacuum may be created in the chamber as the hot air is cooled further while the door is left locked. In this case, a screwdriver may be used to pry open the autoclave door with care; however this is not recommended for beginning students. Alternatively, introduce steam into the chamber for a few seconds after securely locking and pressurizing the door will bring back a positive pressure in the chamber.
  9. Again, remember to wear gloves. Watch out for the boiling liquid and handle the hot autoclaved items with great care so as not to burn oneself. Do not touch the hot chamber wall. Now is the time to make sure that the sterile side of all the sterilized items are not open to invasion by contaminants. For example, plug the mouth of shaker flasks if the plugs have come off during autoclaving. Check all connections on a fermentor jar to make sure that they all remain sealed and that no tubings have been broken. Reconnect them quickly whenever possible. Remove all autoclaved items from the chamber.
  10. Clean up any spills and close the autoclave door when done. Do not pressurize the door.

Summary of Procedures

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Aseptic Culture Techniques -- Use of a Steam Autoclave
Forward comments to:
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
301-405-1910 (voice)
301-314-9126 (FAX)
e-mail: nsw@umd.edu