Autoclave Sterilization

Overview

Autoclaves heat their contents to 121°C, which is 21°C over the boiling point of water. To prevent the solutions from boiling over/vaporizing, the autoclave chamber is pressurized during this process.

Most buffers and other solutions used in any lab are sterilized before use to prevent bacterial and fungal growth during storage. There are two basic techniques for sterilizing solutions: autoclaving and sterile filtration. Many buffers and other salt solutions are autoclaved because filtration of large volumes is time-consuming and expensive. However, before autoclaving any solution you should always check whether it contains any heat-labile ingredients (Media Recipes). If it does, the heat-labile substance will usually have to be prepared separately, filter-sterilized, and added to the remainder of the solution after autoclaving. Also, certain combinations of compounds that are stable when autoclaved on their own can react at the high temperatures in the autoclave to produce compounds that inhibit microbial growth (e.g. Mg and glucose)

Autoclave Most buffers and salt solutions
Rich bacterial and yeast media broth
Do Not Autoclave Buffers with detergents (SDS) – they will boil over
Organic solvents that are volatile and/or flammable (ethanol, acetone, phenol, chloroform)
Heat labile ingredients (vitamins, hormones, antibiotics, proteins), HEPES solutions
DTT (dithiothreitol) or BME (beta-mercaptoethanol)
Among many others...

Be sure the containers you are autoclaving things in are also safe: bottles should be made from borosilicate glass or autoclavable plastic – be sure that any plastic items you put in the autoclave are really autoclavable (polypropylene is OK, polystyrene is NOT). Pipet tips, microfuge tubes, and their storage containers generally are autoclavable, if unsure please ask about other plastics before you experiment and make a mess.

General autoclave use:

  1. Leave at least a quarter of the container volume as free space (otherwise your solutions will boil over).
  2. Place containers in autoclavable pans to catch liquids from any breaking containers.
  3. Make sure all caps are loose and taped on at least on one side (including tinfoil caps)
  4. Use autoclave tape to tape on caps, also put a small bit of autoclave tape on any other items you autoclave. The tape will change color during autoclaving, thus signaling to any future user that the item has really been sterilized.
    • Note: Autoclave tape only shows that the tape has been exposed to heat (80°C). No information on time, steam penetration or temperature/pressure can be inferred.
  5. Tighten autoclave doors thoroughly but not so much that you can’t get it back open; note that this only really applies to older autoclaves, most newer autoclaves will seal themselves.
  6. Select the appropriate program:
    • minimum autoclave time should be 20 min
    • use longer times for larger volumes
    • use the “liquid cycle” for any load containing liquids
    • use the “dry cycle” only if there are no liquids in the load (autoclave will exhaust faster with this program, causing any liquids to boil over)
  7. Make sure the pressure is down completely (check gauge) before opening.
  8. Open the door VERY SLOWLY and STAND BACK unless you want your face scalded
  9. Wear heat-resistant gloves to unload the autoclave
  10. CLEAN UP ANY SPILLS, empty any liquids from autoclave pans down the drain, rinse well

Other considerations:

Secondary Containers:
Only use polypropylene plastic plans or stainless steel containers as secondary containers. Do not use polyethylene or polystyrene as these plastics will melt when autoclaved. Note that plastic containers increase the time needed for perfect sterilization as plastic is a good insulator. As a rule, add 5 min to run when using a plastic container.

Volume:
An important consideration for liquid media is the higher the volume, the longer the sterilization time. Generally, the volume of liquid per container is more important than the total volume per load. For example, a 2 liter flask containing 1 liter of liquid takes longer to sterilize than four 500ml flask each containing a volume of 250ml of liquid.

Packing the autoclave:
Avoid overloading the chamber with material; space must be available for the heat/steam to penetrate everything. Room should be left between flasks, bottles, and other containers to allow proper steam circulation.

General time guidelines:
The following is recommended times for autoclaving liquids (volume is per container used):

75-200ml 20 minutes
200-500ml 25 minutes
500–1000ml 30 minutes
1000-1500ml 35 minutes
1500-2000ml 40 minutes

Remember to modify these times as needed. When in doubt autoclave for 1 hour.

Autoclave locations:

Our lab is authorized to use the autoclaves in MBB, Room 2.316.

The code is 6842

If there is any problems with these autoclaves please notify senior lab personal and/or the MBB building manager.
The current building manager is Rob Newton rob_newton@austin.REMOVE_THIS.utexas.edu.

 Barrick Lab  >  ProtocolList  >  AutoclaveSterilization

Topic revision: r6 - 03 Jun 2014 - 17:14:14 - Main.CraigBarnhart
Lab.AutoclaveSterilization moved from Lab.AutoclaveUse on 23 May 2014 - 19:03 by Main.JeffreyBarrick - put it back
 
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