Use of Autoclave: Sterilization by moist heat

The microbial population is often controlled by the use of physical and chemical agents. Chemical agents include various disinfectants and antiseptics. While physical agents such as temperature, osmotic pressure, and radiations (gamma rays, cathode rays, X-rays, and ultraviolet rays). 

Temperature is the most economical and easy to use method to control microbial growth. Heat above a certain limit can kill microbes and their spores while below a certain limit has a static effect on the microbial population. In microbiological laboratory heat killing of microbes is extensively used either in the form of moist heat or dry heat. The autoclave is the best example of moist heat. Moist heat can denature proteins by breaking H-bonds in the proteins ultimately disrupt plasma membrane and denature nucleic acid. The susceptibility of various organisms to high temperatures can be determined by thermal death time which is the time required to kill a given number of cells or spores at a given temperature. 

Saturated steam under pressure provides an excellent means of sterilization of inflammable substances and culture media. It’s a heavy-duty iron or steel cylinder with various sizes. It can be vertical or horizontal. It has the facility of electric or gas burner to boil water. There is also a provision of an outlet valve for air that can control steam pressure and is also equipped with a safety valve as well as pressure and temperature gauges. 

Procedure

  1. Pour distilled water in the autoclave to a recommended point electric coil should be immersed in water and the water level should not be more than the bottom of the bucket/holding pan.
  2. Place the media flasks in the holding pan inside the autoclave. Screwcap bottles should not be tightly closed.
  3. Tightly close the lid of the autoclave.
  4. Turn on the electric heater or gas burner. Make sure the steam outlet valve is closed. 
  5. Wait until the air is ejected from the valve and steam passes out.
  6. Close both valve outlets, and allow them to produce steam at maximum. Monitor temperature and pressure gauge when a temperature of 121°C and pressure of 15 PSI is reached to reduce the heat.
  7. Allow the pressure and temperature to maintain their specified limit.
  8. After 15-20 min, turn off the heating system and open the steam valve and wait until pressure fell zero.
  9. Open the safety clamps and lid of the autoclave and wait for the temperature to cool down a bit.
  10. Remove media and pour into Petri plates and maintain remaining media at the refrigerator.
  11. Incubate one petri dish at a temperature of 37°C for 24h to determine the efficiency of sterilization.

Precautions

  • Ensure all the air is driven out before closing the air valve.
  • Never autoclave heat-sensitive solutions like sugar or serum.
  • Never tightly close the flasks or bottles to be autoclaved.
  • Never shift glassware directly to the refrigerator immediately after autoclaving.

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