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Ohio Water Microbiology Lab

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Ohio Water Science Center

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OWML: QA/QC

Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory

 

Autoclaves
autoclaveSterilization is the process that eliminates living organisms from substances or objects. The Ohio Water Microbiology Laboratory (OWML) is equipped with three autoclaves for sterilization of glassware, reagents, media, and disposables—two medium-sized autoclaves (Market Forge) that are operated in the side laboratory and one large autoclave (Consolidated) that is operated in the warehouse.

  • Dishes that need to be sterilized are wrapped in aluminum foil or kraft paper and placed in the autoclave for moist heat sterilization. Clean and sterile dishes are stored in closed cupboards until use.
     
  • The autoclaves are operated at 15 lb/in2 steam pressure, producing an inside temperature of 121 to 124oC (American Public Health Association, 2005, Section 9020B). Do not overload the autoclave. Autoclave time depends on the type and amount of equipment as follows:
     
    • Glassware and up to 250 mL of liquid—15 minutes
       
    • 500 to 2,000 mL liquid—30 minutes
       
    • Greater than 2,000 mL to 6,000 mL liquid—15 minutes per 1,000 mL
       
    • Greater than 6,000 mL liquid—90 minutes
       
    • Carbohydrate-containing media—15 minutes (no more than 250 mL volumes)
       
    • Pathogenic organisms—30 minutes, allow autoclave chamber pressure to decrease, then run for a 60 minute cycle
       
    • Contaminated materials and discarded cultures—45 minutes
       
    • Heat-sterilizing tape is used with each run to identify supplies that have been properly sterilized and checks the performance of the autoclave. The performance is also checked monthly by using spore indicators and recorded in the LIMS.
       
    • If the autoclave does not reach the specified temperature or fails the spore indicator test, the autoclave is serviced and all glassware and reagents that were insufficiently sterilized are re-sterilized.

     

  • For the two medium-sized autoclaves, general maintenance is as follows:
     
    • The autoclaves are operated using a mixture of deionized water and tap water.
       
    • At the end of the week, autoclaves are drained. Twice a month, autoclaves are cleaned with Liqui-nox, rinsed with water, and drained. The condensate holding tank is drained daily or as needed. The cleaning date is recorded in the LIMS.
       
    • Twice a year, a contractor inspects and calibrates the autoclaves and performs preventive maintenance. Preventive maintenance dates are recorded in the LIMS.
       
    • Twice a year, the chambers are cleaned with 10% muriatic acid and flushed well with water. Cleaning dates are recorded in the LIMS.

     

  • For the large autoclave, general maintenance is as follows:
     
    • Once a month, the chamber is cleaned with water and liquinox. Cleaning dates are recorded in the LIMS.
       
    • Twice a year, a contractor performs preventive maintenance and inspection, cleans and services the generator, cleans the door gasket and head ring, applies graphite to the door gasket, oils the door hinge pins, and lubricates the door hub. Preventive maintenance dates are recorded in the LIMS.
       
    • Twice a year, the chamber is cleaned with 10% muriatic acid and flushed well with water. Cleaning dates are recorded in the LIMS.
   

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