OWML: QA/QC

Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory

Autoclaves
Sterilization 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
     
  • Operating temperature and pressure are checked once a week. 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, service the autoclave and re-sterilize all glassware and reagents that were insufficiently sterilized.

   

• For the two medium-sized autoclaves, general maintenance is as follows:
   
  • The autoclaves are operated using deionized 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, have a contractor inspect and calibrate the autoclaves and perform preventive maintenance. Preventive maintenance dates are recorded in the LIMS.
     
  • Twice a year, clean the chambers with 10% muriatic acid and flush well with water. Cleaning dates are recorded in the LIMS.

   

• For the large autoclave, general maintenance is as follows:
   
  • Once a month, clean chamber with water and liquinox. Cleaning dates are recorded in the LIMS.
     
  • Twice a year, have a contractor perform preventive maintenance and inspection, clean and service the generator, clean the door gasket and head ring, apply graphite to the door gasket, oil the door hinge pins, and lubricate the door hub. Preventive maintenance dates are recorded in the LIMS.
     
  • Twice a year, clean the chamber with 10% muriatic acid and flush well with water. Cleaning dates are recorded in the LIMS.