Aseptic Technique and Good Cell Culture Practice

Aim

To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines.

Materials

1. Chloros / Presept solution (2.5g/l)
2. 1% formaldehyde based disinfectant e.g.Virkon,Tegador
3. 70% ethanol in water (Prod. No. R8382)
   

Equipment

1. Personal protective equipment (sterile gloves, laboratory coat, safety visor)
2. Microbiological safety cabinet at appropriate containment level
3. Cell Culture Plates(6 well plate, 24 well plate, 96 well plate)
   

Procedure

Sanitize the cabinet using 70% ethanol before commencing work.
Sanitize gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds before commencing work.

Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.
Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding.

Discard gloves after handling contaminated cultures and at the end of all cell culture procedures.
Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids, cell culture plates) should be wiped with tissue soaked with 70% ethanol prior to use.
Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.
Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.

After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving.
Cell culture discard in chloros (10,000) ppm must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.
Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or Virkon or fumigate the cabinet according to the manufacturers instructions. However you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor.

Source: Sigma-Aldrich