Disinfection

Methods designed for the disinfection/decontamination of culture waste, work surfaces and equipment represent important means for minimizing the risk of harm.
The major disinfectants fall into four groups and their relative merits can be summarized as follows:

• Hypochlorites (e.g. Chloros, Presept) Good general purpose disinfectant
  Active against viruses,Corrosive against metals and therefore should not be used on metal surfaces e.g. centrifuges. Readily inactivated by organic matter and therefore should be made fresh daily Should be used at 1000ppm for general use surface disinfection, 2500ppm in discard waste pots for washing pipettes, and 10,000ppm for tissue culture waste and spillage
• Phenolics (e.g. Sudol, Hycolin)
  Not active against viruses
  Remains active in the presence of organic matter
• Alcohol (e.g. ethanol, isopropanol)
  Effective concentrations 70% for ethanol, 60-70% for isopropanol
  Their mode of activity is by dehydration and fixation
  Effective against bacteria. Ethanol is effective against most viruses but not nonenveloped viruses. Isopropanol is not effective against viruses.
• Aldehydes (e.g. glutaraldehyde, formaldehyde)
  Aldehydes are irritants and their use should be limited due to problems of sensitization
• Glutaraldehyde may be used in situations where the use of hypochlorites is not suitable e.g. cleaning of centrifuge bowls or materials constructed of stainless steel that may be attacked or corroded by using hypochlorite solutions.

Waste Disposal

Any employer has a ‘duty of care’ to dispose of all biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture waste can be decontaminated and disposed of safely(especially the solid waste, such as flasks, centrifuge tubes, contaminated golves etc). One of the most important aspects of the management of all laboratory-generated waste is to dispose of waste regularly and not to allow the amounts to build up. The best approach is ‘little and often’. Different forms of waste require different treatment.

Tissue culture waste (culture medium) - Inactivate overnight in a solution of hypochlorite (10,000ppm) prior to disposal to drain with an excess of water
Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight before disposal by autoclaving and incineration
Solid waste, such as flasks, centrifuge tubes(such as 15ml Centrifuge Tube, 50ml Centrifuge Tube), contaminated gloves, tissues etc. should be placed inside heavy duty sacks for contaminated waste and autoclaved prior to incineration. These bags are available from Bibby Sterilin and Greiner.
If at all possible waste should be incinerated rather than autoclaved.

Source: Sigma-Aldrich

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