Culturing Cells Post Sort

For anyone doing sorting applications, I'd like to clear up a common misconception.  Just for the record, cell sorters are not sterile.  The instrumentation is not housed in a biosafety cabinet or hood, and therefore it is not possible to sort in a STERILE environment.  The cells sorters are operated under asceptic techniques that minimize the potential for contamination.  The sheath fluid (PBS) passes through a 0.22um filter that is changed every few months, and therefore, the sheath fluid is clean.  Before every sort, a fresh aliquot of 10% bleach is run through the sample introduction probe to ensure there is no contaminants in that line.  However, tubes are opened in a non-sterile environment, and collection tubes remain open for, sometimes, hours in a non-sterile environment.  The only way to combat potential contamination is to use antibiotics in your culture post sort for a limited time.  Since many bacterial strains are resistant to pen/strep, we recommend using Gentamicin.  A wide variety of concentrations using Gentamicin in tissue culture have been proposed, anywhere from 10ug/ml up to 2000ug/ml.  We propose using a higher concentration for a period of 3 days post sort to kill any low level contamination that may be present in the culture.  A concentration of 100ug/ml in tissue culture media should be sufficient.  Gentamicin is available from Sigma-Aldrich as their Hybri-Max, 50mg/mL, sterile filtered solution, which can be added as 1ml to a 0.5L bottle of media.  We are continually evaluating methods used in the facility for decontaminating the instrument and the environment around the instrument.  If you have any questions or suggestions, please feel free to send them my way.