From: Derek Davies (daviesd2@icrf.icnet.uk)
Date: Thu Jun 11 1998 - 05:59:19 EST
Hello Joanne, We regularly sterilise the sheath lines with 70% ethanol - normally before every sterile sort which means almost on a daily basis. We have a spare sheath tank for the ethanol and while this is on the machine the PBS tank is left to soak in a 5% (approx) solution of "Roccal", a disinfectant. It is also given a good shake to ensure all areas come into contact with the disinfectant. This is then thoroughly rinsed out with sterile deionised water and the tank filled with sterile PBS before putting back on the machine. On exiting the sheath tank the PBS passes through a sterile 0.22u filter which is replaced on a daily basis. PBS is allowed to run through all lines for at least 15 mins before sorting commences. While the ethanol is on the machine a "sample" of alcohol is run therough the sample line and all surfaces are washed down. To some of our users who are desperate to get onto the sorter for as long as possible, it may seem like overkill but we rarely have contaminations that can be traced to the machine. Periodically it is also worth changing all the sheath fluid lines. If you are getting contamination from the plated sheath fluid can it be narrowed down ie is the fluid you are using sterile when it goes in the tank? When it comes out? After it has passed through the nozzle? There are severla possible areas of contamination. Hope this helps - good luck sorting it out! Derek On Mon, 8 Jun 1998, Joanne Yetz-Aldape wrote: > Hello, Flowers, > I am having some difficulties with sterility of cells > sorted on my FACStar PLUS. After running 10% bleach and 70% ethanol > through the system, one investigator still reports having bacterial > contamination in the sheath flow-through, plating the sheath in > gentamicin. Another investigator reported that her cells were > overrun with bacteria after 4 days in culture with pen/strep, when > the sorter had been cleaned in the same way, but after one other > cell prep had gone through. The weird thing is that I have been > sorting this one cell prep every week for quite a > while, and there is no contamination in these cultures after 2 weeks > with gentamicin. Could there be a bug I can't remove from the sorter > that is resistant to pen/strep, and is there a critical level of > gentamicin that works in this one situation where the sortied cells > seem clean? Is there some detergent that I could use to purge my > system, or is this something that should just be treated with > antibiotics? I do add gentamicin to my sheath tank, also. Are there > any special sterility problems associated with the turbo sort option?? **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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