Summary Document
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Hi everyone, thanks to all for your replies.
Kylie Price
Staff Scientist Flow Cytometry
The Malaghan Institute of Medical Research
The Central Services Building
Victoria University Campus
Entrance 7
Kelburn Parade
Wellington
New Zealand
Ph: 64-4-4996914, ext 850
Fax: 64-4-4996915
E-mail: kprice@malaghan.org.nz
Web: http://www.malaghan.org.nz
ORIGINAL QUESTION:
I have been asked to sort some bacterial cells for a client. We have never
sorted bacterial cells at our institute and I was wondering if anyone has
SOPs for this. We will initially be sorting E.coli.
My other concern is about contamination. It seems strange to be putting
bacteria into your sort lines, would this be a problem for future sterile
sorts or is there a different sterilisation process that can be performed?
SUMMARY:
* We usually use a 70um tip although a 50um should also work. In part
depends on how fast you need to sort. With our MoFlo we would go about 30K
cells / sec with the 70 at 60psi.
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* We have sorted a lot of bacteria - E. coli, Pseudomonas (plant pathogen),
Erwinia using a MoFlo. We usually trigger on SSC log and also use log FSC.
We have had no particular problems doing a sterile sort even an hour after
finishing the bacteria. We clean the sample lines with detergent (Contrad
NF 70) then sterilize the sample lines with Exspore (our preference) or
bleach.
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* I have sorted bacteria and yeast fairly regularly through my moflo and
find that if you run a 20% bleach solution thru the machine for 10 minutes
followed by water for 30 minutes, you should clean the lines effectively and
get rid of the bleach so as to not kill your next live sort. I always
advise my client to have antibiotics in their cell suspension before and
after sorting and have them wash the sorted cells before performing
additional assays on them.
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* We routinely do sterile sorts of bacteria. Just go through the normal
sterilization procedure after you run them and you should have no problem.
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* A few years ago I was running the Cellular Analysis Facility at UNSW in
Sydney. I sorted animal, human, bacterial and yeast cells as well as
giardia and malaria parasites. In one day I sorted and analysed samples for
9 different clients, each with a different type of cell. During the 3 years
I ran the facility I did not have a single case of sample to sample
contamination. To ensure my lines were clear I ran bleach for 2 minutes
followed by distilled or deionised water for 2 minutes. Admittedly I had a
MoFlo with a manual sample stage, which is a simple construction and easy to
clean, but in my current position I am using a FACSAria which is fully
automated, and I am not having any reports of sample to sample
contamination. Admittedly I am only sorting human and mouse cells.
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