[Cytometry] Risk of Contamination After Sorting Bacteria

Gerald Gregori gerald.gregori at univmed.fr
Wed Nov 18 11:15:32 EST 2009

Hi Rachael,
in our core facility we are used to run bacteria on our Moflo, among 
other bugs (both prokaryotes and eukaryotes).
There is no problem with cross contamination of samples as far as you 
carefully clean the instrument.
Here is the procedure we use :
- After running a sample of bacteria we are used to backflush the  
sheath  fluid  through the sample line (20 to 30 seconds). This cleans 
most of the bugs in the line. It is incredibly  efficient.
- The major source for contamination on our Moflo, equipped with a 
manual sample station, is at the level of the valve to sart or stop the 
sample. Therefore we are used to run for few minutes a solution of 
Bleach at 10% in distilled water.
- Then we run distilled water to rinse the bleach.
I eventually decided to avoid the solution of Ethanol 70% in distilled 
water as Ethanol can be a source of Carbon for some bacteria.

Now, as far as E. coli is concerned, there are some strains which are 
indeed pathogens, some others are not. You have to ask your user for that.
If the strain is not a pathogen one (Classe 1) then you can run your 
sample (you just need to wear gloves, a coat, and to close the front 
door of the Moflo to keep the cells confined...particularly if you sort 
in case of aerosol formation). After the sorting or the analysis, you 
have to wash the interior of the Moflo with a solution of Ehtanol 70% 
(to clean), and then some bleach solution (10%) to sterilize, and 
eventually with distilled water to remove the bleach.

If you have to work with pathogens, it is a different story. You need to 
have a special equipment  (safety-hood, or class II lab) .

Good luck.

PS : A trick to know when you run bacteria is that in most of the cases 
the abundance is very high (10^6 to 10^9 bacteria/ml). Therefore you 
must run the sample with a pressure differential very low (.1 to .2 at 
the most). If you run too much sample at once, you send too much 
bacteria and the likelihood to have doublets or aggregates becomes higher.

Rachael Walker wrote:
> Hi Everyone,
> I have been asked if I would analyse and sort e.coli on my MoFlo 
> sorter.  I have never sorted bacteria before and am worried about 
> contaminating other mammalian cells lines that go through the machine 
> afterwards. 
> I was wondering whether other people regularly sort bacteria and 
> mammalian cells on the same machine, if they have had any contamination 
> problems and how they disinfect after sorting?  Also is there any risk 
> to my health by sorting e.coli?  Should it be counted as a Class II sort?
> Many thanks
> Rachael

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