[Cytometry] Bacterial contamination in Moflo

Box, Andrew ACB at stowers.org
Wed May 4 12:57:42 EDT 2011


Hi Rachael,

I would say you need to replace all the tubing that contacts sheath fluid, soak the nozzle assembly in 10% bleach and autoclave anything possible that touches sheath fluid.  When was the last time you had a PM?  There are several valves involved, for example, in the debubbling mechanism that should probably be replaced.  These types of small parts are often overlooked and most of this stuff gets replaced during a PM.  If you're on a tight budget you might even be able to buy the PM kit from Beckman and just put the things on yourself, it's not too hard to do and travel/labor is most of the cost service - although PM's are often offered at a flat rate so it might not be too bad.

Our moflo has been fitted with a custom fluidics setup that we designed such that everything touching sheath can be autoclaved, but short of doing this I would say you just need to replace as many things as you can since it can be tough to remove bacteria from nooks an crannies in the various tubes/valves involved in the system - even with bleach or ethanol.  On days where we don't start with a freshly autoclaved fluidics setup we rinse the system with sterile H20 on shutdown the night before, this helps prevent growth as well as reduces salt crystal build up.  I should also say we don't use the sheath filter that comes with the system, instead we use a 2 um PEEK inlet filter (IDEX part number A-551) as a prefilter inside the sheath tank, then a 0.22 um dental filter (Pall Medical part number 5030105) as an inline filter connected with luer locks.  We use a new one of these dental filters every day.  This filter combination seems to work well and we see a background event rate (with threshold set low using 3 um beads) of < 1/sec.

Good luck!

Andrew


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Rachael Walker
Sent: Monday, May 02, 2011 10:49 AM
To: Cytometry at lists.purdue.edu
Subject: [Cytometry] Bacterial contamination in Moflo

Dear All

I have been having a recurring bacterial contamination within my MoFlo 
for several months now.  It will be fine for weeks and then it appears 
again.  I regularly leave the machine with ethanol in all the lines for 
the weekend but this doesn't seem to be eliminating the problem.  I have 
changed everything I can think of including all lines, the nozzle 
assembly, sheath filter and autoclaved the sheath tank and the sample 
probe.

When I first had this problem I discovered a biofilm looking like rust 
at the top of the nozzle assembly, and when swabbed this showed positive 
for bacteria.  I have changed nozzle assemblies 3 times since then for 
various reasons and I have changed my nozzle as well (which is regularly 
sonicated in detergent).  I put the last nozzle assembly in place about 
6 weeks ago and already it is showing signs of this brown biofilm inside 
it.  Does anyone else see this in their Moflo nozzle assemblies?  The 
only things to have gone through that nozzle assembly are PBS (10x from 
VWR, diluted and autoclaved in house), facs rinse, 70% ethanol and 
cells.  I occasionally run bleach and water through the sample lines.

I managed to run a sorter for nearly 4 years without contaminations and 
now I am plagued with problems.  Are there any dead spaces within the 
machine that I don't know about apart from at the top of the nozzle 
assembly?

I would be grateful for any advice.

Many thanks
Rachael

-- 


Dr Rachael Walker
Flow Cytometry Core Facility Manager
Wellcome Trust Centre for Stem Cell Research
University of Cambridge
Tennis Court Road
Cambridge
CB2 1QR

01223 760227

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