[Cytometry] Bacterial contamination in Moflo

Rachael Walker rvw24 at cam.ac.uk
Wed May 11 09:03:54 EDT 2011


Dear All,

Thank you so much to everyone who emailed me with hints about the 
bacterial contamination in my MoFlo.  The machine has now been bacteria 
free for 2 days and hopefully it will stay that way.  I am so grateful 
to all of you who took the time to help me and I am sorry that I have 
not had chance to personally write back to you.

I thought I might share some of the hints that  people have given me and 
how I solved the problem (I hope).

I found last week that both my MoFlo and Aria were still showing signs 
of bacterial contaminations so I took samples from every part of the 
machine that I could, the sheath tank, the filter, backflush, test 
streams etc.  All the samples came back positive on both machines.  
Which led me to think that it must be something being introduced into 
the machines ie. sheath fluid, contaminations in the room or something 
in the pressurised air used in the MoFlo.  I had been having 
intermittent problems with bacteria over the past few months, and was 
blaming people's samples and dead spaces.  I had been checking the odd 
bottle of PBS (made from 10xPBS and autoclaved in house) but they were 
coming out negative.  It wasn't until after bleaching and running virkon 
through the machine and still getting positive broths (tryptone soya 
broth, the contaminations usually come up overnight), that I started to 
sample every bottle of PBS.  I have since discovered that the occasional 
bottle of PBS was contaminated was bacteria, but this will explain the 
intermittent problems and how I was cleaning and cleaning and cleaning 
and still getting problems and why both machines were contaminated.  I 
have had all the lines on my MoFlo changed, changed filters on both 
MoFlo and Aria and bleached and bleached and bleached the machines.  I 
autoclaved the sheath tanks - word of warning MoFlo steel one are fine 
to autoclave (minus the pressure gauge) but the Aria ones shrink when 
autoclaved - whoops.

I have now moved back to FACSFlow as my sheath on the recommendation 
from several people who said that is it good to use for several weeks 
after a bacterial problem and I will continue to use it until I can be 
sure that the PBS I put in is sterile. 

I also found a low level of bacteria in the detergent tank (that I have 
not used at all) that is attached to the smart sampler, so those lines 
have been changed, and the tank filled with 70% Ethanol.   I would urge 
anyone with a smart sampler to check that tank to make sure there 
arent't things growing in there as well.  I don't think this was the 
main cause of the contaminations.  I did also find bacteria in my 
detergent wash bottle, so that's gone in the bin.

A couple of things that I have learnt along the way, Virkon stays in the 
machine along time and not only kills bacteria but will kill your cells 
as well :-) So be careful using virkon and make sure you replace the 
sheath filter after using. 

I had a phone call from a gentleman in Bath or Bristol (sorry I can't 
remember your name) who suggested soaking the nozzle in trypsin as this 
should help to break down any biofilms on it.  I did this to the nozzle 
and the nozzle assembly and I think  it has really helped - thank you 
for the suggestion and I am sorry if I sounded grumpy on the phone, I 
had just discovered the contamination was in my Aria too so I wasn't 
very happy.

I still don't know why the MoFlo nozzle assembly plastic cone has a 
brown layer on it, I suspect its oxidation of the charging wire.  I have 
been sampling my nozzle assembly and that seems to be clear of any 
contamination.

I will be cleaning the whole system with bleach about once a month in 
the future as that is what most people have recommended that I do.

Thanks again for your help and I will be keeping my fingers crossed that 
I have found the source of my contamination problems and my machines 
will now be bacteria free.

Rachael



Box, Andrew wrote:
> 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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