How to deal with ARIA contamination?

Reinier r.vanderlinden at erasmusmc.nl
Thu Jan 31 09:45:49 EST 2008


Hi Akos,
I had the same problems.
I use nitrogen gas from a 175 l LN2 Container for the auxiliary air supply.
(The pressure of the LN2 contaiiner is set to 100 PSI)
It is dry sterile and no O2 depending organisms will grow in the pressurized
part of the instrument.
I never used the bubble filter.
During the long clean the plenums aren't completely filed with beach or 70%
ETOH , so to get rid of the contamination I disconnected the Plenum tubing
and filed the plenums completely with FACS Clean let them soak for a while
and did the same with 70% ETOH and DI. 
Since I use HBSS without any preservative for sheet fluid I routinely run
70% Ethanol through the sheath path including the sheet filter (I replace
the sheet filter every 6 months.) with the stream and sample line back flush
on for a while twice and do the same with DI water and sheath fluid. 
It takes 60 minutes to start up the Aria but I don't have contaminations any
more.
Reinier van der Linden
Erasmus University
MGC- dept. of Cell Biology and Genetics (H Ee 1002)
Dr. Molewaterplein 50
3015 GE Rotterdam
the Netherlands

Tel: +31-10-4087854 / +31-10-4087490
email: r.vanderlinden at erasmusmc.nl
  _____  

From: akos.szilvasi at novartis.com [mailto:akos.szilvasi at novartis.com] 
Sent: Wednesday, January 30, 2008 15:09
To: cyto-inbox
Subject: Re: How to deal with ARIA contamination?

We did all of those you suggested, Simon. The air bubble filter landed in
the garbage a long-long time ago. That dramatic bleaching (10% bleach in
both the sheath and the dist. water tank followed by repeated start ups and
shut downs) solved the contamination only temporarily, say, 4 weeks or so.
Even in between we occasionally could grow a couple of colonies from the
sheath sample. 

According to BD the house air can be a source of contamination. Strangely
there is no barrier built in the outside compressed air source but (if I
understood correctly) there is a filter for the sorter's own compressor.
Next I will have to find an air filter for the house air (even though the
line has two huge cases attached to - a moisture and an oil trap). 

Thanks for all the responses (on and off line). 

Akos

__________________________ 
Akos Szilvasi
NIBRI Core Laboratory Services
manager
USCA, 601-5301
Novartis Institutes for BioMedical
Research, Inc.
100 Technology Square
Cambridge, MA 02139
USA
Phone: +1 617 8717177
Email :  <mailto:akos.szilvasi at novartis.com> akos.szilvasi at novartis.com







Simon Monard <smonard at ibmc.up.pt> 
01/29/2008 08:08 PM 

To
akos.szilvasi at novartis.com 

cc
Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu> 

Subject
Re: How to deal with ARIA contamination?




Hi Akos

I had some probelms with my Aria for a while. Drove me nuts. I ended up
doing a
few things. I ditched that bubble filter entirely. I filled all the tanks
one
at a time with 10% bleach and did a ton of tank primes, basically trying to
give every part of the fluidics a good bleaching. I ran bleach though the
sheath path with the stream on for a while. Then rinced out the tanks real
good
with DI water and did the same with DI water, running it though all the
lines
for a while. Then I replaced all the fluid filters with new ones  (you could
rinse then really well and autoclave them, the connections can get a bit
leaky
after autoclaving). I then ran though a bunch of autoclaved sheath fluid. I
now
autoclave my sheathfluid and haven't had any contaminations since. Doing
this
took most of the day. I had tried using less drastic measures before but the
contamination kept coming back. There are so many places bacteria can lurk
in
the Aria there being so much tubing. The bubble filter needs to go as it
never
gets sterilized if you isolate it when running ethanol as BD suggest. For
many
years I had no contaminations at all with Vantages etc.
You do need to make sure you get rid of any traces of bleach as cells don't
get
on very well with it. I make up my own sheath fluid as 10x stock, dilute it
to
1x and autoclave it in the spare sheath tank.

Before asceptic sorts I run 70% ethanol though the sheath path for a while
(not
the sheath filter) and do an bunch of fills after switching back to sheath
fluid. I have done many sorts now with no problems even without having
antibiotics in media afterwards.

Best of luck

Simon

Quoting
akos.szilvasi at novartis.com:

> Dear FLOWers,
>
> We have a less than two years old (Special Order - UV equipped) ARIA
> sorter that had an unrelenting sheath fluid contamination problem. The
> recurring mysterious contamination existed from the arrival of the sorter.
> We reported it multiple times. BD replaced a few parts of the fluidics
> system but it kept the bugs out of the sheath only for 4-6 weeks at the
> time. Filter replacements, bleaching the system works only temporarily.
> Our other Aria sorter, an older one, never had any such symptoms. We use
> them in an identical fashion.
>
> One lab suggested to follow their solution (this is not an isolated
> occurrence - not matter what they say) to fill the distilled water tank
> with 70% ethanol so that the fluidics shut down procedure would fill the
> lines with alcohol. That sounds good but no one could tell if this trick
> has some detrimental effect on any parts of the fluidics system. Have you
> used alcohol over night in the sorter? Did it cause any problem on the
> long run?
>
> Any other solution? BD offers to replace the whole fluidics system for $
> 13,000.
>
> Best regards,
> Akos
>
> __________________________
> Akos Szilvasi
> NIBRI Core Laboratory Services
> manager
> USCA, 601-5301
> Novartis Institutes for BioMedical
> Research, Inc.
> 100 Technology Square
> Cambridge, MA 02139
> USA
> Phone: +1 617 8717177
> Email : akos.szilvasi at novartis.com
>
>
>
> _________________________
>
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-- 
Simon Monard
Cytometry Lab Manager
Rua do Campo Alegre, 823,
4150-180 Porto - Portugal
Tel +351 226 074 900 ext 3102 . Fax +351 226 099 157
Cellphone # 919 036 680

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