ARIA Answers

"BD should have upgraded your flow cell from a clear cell to an amber 
coloured one.  The amber coloured flow cell is immune to damage from Etoh.

As to a 'dirty' flow cell.  My first flow cell (clear) was found to be full 
of an amorphous crystaline 'stuff'!  This problem has been eliminated by 
removing the nozzle when the Aria is shutdown, and turning the stream on 
after startup, but before installing the nozzle to ensure anything hanging 
around in the flow cell is pushed out.

I store my nozzle in water with a mild detergent and sonicate before use, I 
give it a good rinse in distilled water before installing it in the instrument.

I only use the standard startup and shutdown to keep the Aria clean.  I 
have had no (0) cases of contamination.  Every now and then I will run 
bleach through the sample line for 5 - 10 minutes, particularly after long 
sorts where I have had cell precipitation in the lines.

I ran the MoFlo at the CAF for 3 years without a single case of 
contamination simply by keeping my cleaning protocols simple." ---> Rob

"We had a lot of initial sterility problems with our Aria which
we have had nearly 2 years. We never use the EtOH procedure for
sterilising the Aria as it causes instability in the stream which takes
too long to sort itself out especially when we want to start sorting at
about 10.00am. We have replaced the DI Water with a disinfectant called
Disifin which is non corrosive to the connectors and sheath tubing etc.
We therefore do the Fluidics Shutdown with Disifin rather than DI Water.
We have also replaced the bubble filter with a stainless steel
autoclavable filter which is replaced each day. This is extra protection
for the Pall Filter which sometimes seems to break down even though we
autoclave it monthly and change it every 3 months." ----> Michael

"can't address question 1, but I can tell you what I've been doing for
the FACSAria.  To sterilize for sorting I do a "Long Clean with Bleach"
with 10% bleach, then two "Startups".  Then I reconnect the bubble
filter and do one more startup, I don't know if it's necessary, it's
just the way I've started doing it.  I never run EtOH through, either.
For shutdown, I've been doing 2 "Shutdowns".  For the first, I use 50%
Contrad 70 and let it sit in the flow cell for a few minutes, then I use
DI water for the next 3 cleaning solutions.  Occasionally, if my flow
cell is looking dirty, I do a "Clean Flow Cell" with 100% Contrad 70,
and let it sit for a while, up to overnight, but you don't want to do
that too often.  Then I do several "Clean Flow Cells" with DI water to
rinse it out.  When I'm not sorting, my startup is 1 "Shutdown" and then
2 "Startups".

This is something that I put together on advice from my engineers and an
application specialist.  It's been working for me for about a year, with
no complaints about any contamination issues.  I do recommend that my
customers use antibiotics to be sure. " ----> Robin

"For the Aria you should look into buying some Contrad 70. It's a detergent
that was recommended to us by the service engineer from BD who installed our
machine. It does a good job of cleaning the flow cell and normally works well
if you have a clog. For shut down I've been running 10% bleach for about 2
mins on 11, the di h2o for at least double the time normally about 10 mins
also at a flow rate of 11. Do fluidics shutdown and use 5% contrad for the
FACSrinse and Di h2o parts, then repeat the shutdown and use di h2o at the
end and let that stay there. You probably don't need to use bleach as much as
you have been, and the long clean with bleach probably only needs to be done
once a month. Also I wouldn't recommend using beach a short time before you
sort because it can take a while to clean that out with di h2o. BD has a new
flow cell that supposed to be ok with ethanol but apparently other parts
aren't so don't use that. Our service engineer has said before not to use
germicidal grade bleach, that can do some damage somehow and just to use the
regular store bought kind. If you're interested I can send you what I do for
a monthly cleaning. I started using our Aria in Sept. '04 and so far we
haven't had any issues with contamination. The flow cell needed to be
replaced in January this year as it was developing some cracks but there was
a new one that it was going to be replaced with anyway. In general we haven't
had many problems aside from the fuses in the fluidics cart blowing which has
been replaced and is working fine now." ---> Bryan

"I also have an Aria and have been running it consistantly for over a year 
now. My shut down procedure is simple. After my last run I let the stream 
go for 10 minutes just to clean out any debris on the nozzle. Then I take 
DI water and fill a 5 mL tube. Add one drop of ethanol to the DI water and 
do a clean flow cell. One time and that is all I do. I do not bother with 
the Instrument shut down window or anything like that. I have never used DI 
water instead of sheath fluid but please let me know what others say on 
that because it would be great because of the salt build up you get 
sometimes. I am curious, who told you that ethanol was hurting the machine? 
I have been saying this for awhile now and yet no one else had heard of it 
that I spoke to." ---> Rebecca

"Primary cleaning



Every six months or so, I bypass the flow cell (i.e. join the sheath and 
vacuum lines together) and run though 3l 0.1% detergent (I use DECON90), 
this is followed by  15l  water, 3l 70% ethanol and 6l PBS.



The rinse bottle is 70% v/v ethanol/water (on the Elites, this flushes the 
system on auto shutdown).



On the day of a sort, I do the following;



i)		       Swap the sheath and rinse bottles

ii)		      Power up the instrument

iii)		      Go to the valve control menu and select Flow cell Vac 
stop. To OFF this flushes the fluidics system with 70% ethanol

iv)		     After 10 mins, select Flow cell Vac stop. To ON

v)		      Stop the sheath flow (on the elites this is the 
vacuum button)

vi)		     Take off the rinse bottle and empty the remaining 70% 
ethanol into the sheath bottle- the sheath bottle is now the rinse bottle.

vii)		     Wash the rinse bottle with sterile dh20 and refill 
with sterile PBS -the rinse bottle is now the sheath bottle

viii)		    Put the rinse bottle on as the sheath bottle

ix)		    Flush through with 3l sterile PBS as in steps (iii)-(iv)

x)		     Refill with sterile PBS

xi)		    Sort.



So far, no contamination." ----> Arnold

"Aria I would be interested to know who recommended that you don't
use ethanol as the BD people here in Sydney have said nothing like
that to us. Unless it was related to the second generation flow cells
(clear with removal grounding plate and ceramic nozzles) - that one
certainly deteriorated very quickly and BD replaced it with the third
generation flow cell which supposedly is not sensitive to ethanol
(this is the brown tinted one without the removal grounding plate).

Our third-generation flow cell (brown) was installed in early
December and we have been having problems with flurosecence signals
levels since mid-Jan. Been though all sorts of procedures but are
going to have the flow cell replaced on Monday - latest theory is
that is "dirty" - this seems to be a theory that has come back from
Europe after some on the engineers went to training over there - I
don't know how true it is.

We actually run three shutdowns at the end of each day - bleach (x2)
in the first one, extran in the second and millipore water for the
third." ---> Adrian

"If you have a FACS Aria with a dark Amber flow cell, it is compatible with
70% EtOH for sterilization. The earliest Aria's had a clear flow cell that
was compatible with EtOH, but I believe at some point, the manufacturer
that supplies them changed the composition of the flow cell body. Thus,
contact with EtOH caused them to become opaque. The new dark Amber flow
cell housing and dark Amber plenums are EtOH compatible.

One thing to be cognizant of: Many people make the assumption that DI water
is sterile and were using it straight from the faucet for the DI Tank.
Unless your DI water source also goes through a Millipore filtration unit,
or you cold filter sterilze it through a 0.2 micron Millipore or Nalge
filter, or autoclave it, assume it to be potentially contaminated. A few
Aria users would disinfect the Aria with bleach, only to recontaminate it
with bacteria-laden DI water.

Attached is a protocol that I believe was developed by Steve Perfetto at
the Laboratory of Vaccine Development at the NIH in Bethesda, Maryland,
USA. He maintains a website with information on Aria sterile sorting.

(See attached file: Decontamination Procedure for the FACSAria.doc)

One thing I do if the instrument is heavily contaminated is to fill all the
containers with bleach. remove all the PALL filters and discard. Prime each
tank using the software to get bleach through all tubing lines and to
disinfect the liquid level sensors. Select the various cleaning cycles from
the Instrument>Cleaning submenu. This will cause the solenoid valves to
open during the various cycles to clean all the lines. Rinse all tanks3X
thoroughly with sterile, DI water. Drain residual level sensor fluid lines
of bleach. Refill tanks with the respective fluids, then do "Prime after
tank refill" 2-3X. Do the startup with the DI water on the Sheath input
port 5-7 times to remove all the bleach. Replace the DI water tank with the
Sheath Tank. Install new 0.2 micron PALL filters on the wetcart. Do Startup
using Sheath at least 5X to remove all the DI water" ---> Steve

thanks to all and all thanks



Geza Paukovics B.MLS.
Senior Research Assistant
The Macfarlane Burnet Institute for Medical Research and Public Health
Site address:
Alfred Medical Research & Education Precinct (AMREP),
corner Punt & Commercial Roads, Prahran 3181 or
GPO Box 2284, Melbourne 3001
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Sidney Myer Fund Flow Cytometry Unit  \_.--._/	*	* | | |  *
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Monash Central School of Medicine (AMREP)
Flow-Cytometry and Sorting Suite Co-ordinator

tel: (+61 3) 9282 2246 (Burnet) or 9903-0601 (Monash)
fax:(+61 3) 9282 2100
email: paukovic@burnet.edu.au 

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