dWater as sheath plus ARIA question

Arnold Pizzey rmgc300 at ucl.ac.uk
Thu Feb 16 05:27:40 EST 2006



Hello Geza.

In our Elites I see no major differences between ddh20 and buffer as a 
sheath fluid in terms of sensitivity/resolution; however, water becomes 
contaminated much quicker -I guess that the opportunistic organisms are 
geared more towards puddles of rainwater than puddles of seawater!



All in all, I would go with some sort of buffered solution for the sheath I 
see no corrosion on the Elites, and I guess that most manufactures use 
salt-resistant materials for their hardware

If you are worried about corrosion, I'd take it easy with that bleach 
though -I use hypochlorite very occasionally when I suspect that the 
sample-side plumbing may have some protein build-up.

Incidentally, I have found that whilst urea solution is great for removing 
protein contamination, it's also great for dissolving the urea glue holding 
together the flow cell......

I've been sorting on our machines for around 17 years and have tried all 
sorts of protocols for machine sterilization. The one that works for me is 
as follows;



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.




At 17:09 14/02/2006 +1100, you wrote:
>Deer fellow Flow Figures,
>
>I have two questions to pose:
>
>1. Are there any groups or labs who have substituted distilled water for 
>their sheath fluid in their analysers. I vaguely remember some members on 
>the list discussing this. Has anyone implemented it?? Has anyone done a 
>study on the changes of refractive index of the stream (well within the 
>cuvette) and the effects on light scatter properties. Correspondingly how 
>does it affect their PMT sets. How is the fluorescence affected? Okay so 
>that was a few more than just one question.... In our joined departments 
>we have 1x FACScan 2xFACSCalibur (4 colour) and I wish to remove or reduce 
>the effect of salt corrosion both in the machine in general but most 
>importantly around the flow cell. We just had to replace a flow cell and 
>they're not cheap. We currently use 1. commercial IsotonII from Coulter 2. 
>in house sheath fluid (180mg NaCl/20L distilled water)
>
>2. We recently acquired 2XFACSArias. Can all ARIA users please expose 
>their shut down and sterilisation procedures. We were recommended not to 
>use ethanol (for sterilization) as it's detrimental to the flow cell. I 
>have been using a 1% bleach solution running for 10-15minutes before a 
>sort for quick sterilization. For the long aseptic sort clean I use 1% 
>Bleach in the ethanol tank instead of ethanol and also bi-pass the 
>de-bubbler and the sheath filters as recommended in the instructions. I 
>have had no problems with sterility up to now. One of the ARIAs, only 
>after 3months developed a "dirty" flow cell which had to be replaced. I 
>was stunned, aghast, amazed as my cleaning protocol is quite stringent. 
>Quickly, it entailed running 1% bleach solution for 10-15minutes before 
>and after a sort or analysis. Followed by 10mins of millipore filtered 
>water. I then shut the instrument down 2x (Instrument shutdown menu) with 
>the last two washes of the flow cell being 1%bleach (2x) the first time 
>and millipore filtered water (2x), for the second shutdown. Post the flow 
>cell replacement I changed the shutdown and post sort cleaning to a 
>detergent (1% Extran solution).
>
>Let's share information...
>
>cheers and beers
>
>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
>							 .***.		 ***
>AIDS Pathogenesis Research Unit	   _ _|\       * | | | *	* | |
>VHPF Flow Cytometry Program Group    /       \    *   * | | | *    * | | |
>Sidney Myer Fund Flow Cytometry Unit  \_.--._/  *	 * | | |  *
>								     O 
> ***	     ***
>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 at burnet.edu.au





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