Hi Andrew I do a lot of microbiological measurements on our EPICS ELITE. On our slightly modified instrument we have actually a light scatter detection to go down to approximately 300nm latex beads on narrow forward angle light scatter and 190 nm latex beads on side scatter to cluster separate from the background. I remember when Stefan Andreatta from Innsbruck copied on this mail (who actually now uses a MoFlo) came to see me in 1998 to discuss microbial cytometry. He brought some water samples to try out on the Elite and we found a couple of clusters that did only light up with the nucleic acid stains but remained below our light scatter detection limit which could have probably been virus particles. However we also found a couple of clusters that had distinct light scatter characteristics but did not take up the DNA stains. As we did neither fix nor sort the cells at the time we could not conclude what they were, but it emphasises the importance on multi-parameter triggering. I had similar experiences with elementary bodies of chlamydia who also had insufficient light scatter signal, but they did not stain for DNA either. However, their fluorescence from the antibody staining was giving very good clustering, only detected by using multi channel trigger or threshold signals. In fact the antibody staining shows very nice clustering of multiple intensity units indicating either aggregation or actually size variation and can be used for very accurate enumeration. Staining was achieved in a small volume (10ul culture + 10ul directly labelled anti-MOMP-FITC) followed by a dilution in 2ml buffer. Hope the information is of use Gerhard Nebe-von-Caron Research Scientist Applied Science & Technology Group SEAC - Safety and Environmental Assurance Centre Unilever Research, Colworth Laboratory, Sharnbrook, UK - MK44 1LQ Tel: +44 (0)1234 264822, Fax: +44 (0)1234 222552 E- mailto:Gerhard.Nebe-von-Caron@unilever.com PS Hi Stefan Vielen Dank noch mal fuer das Pedderson Buch. Sebastian (jetzt 10 1/2) hat gerade vor ein paar Tagen deinen Brief aus dem Buch gefischt. Aus dem Rueckbesuch ist ja leider immer noch nichts geworden. -----Original Message----- From: Andrew Beernink [SMTP:ABeernink@novasite.com] Sent: Wednesday, October 24, 2001 5:36 PM To: Cytometry Mailing List Subject: Hi guys & gals- Has anybody sorted virus? Any suggestions re: 1) safety, and ) labelling/staining techniques? I'm assuming you'd trigger off a fluorescence signal (Ab label), with a Hoe342 or other xNA "counterstain" to discount drops with multiple particles. This would be done on a MoFlo (488, I70 Spectrum, I302), if it's doable! Thanks for any input! Andrew Andrew Beernink Research Scientist - Flow Cytometry Novasite Pharmaceuticals, Inc. 3520 Dunhill St. San Diego, CA 92121 (858) 638-8584 (858) 597-4943 fax
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