Re: Bacteria and flow cytometry
/G=Gerhard/S=Nebe-von-Caron/OU=1890CHPE/O=TMGB.URC/@LANGATE.gb.sprint.com
Fri, 17 Nov 1995 04:46:00 -0500
Depends very much on your staining conditions. Never add the
stains whilst heatstressing or sonicating as that perturbs
the membrane. You should also avoid solvents like DMSO.
Above 0.05% they can lead to similar problems. I therefore
recommended Molecular Probes to sell their kit as powder
allowing to adjust the concentrations accordingly.
Unfortunatelly their dyes are not stable in aqueus
solutions.
CFDA is well known to stick to dead cells as well,
particular in yeasts where it seem to accumulate in
vacuoles. The same can be true for membrane potential dyes
depending on their concentration. If you add to much they
stain live and dead bugs.
Using CFDA and PI on Lactobacillus plantarum we also get
double positives, most of them actually cell aggregates.
There is also a dim PI stain especially after heat treatment
which is probably staining occuring outside the cell
membrane that is probably nonspecific. You can also get it
with starvation and it occurs before the cell is
depolarized.
Gerhard Nebe-v.Caron
Unilever Research, Colworth Laboratory Sharnbrook,
Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
E.mail: gerhard.nebe-von-caron@urcgb.sprint.com
______________________________ Reply Separator _________________________________
Subject: Bacteria and flow cytometry
Author: harmane@phibred.com at INTERNET
Date: 16/11/95 20:19
We are working with bacteria with a special interest in viability staining.
We have used Rhodamine123, CFDA, PI and Molecular Probe's viability kit
called LIVE/DEAD BacLight.
An interesting phenomenon that we initially noticed with fluorescent
microscopy and later confirmed with flow cytometry is the presence of a
population which stains a color that has properties of both live and dead
stains. We find that as cell cultures are stressed (chemical, heat,
radiation) the level of these 'intermediate' colored cells increases. We
have found that there seems to be a correlation between the relative amounts
of stress and the population of intermediate colored cells.
This has been shown with a number of different genera and species. There does
seem to be some differences in the amount of the intermediate population with
different cultures, but their presence seems to be universal.
If there are other researchers out there doing work like this and can offer
any theories or explanations for this phenomenon, we would very interested in
hearing from you.
Bill Rutherford (Rutherfor@phibred.com)
Beth Harman (Harmane@phibred.com)
Lyse Norian (Norianla@phibred.com)
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
