Cell counting by flow cytometry

Dear All,

We are currently attempting to count Haloferax cells, an extreme Archaeal
halophile from the Dead Sea, in order to determine cell numbers in varying
environmental conditions. This organism is tolerant of elevated magnesium
chloride concentrations, living in a saline solution of 2.1M NaCl, 0.4M other
salts and the MgCl2 range 10mM - 1.5M.

Currently we have stained our cells using Sybr Gold and have viewed a stable
population of cells in the flow cytometer. However we have not been able to get
a definitive cell count as the counting beads agglomerate in the high salt
medium. We intend to use flow rate to determine absolute cell number unless we
can find some beads which work under these conditions.

We would be grateful for any advice that you can give, especially in answering
these questions;

1. Are there any methods other than the addition of SDS to prevent bead
agglomeration - are there beads that do not agglomorate in saline solutions?

2. Are there any other recommended stains which are better than Sybr Gold for
small bacterial/archaeal cells?

3. Do people still use a calibrated flow rate to count absolute numbers of
cells?

-- 
Sue Clark
Flow Cytometry Technician
Institute for Cancer Studies.
Division of Genomic Medicine.
University of Sheffield.
G Floor. Medical School.
Beech Hill Road
Sheffield
S10 2RX

Tel 0114 271 3023/271 2968