On Tue, 22 Jul 1997 21:53:31 -0700, Simon Geraghty
<sgeragty at ozemail.com.au> wrote:
>I need a rapid microscopic method for determining gram positive cell
>viability. Apparently flourescence microscopy can be used for this. Has
>anyone had any experience with this type of method or are there any
>references?
I recommend using one of the following tetrazolium salts:
(TTC) Triphenyl tetrazolium chloride
(INT) 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium
(CTC) 5-cyano-2,3-ditolyl tetrazolium chloride
These compounds, acting as alternate electron acceptors,
are reduced and fluoresce in the presence of cellular respiration.
The technique is applicable to a wide range of cell types.
P.S. optionally, you can enhance detection of metabolic activity
by adding an appropriate subsrate, (i.e. pyruvate, succinate).
Some references:
Pyle BH, Broadaway SC, McFeters GA. A rapid, direct method for
enumerating respiring enterohemorrhagic Escherichia coli O157:H7
in water. Applied Environmental Microbiology 1995 Jul;61(7):2614-2619
(Gordon McFeters has published several excellent papers in this area)
Gribbon LT, Barer MR. Oxidative metabolism in nonculturable
Helicobacter pylori and Vibrio vulnificus cells studied by
substrate-enhanced tetrazolium reduction and digital image
processing. Applied Environmental Microbiology 1995
Sep;61(9):3379-3384. (Used substrate enhanced INT)
Silva MT, Sousa JC, Polonia JJ, Macedo PM.
Effects of local anesthetics on bacterial cells.
Journal of Bacteriology 1979 Jan;137(1):461-468
(Used TTC with gram positives: Bacillus cereus,
B. megaterium, B. subtilis, and Streptococcus faecalis)
best wishes,
John Hegarty
Environmental Microbiology/Penn State University
M.Sc. Candidate
Email: goo at epix.net
