Viability and Flourescence

goo at epix.net goo at epix.net
Tue Jul 22 22:32:57 EST 1997

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 

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

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