epifluorescence microscopy

John Hegarty goo at epix.net
Mon Mar 30 23:16:10 EST 1998

ANTONIO H T MACHADO <ahmachado at ip.pt> wrote: 
> hi, i'm a marine biology student and i have a paper to do on marine
> microbiology, and i need to know why the direct-count method using a
> fluorescent dye and the epifluorescent microscope to count aquatic 
> bacteria has been widely adopted, this is, why the use of 
> epifluorescence microscopy in marine microbiology?
> which are its advantages? why is it so commonly used?

re:  concerning use of  acridine orange and DAPI staining for viability

The use of Acridine Orange (AO) for viability counts is an issue which has
bounced around in the scientific community for yer\ars.  Fluorescence of a
cell culture when stained with AO varies depending on the ratio of DNA to
RNA.  Therefore, some researchers have suggested that the color that a cell
stains reflects its physiological state.  The problem which this assumption
is that staining properties depend on the staining method, particularly if
a destaining technique is used.  In addition, the relationship between cell
color and physiological state applies to pure cultures, grown in batch
culture only.  Since marine samples (of which you are interested) represent
mixed cultures of orgainisms with different cell permeabilities each, there
is a general concensus that AO and DAPI should not be used for viability
determination in natural samples.

There are some techniques which can be used to address this issue. 
Tetrazolium salts can functions as alternate electron acceptors and
therefore may be used as a viable stain.  The two most commonly used
tetrazolium salts are INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl
tetrazolium chloride) 
and CTC (5-cyano-2,3-ditolyl tetrazolium chloride).  In general these are
reduced (and hence form colored formazan crystals deposited inside the
cell) by respiring organisms.  A second approach to viable counts is the
use of dyes which indicate membrane pemeability and potential.  A third
approach involved the use of naladixic acid for DFC (direct viable counts).

Suggested reference:
Smith, JJ. Howington, JP., McFeters, GA. (1994) Survival, physiological
response and recovery of enteric bacteria exposed to a polar marine
environment. Applied Environmental Microbiology Aug;60(8):2977-2984

Katherine H. Baker Ph.D.
Environmental Microbiology
Penn State University
khb4 at psu.edu

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