Epiflourescence Microscopy

Aquatic Environment Microbiology
Part 2
Atmospheric Waters
Surface Waters
Ground Waters
Stored Water
Water Cycle
Study of Aquatic Microorganisms
Determination of Bacterial Numbers
Total Viable Count
Total Cell Count
Determination of Bacterial Mass
Direct Methods
Indirect Methods
Flow Cytometry
Epiflourescence Microscopy
Microbial Communities in Aquatic Environments
Neuston
Pleuston
Plankton
Phytoplankton

Epiflourescence Microscopy

Epiflourescence Microscopy- Direct total counts of bacteria from water samples can be routinely obtained using epifluorescence microscopy. The technique employs fluorescent stains or fIuorochorome stains.

The most commonly used fluorochrome - is acridine orange. The fluorochrome which is becoming popular nowadays is 4', 6' diamidino - 2 - phenyl indole (DAPI) (King & Parker, 1988). When using fluorescence techniques, the sample is either stained and then filtered onto membrane filters or the cells are stained on the filters.

Fluorochrome - stained - cells counts give an estimate of total living heterotrophic bacterial population in aquatic environments. However, a large proportion of the total cells are in a metabolically inactive state. To get an estimate of metabolically active cells a dehydrogenase stain assay can be employed. In this method, the active dehydrogenase enzyme reduces a synthetic water soluble, membrane permeable, straw colored tetrazolium salt converting it to a pink-red, water insoluble formozan.

The proportion of cells that have accumulated pink formozan through enzymatic dehydrogenation of tetrazolium substrate represents metabolically active cells.

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