Environmental Microbiology of Aquatic and Waste Systems

2.5 Methods for the Enumeration of Microorganisms in the Aquatic Environment 25

bac terial cells are captured on the surface of poly-

carbonate membrane filters, stained with a fluores-

cent dye such as acridine orange, and visualized

using epifluorescence microscopy. The fluoro-

chrome (fluorescent dye) which is currently popu-

lar is 4¢, 6¢ diamidino-2-phenyl indole (DAPI).

When using fluorescence techniques, the sample is

either stained and then filtered onto membrane fil-

ters, or the cells are stained on the filters.

Fluorochrome – stained – cells counts give an esti-

mate of total living heterotrophic bacterial popula-

tion in aquatic environments. However, a large

proportion of the total cells are sometimes 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 per-

meable, 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.

It has also been used to characterize planktonic

procaryotic populations. An image analysis system

may be used to digitize the video image of auto-

fluorescing or fluorochrome-stained cells in the

microscope field. The digitized image can then be

stored, edited, and analyzed for total count or indi-

vidual cell size and shape parameters, and results

can be printed as raw data, statistical summaries, or

histograms (Sieracki et al. 1985 ).

3. The confocal laser scanning microscope
   The confocal laser scanning microscope (CLSM or
   LSCM) is now often used in biology (Prasad et al.
   2007 ).
   It is called “confocal” (meaning the same focus)
   because the final image has the same focus as the
   point of focus in the object. When an object is
   imaged in the fluorescence microscope, the signal
   produced is from the full thickness of the specimen;
   on account of this, most of the image is out of focus
   to the observer. The pinhole aperture of the confo-
   cal microscope blocks out this out-of-focus light
   (dotted lights in Fig. 2.4) and thus light from above
   and below the point of focus in the object. Filtering
   away some of the light reduces the amount of light
   and thus also reduces the “visibility” of the focused
   part of the specimen. To make up for this, laser
   beams are used which produce extremely bright
   light at a fixed wavelength. Highly sensitive photo-
   multiplier-detectors (PMTs) are used to pick up and
   multiply the reduced laser beam striking the image.
   The laser is focused on a fixed portion of the speci-
   men (a square or rectangle) at a time using a

Objective

Emission / Barrier

Filter

Fluorescence Light

Excitation to Eyepieces

Filter

1

2

3

6

4

5

Excitation

Lamp

Lamp

Housing

Mirror

Collector

Lens

Microscope Slide

with Sample

Dichroic

Mirror Key

Emitted

Light Rays

Excitation

Light Rays

Fig. 2.3 Setup illustrating the principle of the epifluorescence microscope (From http://international.abbottmolecular.com/
DiagramoftheFluorescenceMicroscope_8843.aspx. With permission)