Counts of microorganisms may be made by direct microscopic techniques or through indirect methods such as culturing.
11.5.1 Direct Counts
In some samples, microorganisms can simply be counted under the microscope. Counting chambers—specialized microscope slides with wells holding a fixed volume—are available for this purpose. Some, such as the Petroff-Hauser cell (Figure 11.9), also have a grid marked on them. An example of a chamber with a deeper well used for larger organisms or associations such as filaments or floc is the Sedgwick-Rafter cell (Figure 11.10); it has an area of 1000 mm2 and a depth of 1 mm, thus holding a volume of 1.0 mL. Fresh live samples are used when possible, especially for protozoa and metazoa, so that activity such as motility or feeding can be observed.
Alternatively, a fixed volume (usually 1 drop, or about 0.05 mL = 50 mL) can be added to a regular slide, and a coverslip (usually square, 22 to 25 mm on a side) placed over it (spreading the sample under the coverslip). The number of cells seen in one field (the visible area seen through the eyepiece, usually about 1.6 mm in diameter at 100 x and 0.16 mm at 1000 x) under the microscope is then multiplied by the ratio of the area of the coverslip to the field area to get the concentration (count per volume).
Example 11.2 An average of 3.2 ciliated protozoans were seen per field when 10 fields were counted at 100 x. If the sample size was ^0.05 mL, the microscope field at that magnification is 1.6 mm in diameter, and a 22 x 22 mm coverslip was used, what is the concentration of ciliates present?
Answer Area of field Af = pr2 = p(1.6/2mm)2 = 2.01 mm2/field Area under coverslip Ac = 22 mm x 22 mm = 484 mm2/coverslip
Conc. of ciliates = 3.2/field x-r---0.05 mL/coverslip
For observation of highly motile protozoa, it is sometimes desirable to slow down their activity. This can be done by adding a compound such as methylcellulose to increase viscosity, or by using an inhibitory compound such as nickel sulfate.
Although small metazoa such as rotifers and nematodes can often be enumerated along with the protozoa, larger forms may require specialized techniques. Some, in fact, may actively avoid being drawn into a small sampling or subsampling device, such as a pipette. Others, as well as larger associations of prokaryotes such as large floc, may simply not fit through the opening of fine tip pipettes.
Because of their silica shells, a special technique can be used for diatoms. After washing of the sample with distilled water, organic matter is destroyed by heat or an acid-oxidation step, leaving the shells for counting.
Note that using approximations from the above example, 5000 cells/mL (500 mm2/ 2 mm2 divided by 0.05 mL) would be needed in a sample for there to be one cell on average per microscope field at 100 x. For prokaryotic cells, which are usually too small to count at low magnification, concentrations of above 106mL_1 (2 per field at 1000 x) are usually necessary for direct counting. If there are too many cells to count (>108mL~1), the sample might be diluted first. If the sample contains too few microorganisms, filtration might then be used to concentrate them on a filter surface for viewing.
However, even with dilution or concentration, this simple counting approach is limited to samples with little extraneous material that would prevent clear viewing and that contain individual, dispersed cells rather than cells in flocs or biofilms. To allow direct counting in samples such as activated sludge (flocculated), biofilms, and soils, a variety of stains and probes have been developed, including many that are fluorescent. These allow differentiation between inert particles and cells, between living and dead cells, or even among specific strains of organisms.
One such approach uses a diacetate ester of fluorescein, which is able to pass into the cytoplasm of cells. Once inside, the ester is rapidly hydrolyzed by nonspecific esterase enzymes. The free fluorescein (which, as its name suggests, is fluorescent) that is released is trapped in the cell, thereby making it readily visible with a fluorescence microscope.
The acridine orange direct count (AODC) uses another fluorescent dye that passes into the cytoplasm and then binds to nucleic acids, giving an orange or green color. This does not require the activity of enzymes within the cell and thus may give a higher, total count, including both viable and nonviable (dead) cells.
One problem with the AODC method is that clay particles also may appear orangish, potentially interfering with cell counts. This has contributed to the increased popularity of a blue fluorescent dye, 4',6-diamido-2-phenylindole (DAPI), which binds more specifically with DNA.
If antibodies to the surface of a specific organism can be produced, the fluorescent antibody technique can be used. In this method the antibody is conjugated with (chemically attached to) a fluorescent dye. When this preparation is added to the sample, it attaches to the cell so that the surface of the target organisms fluoresce.
Genetic probes can also be used to stain specific groups of organisms for counting. The fluorescent in situ hybridization (FISH) technique (Section 10.4.4) is one popular example.
Another alternative is the use of metabolic stains. These typically utilize a dye that is reduced by (accepts electrons from) a cell's metabolic activities to form a colored product. A number of tetrazolium dyes such as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tet-razolium chloride (INT), for example, are capable of passing through the cell membrane and then accepting electrons by way of dehydrogenase enzymes. The result is conversion to dark red to deep-purple formazan crystals that are trapped within the cell. Thus, a count of respiring cells can be made. The formazan can also be eluted with isopropyl alcohol and an indirect measure of cell concentration (number or mass) made from a spectro-photometric measurement of the intensity of the color released.
Filamentous bacteria (including actinomycetes, many cyanobacteria, and some gram negatives, such as Sphaerotilus and Beggiatoa), fungi, and algae can pose a problem in quantification in that a single "count" might be composed of only one cell a few micrometers long or might contain many cells extending to a length of 1 mm or more. In some cases the number of cells in the filament may be counted or the filament length measured, perhaps for conversion to a biomass estimate (similar to the calculations of Section 11.5.3). Similarly, counts of colonial protozoa and algae may require some enumeration of individuals or a measure of the size of the colony. For bacterial associations such as floc, individual counts may be impossible, but the size of the association may be measured.
Image analysis is a tool that can be combined with several of the techniques described above. A digital image of the microscope field is captured and can then be analyzed using computer software to pick out and quantify specific size (including filament length or floc size), shape, and/or color (including from stains and fluorescence) objects. This information can then be provided as total or differential counts, and can also be used to estimate cell volume (and hence mass; Section 11.5.3).
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