Total Number of Microbial Cells

Total number of cells (live and dead cells) can be measured by using special counting chambers such as the Petroff–Hauser chamber for bacterial counts or the Sedgewick–Rafter chamber for algal counts. The use of a phase-contrast microscope is required when nonphotosynthetic microorganisms are under consideration. Presently, the most popular method consists of retaining the cells on a membrane filter treated to suppress autofluorescence (use of polycarbonate filters treated with Irgalan Black) and staining the cells with fluorochromes such as acridine orange (AO) or 40,6-diamidino-2-phenylindol (DAPI). The microorganisms are subsequently counted using an epifluorescence microscope (Kepner and Pratt, 1994).

An advantage of DAPI is its stable fluorescence. A wide range of other fluorochromes are available for many applications in environmental microbiology studies. These include, among others, PicoGreen, SYBR-Green 1 and 2, Hoechst 33342, YOYO-1, and SYTO dyes (green, red, and blue) (Neu and Lawrence, 2002).

Scanning electron microscopy (SEM) has also been considered for measuring total microbial numbers. Electronic particle counters are also used for determining the total number of microorganisms in a sample. These instruments do not differentiate, however, between live and dead microorganisms, and very small cells may be missed. Flow cytometers are fluorescence-activated cell sorters and include a light source (argon laser or a mercury lamp) and a photodetector, which measures fluorescence (use correct excitation wavelength) and scattering of the cells. They sort and collect cells with predefined optical parameters. They are often used in the biomedical and aquatic microbiology fields (Paul, 1993). They have been used to sort algal cells and to distinguish between cyanobacteria from other algae, based on phycoerythrin (orange) and chlorophyll (red) fluorescence. They can help identify microorganisms when combined with fluorescent antibodies.