Measurement of the Number of Viable Microbes on Solid Growth Media

This approach consists of measuring the number of viable cells capable of forming colonies on a suitable growth medium. Plate count is determined by using the pour plate method (0.1–1 mL of microbial suspension is mixed with molten agar medium in a petri dish), or the spread plate method (0.1 mL of bacterial suspension is spread on the surface of an agar plate). The results of plate counts are expressed as colony forming units (CFU). The number of CFU per plate should be between 30 and 300. Membrane filters can also be used to determine microbial numbers in dilute samples. The sample is filtered and the filter is placed directly on a suitable growth medium.

                                     

Culture-based methods have been routinely used in soil, aquatic, and wastewater microbiology, but they reveal only about 0.1–10 percent of the total bacterial counts in most environments (Pickup, 1991). Indeed, some microorganisms (e.g., E. coli, Salmonella typhimurium, Vibrio spp.) can enter into the viable but nonculturable (VBNC) state and are not detected by plate counts, especially when using selective growth media (Koch, 2002; Roszak and Colwell, 1987). The VBNC state can be triggered by factors such as nutrient deprivation or exposure to toxic chemicals. This phenomenon is particularly important for pathogens that may remain viable in the VBNC state for longer periods of time than previously thought. The VBNC pathogens may remain virulent and cause disease in humans and animals.

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.

Plasmids

A plasmid is a circular extrachromosomal circular DNA containing from 1000 to 200,000 bp, and reproducing independent from the chromosomal DNA. Plasmids are inherited by daughter cells after cell division. Plasmid replication can be inhibited by curing the cells with compounds such as ethidium bromide. Some of the plasmids may exist in a limited number (1–3) of copies (stringent plasmids) or relatively large number (10–220) of copies (relaxed plasmids).