Staining using DNA-specific fluorochromes, such as N,N, N′,N′-tetramethylacridine-3,6-diamine (acridine orange) or 4′,6- diamidino-2-phenylindole (DAPI), followed by epifluorescence microscopy, has been used to count total microbial cells in aquatic samples. Other fluorochromes, such as 5-cyano-2,3-ditolyl tetrazolium (CTC) or SYTO 9/propidium iodide, can be used for viable or total bacteria cell counts.
Direct total cell counts of bacteria in water or wastewater usually exceed counts obtained via heterotrophic plate counts (HPC) and most probable number (MPN) methods because they preclude the errors caused by viability-related phenomena, such as selectivity of growth media, cell clumping, and slow growth rates or errors inherent to the methods. Some water samples might contain large amounts of dead or nonculturable cells.
The method described in this section uses acridine orange as the staining agent.