An important tool to investigate bacterial communities is the most probable number (MPN) technique. The MPN technique can be used on a wide variety of samples for example soils, water, food and agricultural products. The methodology of the MPN technique is dilution and incubation of replicated cultures across several serial dilution steps in a liquid substrate. For the most diluted culture that displays actively growing cells, the number of cells needed to promote growth can be calculated and this can be converted to a measure of viable cells in the original sample.
Dilution Culture is suitable to use for cultivation of bacteria from natural aquatic samples. Cultures are diluted in two fold steps from the original samples to nearly extinction in sterile seawater depending on source. The dilutions are made in 96-well plates; growth is determined by epifluorescence microscopy and spotting on agar plates.
The use of two systems for the detection of bacterial growth enables one to distinguish both colony forming and non-colony forming bacteria. When using the MPN technique for testing the viability of bacteria in seawater the results are striking: MPN almost always indicates a higher number of viable cells than colony forming units (CFU) which distinctly shows that agar plate-based enumeration underestimates the number of live cells. This has been known for a long time as the great plate anomaly.
Picture shows SYBR GOLD stained bacteria detected with epifluorescence microscopy, sample originating from the Baltic Sea.