Direct counting techniques do not rely on cell population
growth. The oldest technique is microscopy where the individual cells are
magnified to become visible to the naked eye. This procedure has developed from
the original brightfield techniques through staining and phase-contrast
More recently epifluorescence and immunofluorescence adaptations of cell
labelling are used in conjunction with cytometry e.g. laser scanning/flow
cytometry. In both technologies the trigger for a count is derived from single
cells. Digital microbiology! These counts may also be confirmed visually using
Culture techniques rely on the growth of a microbial population to levels that
are visible. This is achieved under specific conditions, e.g. time,
temperature, oxygen content and pressure in liquid or on solid media containing
specified nutrients. On solid media after incubation, the resultant colonies
may be counted.
This technique assumes that each colony is derived from an individual cell and
that the incubation conditions allowed the recovery of all cells present.
Despite these limitations, plate count techniques remain the 'gold standard' in
microbiology. Sensitivity may be improved with the use of filtration and labour
can be reduced by the use of ready-to-use media.
Serial dilutions can be avoided by the use of spiral platers or other automated
plating techniques. The task of counting the colonies present can be achieved
with the use of automated plate readers or image analysers. Counts may also be
achieved in liquid media using Most Probable Number (MPN) techniques.
Reporter assays assess the microbial population size through the metabolic
activity of the cells. The population does not necessarily have to be growing.
Such techniques include colorimetry; impedance/conductance; ATP (adenosine
triphosphate) and turbidometry.
Optical systems such as colorimetry measure the colour changes in dyes added to
the growth medium as they are metabolized by microorganisms. The technique can
be correlated to colony forming units (cfu), such as with the BioLumix
Turbidimetric systems also rely on changes in the optical characteristics of
the growth medium. In this case, changes in the microbial cell population are
monitored by measuring the opacity of the growth medium, which will become more
opaque as the cell numbers increase.
Microbial metabolism produces highly charged extra-cellular end products,
changing the impedance/conductance of the growth medium. It is this change in
electrical activity that is utilized in these techniques to report on the
growth characteristics of microbial populations.
Intra-cellular molecules can also be utilized in reporter assays. The
bioluminescent reaction produces light in direct proportion to the level of ATP
present. Microbial ATP can be specifically targeted by differential extraction
to report on the loading of microbes in a variety of product and environmental
samples. In practical use ATP sytems are usually used as a screening
[presence/absence] test rather than as a counting device.