Methodologies Used in Microbiological Tests
HNU-WATERSS Laboratory
Because the analysis is always based on a very small sample taken from a very large volume of water, all methods rely on statistical principles.
Multiple tube method
One of then oldest methods is called the multiple tube method. In this method a measured sub-sample (perhaps 10ml) is diluted with 100ml of sterile growth medium and an aliquot of 10ml is then decanted into each of ten tubes. The remaining 10ml is then diluted again and the process repeated. At the end of 5 dilutions this produces 50 tubes covering the dilution range of 1:10 through to 1: 10000. The tubes are then incubated at a pre-set temperature for a specified time and at the end of the process the number of tubes with growth in is counted for each dilution. Statistical tables are then used to derive the concentration of organisms in the original sample. This method can be enhanced by using indicator medium which changes colour when acid forming species are present and by including a tiny inverted tube in each sample tube. This inverted tube catches any gas produced. The production of gas at 37 Deg Celsius is a strong indication of the presence of Escherichia coli
Plate count
The plate count method relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 10 and 100 colonies of the target bacterium are grown. Fewer than 10 colonies makes the interpretation statistically unsound whilst greater than 100 colonies often results in overlapping colonies and imprecision in the count. To ensure that an appropriate number of colonies will be generated several dilutions are normally cultured.
The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc.) in sterile water and cultivating these on nutrient agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count gram-negative bacteria such as E. coli. Typically one set of plates is incubated at 22ºC and for 24 hours and a second set at 37ºC for 24 hours. The composition of the nutrient usually includes reagents that resist the growth of non-target organisms and make the target organism easily identified, often by a colour change in the medium. Some recent methods include a fluorescent agent so that counting of the colonies can be automated. At the end of the incubation period the colonies are counted by eye, a procedure that takes a few moments and does not require a microscope as the colonies are typically a few millimetres across.
Membrane filtration
Most modern laboratories use a refinement of total plate count in which serial dilutions of the sample are vacuum filtered through purpose made membrane filters and these filters are themselves laid on nutrient medium within sealed plates. The methodology is otherwise similar to conventional total plate counts. Membranes have a printed millimetre grid printed on and can be reliably count a much greater number of colonies under a binocular microscope.
Pour plates
When the analysis is looking for bacterial species that grow poorly in air, the initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar which is then poured into bottles which are then sealed and laid on their sides to produce a sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after incubation.
The total number of colonies is referred to as the Total Viable Count (TVC). The unit of measurement is cfu/ml (or colony forming units per millilitre) and relates to the original sample. Calculation of this is a multiple of the counted number of colonies multiplied by the dilution used.
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