Introduction to Basic techniques for microbial enumeration by the most probable number method ( MPN)

The Most Probable Number technique ( MPN) is a quantitative method of analysis that allows determining the MPN of the target microorganism(s) in a sample, by inoculating aliquots of that sample into a series of tubes containing a liquid culture medium appropriate to its growth. The determination of the number of microorganisms is based on the principle that, by subdividing the sample in aliquots, some of the aliquots will contain microorganisms and others will not, depending on the quantity of microorganisms in the sample. The number of aliquots with microorganisms (tubes with positive growth after incubation) and aliquots without micro-organisms (tubes with negative growth after incubation) allow estimating, by probability calculations, the original density of the microorganisms in the sample. This application of the probability theory relies on the assumption that the microorganisms are randomly and homogeneously distributed throughout the entire sample. In the case of liquid samples, this condition may be readily achieved by the careful agitation of the material. In the case of solid samples, it may be achieved by preparing and homogenizing the first dilution and taking aliquots directly from this first dilution. There are situations in which the aliquots of solid samples are inoculated directly into culture broth. Direct inoculation of solid samples is, however, a less common practice and depends on the type of sample.

Since inoculation is done directly into liquid media, the MPN technique has several advantages over the standard plate count method. One of these advantages is the possibility to inoculate greater amounts of the sample by proportionally increasing the volume of culture medium. This gives the technique a significantly higher sensitivity as compared to the plate count method and also increased flexibility to establish the detection limit. Another advantage is that the MPN technique enables the introduction of injury recovery steps, using a non-selective medium for initial inoculation, more favorable to injured microorganisms, allowing for the recovered culture to be transferred to selective media later on. The MPN technique is quite versatile and allows the enumeration of different groups or species of microorganisms by varying the culture medium and incubation conditions. Its main applications are the counts of total coliforms, fecal coliforms and E. coli in water and foods. The technique can also be used in other quantitative microbiological examinations, when the expected contamination of the sample is lower than the detection limit of plating methods or when particles of the food or contained in the food to be examined interfere with the enumeration of colonies in plate counts. Another application is the possibility to adapt and transform qualitative methods into quantitative methods, such as the counts of Salmonella and other microorganisms that are traditionally examined using presence/absence methods.

As for inoculation, the MPN technique can be performed using one of two formats, depending on how the aliquots are distributed. One is the format of the multiple dilution test, in which three or five aliquots of a dilution are inoculated in a series of three or five tubes and, later, in a new series of three or five aliquots of the subsequent dilution, are inoculated in another series of tubes and so forth. The higher the number of dilutions and the number of tubes per dilution, the greater the accuracy of the count. For most of the situations commonly encountered in the micro-biological examination of foods, three dilutions with three tubes per dilution are sufficient to obtain a good estimate of the MPN. The other format is the single dilution test, in which all inoculated aliquots (generally five to ten) are of the same dilution, containing an equal amount of sample.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .