Explain the Serial Dilution Process - Plate Counts?

Dilution factor is the reciprocal of the dilution used. As number of microorganisms in original sample is not known, the original sample is diluted several times in sterile water blanks to ensure that separate colonies are developed and some colony counts will be in the specified range. The process is called serial dilution. The procedure is shown in Figure. Usually a ten-fold serial dilution is used in the process where 1 ml of the sample is transferred to a 9 ml of the diluent to get 1:10 dilution. 1 ml of this is transferred to another tube of 9 ml of diluent to get 1:100 dilution and so on till the required dilutions are obtained.

Figure: Serial dilution process

It is important that the dilution used should produce a limited number of colonies on plate. When many cells are present, overcrowding of cells may not allow many colonies to develop and may also result in many colonies to fuse. This leads to inaccuracies in count. Similarly, too less colonies are not statistically significant. According to Food and Drug Administration, only those plates having 25-250 colonies should be considered for calculation; but most microbiologists use plates with 30-300 colonies to get the microbial numbers in the sample. Plates getting colonies less than 30 are marked too less to count (TLTC) and getting more than 300 are marked as too numerous to count (TNTC) and are discarded.