Single dilution test

The single dilution test is more used for the examination of samples with a low level of contamination, for which there is no reason to inoculate dilutions. One of its main applications is the enumeration of coliforms in drinking water and in fruit juices and carbonated soft drinks.

The standard procedure consists in inoculating five or ten 10 ml aliquots of liquid samples in 10 ml of double-strength culture medium (50 to 100 ml in all), or five or ten 10 ml aliquots of the first dilution of solid samples in 10 ml of double-strength culture medium 5) to 10 g in all). These quantities may vary depending on the expected level of contamination of the sample. For example, in the case of samples with lower levels of contamination, five or ten 100 ml portions may be inoculated in 100 ml of double-strength broth, whereas in the case of samples with higher contamination levels, five or ten 1 ml portions may be inoculated in 10 ml of single-strength broth. The inoculation of five or ten aliquots with amounts multiples of ten have the advantage of having the results tabulated in  MPN Tables. However, any number of aliquots, of any quantity, may be inoculated, provided they are all equal or identical and that the dilution corresponds to one part of sample added to ten parts of broth. In these cases, calculating the results requires formulas instead of tables.

Exactly like with the multiple dilution test, the selection of the culture medium and the way in which the presence of the target microorganism(s) in the inoculated aliquots is to be verified are described in the corresponding specific chapters. Depending on the complexity of the subsequent confirmation tests, the format of five aliquots may be more advantageous for samples with low contamination than that of ten aliquots or the multiple dilution test.

1-Material required for the analyses

-   Materials for preparing the sample.

-  Culture media recommended for the tests to be carried out.

- Laboratory incubator(s) or temperature-controlled water baths set to the temperature(s) specified by the test to be performed, as described in the specific chapters.

2-  Procedure

Properly identify all the tubes that will be inoculated by labeling them with the sample code and the standard abbreviation of the culture medium.

a)  Preparation of the samples.

b) Inoculation. Following the instructions and guide-lines described above, select the amounts of sample most appropriate for inoculation. Inoculate five or ten aliquots of the sample in five or ten tubes or flasks containing culture broth. Add one part of the aliquot to ten parts of broth. Select the broth in accordance with the test to be carried out (described in the corresponding specific chapters). The uncertainty of the volume measurements should not exceed 5% (ISO 6887-1:1999). Change the position of the tubes or flasks as they are being inoculated, to avoid the risk of inoculating the same tube/flask more than one time, or to leave a tube/flask un-inoculated.

c) Incubation. Incubate the tubes under the conditions specified for the test.

d) Verification of the presence of the target microorganism(s) in the tubes. Follow the same instructions and guidelines as those provided for the multiple dilution test.

e) Transfers. Follow the same instructions and guide-lines as those provided for the multiple dilution test.

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 .

International Organization for Standardization (1999) ISO 6887-1:1999.  Microbiology of food and animal feeding stuffs – Prepa-ration of test samples, initial suspension and decimal dilutions for microbiological examination – Part 1: General rules for the prepara-tion of the initial suspension and decimal dilutions. Geneva, ISO.