Serial decimal dilution of the sample

The preparation and inoculation of serial dilutions of the sample are required for quantitative tests, to reduce the number of microorganisms per unit of volume, and make it possible to count them. This series of dilutions is generally decimal or ten-fold for ease of calculation of final results.

The number of dilutions necessary depends on the expected level of contamination and should be such as to allow for, in plate counts, obtaining plates with numbers of colonies varying between 25–30 and 250–300 or between 15 and 150 in yeast and mold counts. In counts by the Most Probable Number Method (MPN) the number of dilutions must allow for obtaining positive tubes at the lowest dilutions and negatives tubes at the highest dilutions.

According to the general procedure described by the Compendium (Swanson et al., 2001), the second dilution is to be initiated immediately upon completion of the first dilution. The duration of the complete procedure, from the preparation of the first dilution until inoculation of all culture media, should not exceed 15 minutes.

According to the general procedure described by ISO 6887-1:1999, the duration of the complete procedure should not exceed 45 minutes and the time interval between the end of the preparation of the first dilution and the beginning of the second and subsequent dilutions should not exceed 30 minutes (except when specified in specific procedures).

For dehydrated or dried foods (except for milk powder, egg powder, and live yeast powder) ISO 6887-4:2003/Cor.1:2004 recommends a resuscitation step before preparing the second dilution. In general, leave the sample to rest for about 30 ± 5 min at laboratory temperature. Do not exceed a temperature of 25°C before preparation of further dilutions. In all cases in which volumes are transferred, the uncertainty of the measurement must not exceed 5%).

How to prepare the second  dilution (10⁻²): Transfer

aseptically 1 ml of the first dilution (10⁻¹) to 9 ml diluent. The diluents are the same as those recommended for the first dilution. In the second dilution there are no special cases in which a different diluent is required from the one used to prepare the first dilution.

Do not dip the tip of the pipette to a depth of more than 1 cm when pipetting the volume from the first to the second dilution. If the first dilution does not contain suspended particles, the material may be agitated before transferring the volume from the first to the second dilution. If there are suspended particles, ISO 6887-1:1999 recommends not to agitate and wait until the suspended particles settle to the bot-tom before transferring the volume. In the case of viscous samples, which adhere to the internal wall of the pipette, ISO 6887-5:2010 recommends dispensing the volume and subsequently wash the pipette with diluent (by aspirating several times) to ensure that all the material be transferred to the second dilution.

How to prepare subsequent  dilutions: Transfer 1 ml of the previous dilution to 9 ml diluent. Before withdrawing the volume to be transferred, agitate the tube vigorously, inverting it 25 times in a 30-cm arc (within 7 s) or using a laboratory vortex mixer (15 s).

References

SILVA, N.D .; TANIWAKI, M.H. ; JUNQUEIRA, V.C.A.;  SILVEIRA, N.F.A. , NASCIMENTO , M.D.D. and GOMES ,R.A.R .(2013) . MICROBIOLOGICAL EXAMINATION METHODS OF FOOD AND WATE A Laboratory Manual . Institute of Food Technology – ITAL, Campinas, SP, Brazil.

Midura, T.F. & Bryant, R.G. (2001) Sampling plans, sample col-lection, shipment, and preparation for analysis. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 2, pp. 13–23.

Swanson, K.M.J, Petran, R.L. & Hanlin, J.H. (2001) Culture methods for enumeration of microrganisms. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 6, pp. 53–67.