Preparation of the first dilution of the analytical unit

To proceed with the analysis, the analytical unit must be diluted and homogenized with an adequate diluent, to allow inoculation into or onto culture media. The recommended diluents and initial dilution ratios vary with the type of sample and the type of test that will be performed, as described below:

Diluents for presence/absence tests

These tests are performed with dilution and homogenization directly in enrichment broth.

Diluents for tests requiring differentiated handling of  the sample

Also for these tests the specific chapters should be consulted.

Diluents for general quantification tests

For these tests the following recommendations apply: The Compendium (Midura & Bryant, 2001) recommends, for general use in the examination of foods, 0.1% Peptone Water (PW) or Butterfield’s Phosphate Buffer.

The section 4.030 of the  Standard Methods for the Examination of Dairy Products (Davis & Hickey, 2004( recommends, for general use in the examination of dairy products, Butterfield’s Phosphate Buffer (called Phosphate Dilution Water) or Magnesium Chloride Phosphate Buffer (called Phosphate and Magnesium Chloride Dilution Water).

The Standard Methods for the Examination of Water & Wastewater (Hunt & Rice, 2005) recommends, for gen-eral use in the examination of water samples, 0.1% Pep-tone Water (PW) or Magnesium Chloride Phosphate Buffer (called Buffered Water) .

ISO 6887-1:1999 and ISO 6887-4:2003/Cor.1:2004 recommend, for general use in the examination of foods, Saline Peptone Water (SPW) or Buffered Peptone Water (BPW). ISO 6887-2:2003 and ISO 6887-3:2003 also recommend Saline Peptone Water (SPW) or Buffered Peptone Water (BPW) for general use in the examination of meat and meat products, and fish and fishery products.

ISO 6887-5:2010 recommends, for general use in the examination of milk and dairy products, 0.1% Peptone Water (PW), Buffered Peptone Water (BPW), Saline Peptone Water (SPW), Ringer’s Solution Quarter-Strength or Phosphate Buffered Solution according ISO 6887-5.

 How to prepare an initial 1:10 (10^-1)   dilution

The initial dilution recommended for most samples is 1:10 (10^-1) obtained by adding m grams or milliliters of the sample to 9 × m milliliters of diluent. For example, for 25 g of sample, add 9 × 25 ml of diluent (225 ml.( There are situations in which the diluent and the initial dilution are different.

How to prepare an initial  dilution different from 1:10

There are special situations in which the first dilution is different from 1:10. To determine the volume of dilu-ent necessary to obtain a predetermined 1:k dilution of the sample, use the v = [(k.m) – m] ratio. For example, to obtain a 1:50 dilution of an analytical unit of 10 g, add [(50  × 10)  − 10] milliliters of diluent (490 ml.( To obtain the same dilution for an analytical unit of 20 g, add [(50  × 20)  − 20] milliliters of the diluent980 ml.

Procedure for the preparation of  the first  dilution of  liquid samples

In the case of liquid foods, transfer the analytical unit directly to tubes or flasks containing the amount of diluent necessary for a 1/10 dilution. Homogenize the sample with the diluent by agitation, inverting the container or package 25 times. To allow for perfect homogenization, use tubes or flasks with screw caps. They should be of a size sufficiently great to ensure that no more than 2/3 of their holding capacity is taken up by the analytical unit  + diluent. There are special cases that require a different initial dilution

Procedure for the preparation of  the first  dilution of  solid or concentrated liquid samples

In the case of solid or concentrated liquid foods, transfer the analytical unit to a sterile homogenization flask or bag. Add to the sample the amount of diluent necessary to obtain a 1:10 dilution. Homogenize the analytical unit with the diluent, which can be achieved by manual agitation, shaking the flask in an inverted position 25 times through a 30 cm arc within seven seconds (concentrated liquids, soluble powders), agitation in a peristaltic homogenizer (better known as stomacher( for 1–2 min (soft foods, pasty foods, ground or minced foods, poorly soluble powders) or in a blender (hard foods). In the case of homogenization using a blender, the  Compendium (Midura & Bryant, 2001) recommends using high speed during the first few seconds and low speed (8,000 rpm) for the remaining time, which should not exceed 2 min. If a more prolonged homogenization is necessary, it is important to prevent excessive heating of the material. For that purpose, the Compendium (Midura & Bryant, 2001) recommends cooling the diluent in an ice bath before use, while ISO 6887-4:2003/Cor.1:2004 recommends not homogeniz-ing for periods longer than 2.5 min. There are special cases that require a different initial dilution.

Procedure for the preparation of  the first  dilution of  samples obtained by surface  swabbing or surface  washing

The diluent retaining the contamination collected with swabs, sponges or surface washing is, in itself, already the first dilution of the sample. The subsequent treatment of serial decimal dilution is performed using this suspension as point of departure.

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.

Davis, G.L. & Hickey, P.J. (2004) Media and dilution water prepa-ration. In: Wehr, H.M. & Frank, J.F (eds).  Standard Methods for the Examination of Dairy Products. 17th edition. Washington, American Public Health Association. Chapter 4, pp. 93–101.