AS 4659.2:2015 – Guide to determining the equivalence of food microbiology test methods Part 2: Quantitative tests
AS 4659.2:2015 – Guide to determining the equivalence of food microbiology test methods Part 2: Quantitative tests.
4.3 Select test organisms
4.3.1 For flofl-wkctibe methods
All samples should be naturally contaminated samples.
4.3.2 For .celec,ive quwi:itaIivt’ methods
A minimum of five strains of the target organism should be selected. The reference culture prescribed as a positive control should be one of the five strains. The other strains may be selected from the following list (in order of preference):
(a) Strains of the target organism isolated by the laboratory from previous samples of the matrix under examination.
(b) Strains of the target organism isolated by reference laboratories or industry sources as representative of the strains found in the type of matrix under examination.
(c) Strains of the target organism traceable to culture collections.
NOTE: Commercial preparations of known concentrations may bc used.
The strains selected should encompass the variation that may be expected to he found within the target organism in the matrix under examination.
The identity of the strains chosen should be determined by appropriate means (e.g. biochemical, physiological, serological, molecular) before performing furiher work. Consultation with a reference laboratory or an expert may he necessary. The source and identity of the strains should be recorded.
NOTE: It is possible, by the selection of tcst strains, to bias the results of the study. Care should bc taken to sclcct representative strains sshich do not possess characteristics which arc Iikcl to lead to a biased result being obtained.
4.4 Select samples of (he matrix
Five typical samples of the matrix should be selected. They should represent as far as possible the range of variation fuund within the defined matrix. The choice of samples will depend upon the matrix, but may consist of hatches with high or low background contamination, samples with different pH values, fat, protein or moisture levels. The aim of the selection is to choose samples which will represent the range of variation expected to be found in the defined matrix. Care should be taken to select samples with characteristics which are expected to yield homogeneous results in the test being validated, Naturally contaminated samples, if available, should he used instead of artificially contaminated samples. Care should be taken to determine the identity, level and homogeneity of contamination.
The significant characteristics of the product should be measured and the results recorded. 45 Preparation (selective quantitative method)
For artificially inoculated samples, the strains chosen should be prepared for use by culturing the strains of the test organism in a non-selective medium (e.g. tryptone soya agar. nutrient agar) to early stationary phase (e.g. IX hour culture) at the optimum growth temperature. A cocktail of strains may be prepared by combining approximately equal numbers of each strain grown as a pure culture. The cocktail of strains should hc added to each matrix sample at two levels:
(a) Approximately 10 times the lower limit of detection of the reference method.
(b) Approximately within one log11 of the upper limit of detection of the alternate method or the highest expected level of’ the target organism.
The number of target organisms added to a sample is determined using a non-selectise medium (e.g. tryptone soya agar). incubated at the optimum temperature for ihe target organism and for an appropriate time to reach the start of the stationary phase.
NOTE: For example:
(a) If the reference method is capable ot detecting lot) target organisms per gram of the matrix ilien – 1000 cells of the selected test organism should he added per gram of the matrix.
(b) If thc highest expected Icc1 of organisms in thc product is I x 10’ efug. then thc second inoculation kscl should bc — I x l0 to — I X tOT efn’g.
(c) Thc number of colony forming units of the test organism may be seritied by a nonselectise method such as a most probable number or by plate count of a sufficient solume of the suspension tO obtain an accurate count.
Serial dilutions of the cultures of the tes organism should be made to citable enumerinion of the test organism and inoculation of the broth to which the matrix sample is added.
NOTE: Commercially asailable preparations of known concentrations of the test organism may he used
The method of preparing the strains for inoculation into the matrix, the method of inoculating the matrix, the method used to determine the level added, and the level added should be recorded.