AS 5013.4:2021 – Food microbiology Method 4: Microbiology of food and animal feeding stuffs — Horizontalmethod for the enumeration of coliforms —Colony-count technique (Iso4832:2006,MOD)
AS 5013.4:2021 – Food microbiology Method 4: Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coliforms —Colony-count technique (Iso4832:2006,MOD).
6.10 pH-meter, accurate to ±0,1 pH unit at 25 C.
6.11 Loop, of platinum-iridium or nickel-chromium, approximately 3 mm In diameter, or disposable loops.
Sampling should have been carried out in accordance with the specific International Standard appropriate to the product concerned. If there Is no specific International Standard. It Is recommended that the parties concerned come to an agreement on this subject.
8 Preparation oltest sample
Prepare the test sample in accordance with ISO 6887 (relevant part), ISO 8261 or the specific
International Standard appropriate to the product concerned. If there is no specific International
Standard, It is recommended that the parties concerned come to an agreement on this subject.
9.1 Test portion, initial suspension and dilutions
Prepare the test portion, initial suspension (primary dilution) and further dilutions in accordance with ISO 6887 (relevant part). ISO 8261 or the specific International Standard appropriate to the product concerned.
9.2 Inoculation and Incubation
9.2.1 Prepare two dishes for the liquid product and/or from each dilution chosen. Transfer, with a sterile pipette (6.4), 1 ml of liquid product or the appropriate dilutions to the centre of each dish. Use another sterile pipette to Inoculate each dilution Into the dishes.
9.2.2 Pour about 15 ml of the VRHL medium (5.3), at 44 °C to 47 °C, into each Petri dish. The time elapsing between the end of the preparation of the initial suspension (or of the 10-1 dilution if the product is liquid) and the moment when the medium is poured into the dishes shall not exceed 15 mm.
Carefully mix the inoculum with the medium and allow the mixture to solidify, with the Petri dishes standing on a cool horizontal surface.
Also prepare a control plate with 15 ml of the medium for checking its sterility.
9.2.3 After complete solidification, pour about 4 ml of the VRBL medium (.3), at 44 C to 47 °C, onto the surface of the inoculated medium. Allow to solidify as described above.
9.2.4 Invert the prepared dishes and incubate them in the incubator (6.2) set at 30 °C or 37 °C (as agreed)for24h±2h.
After the specified period of Incubation (see 92.4), select the Petri dishes with. If possIble, 10 or more colonies and fewer than 150 colonies. Count, using the colony-counting equipment (6.6). the purplish red colonies with a diameter of at least 0,5 mm (sometimes surrounded by a reddish zone of precipitated bile). These are considered as typical colonies of coliforms and do not require further confirmation.
For details of the colony-count technique, see ISO 7218.
Also count and confirm atypical colonies (e.g. of smaller size), and all colonies derived from milk products that contain sugars other than lactose, immediately after the incubation period according to 9.4. Conversion of sugars other than lactose may result in colonies with an appearance that looks similar to the typical coliforms.
NOTE The appearance of a reddish zone o precipitated bile around the colonies depends on the type of col iform and the quality of the medium.
Inoculate five colonies of each atypical type, if available, into tubes of brilliant green lactose bile broth (5.4). Incubate the tubes in the incubator (6.2) set at 30 °C or 37 °C (as agreed) for 24 h ± 2 h. Consider as coliforms colonies that show gas formation in the Durham tube. Take the results into account in the calculation (Clause 10),
10 Expression of results
See ISO 7218.
Given a Poisson distribution of microorganisms In the substrate, the confidence limits of this method vary according to the count of colonies examined from ± 16% to ± 52 % (see Reference [3D In practice, even greater variation may be found. In various collaborative studies, the standard deviation of the repeatability (sr) appeared to be 0.20 log units, the standard deviation of the reproducibility (sR) appeared to be 0,35 log units (see References 141 and ).
More Information about the confidence limits is given in ISO 7218.
12 Test report
The test report shall specify:
— all information necessary for the complete Identification of the sample;
— the sampling method used, if known:
— the test method used, with reference to this International Standard;
— all operating details not specified In this International Standard, or regarded as optional, together with details of any incident which may have influenced the result(s);
— the test result(s) obtained;
— if the repeatability has been checked, the final quoted results obtained.