ISO/NP 21528-3

Microbiology of the food chain — Horizontal method for the detection and enumeration of Enterobacteriaceae — Part 3: Enumeration by most probable number technique

Scope

Warning — In order to safeguard the health of laboratory personnel, it is essential that tests for detecting Enterobacteriaceae are only undertaken in properly equipped laboratories under the control of a skilled microbiologist, and that great care is taken in the disposal of all incubated materials. Persons using this document should be familiar with normal laboratory practice. This document does not purport to address all of the safety aspects, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices
The most probable number (MPN) technique can be used when the number of Enterobacteriaceae sought is expected to be in the range 1 to 100 per millilitre or per gram of test sample. The MPN technique follows the same principles as the detection method, but several dilutions are tested. It is known that wide variations in results can occur using this technique for enumeration. The MPN technique can however be used when low numbers are expected or because of the nature of the product under test, according to the instructions in this annex
Specifications.
Structure of other MPN methods.
This document specifies a method, with enrichment, for the enumeration of Enterobacteriaceae using MPN technique. It is applicable to
— products intended for human consumption and the feeding of animals, and
— environmental samples in the area of primary production, food production and food handling.
This method is applicable
— when the microorganisms sought are expected to need resuscitation by enrichment, and
— when the number sought is expected to be below 100 per millilitre or per gram of test sample.
A limitation on the applicability of this document is imposed by the susceptibility of the method to a large degree of variability (see Clause 11).
NOTE 1 As of 2021, due to a reclassification of the nomenclature, the family of Enterobacteriaceae is a member of the order Enterobacterales. Enterobacterales contains seven families (Budviciaceae, Enterobacteriaceae, Erwiniaceae, Hafniaceae, Morganellaceae, Pectobacteriaceae and Yersiniaceae). Several species, which previously belonged to Enterobacteriaceae, are now part of other families within the order Enterobacterales. These species form characteristic colonies on VRBG, ferment glucose and show a negative-oxidase reaction. Therefore, these species are typed and regarded as Enterobacteriaceae in the ISO 21528 series.
NOTE 2 In a clinical setting, three species represent 80% to 95% of all identified isolates. These are Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Proteus mirabilis is considered part of the Morganellaceae, within the order Enterobacterales. This indicates that for food safety issues it is recommended that the ISO 21528 series detect more than the family of Enterobacteriaceae, but also the other families within the order Enterobacterales.