Revised EN ISO 21528:2017 for Detection and Enumeration of Enterobacteriaceae in the Food Chain
The International Organization for Standardization (ISO) published the revised EN ISO 21528-1 and EN ISO 21528-2 standards in 2017. Part 1 specifies a horizontal method for the detection of Enterobacteriaceae, while Part 2 outlines the enumeration of these organisms in both food and animal feed samples. The revised standards are also applicable to environmental samples in the areas of primary production, food production and food handling.
- Introduction to ISO 21528 Standards
- Procedure for Detecting Enterobacteriaceae as per EN ISO 21528-1:2017
- Overview of Revised EN ISO 21528-1:2017
- Procedure for Enterobacteriaceae Detection and Enumeration in Food Chain per EN ISO 21528-2:2017
- EN ISO 21528-2:2017 Summary
- Culture Media Compliance with Revised ISO Standards
- Related products
Introduction to ISO 21528 Standards
- Part 1 is applicable when the microorganisms sought are expected to require resuscitation by enrichment, and when the number sought is expected to be below 100 per mL or gram of test sample.
- Part 2 is applicable when the number of colonies sought is expected to exceed 100 per mL or gram of the test sample.
Part 1 edition cancels and replaces the first edition, EN ISO 21528-1:2004. Similarly, Part 2 cancels and replaces the first edition of EN ISO 21528-2:2004. Compared to the previous edition, the main changes in Part 1 are considered as major, and in Part 2 are regarded as minor.
Part 1: Detection of Enterobacteriaceae
Day 2 - Plating out
by surface inoculation on Violet red bile glucose (VRBG) agar
Incubation at 37 °C ± 1 °C (or 30 °C ± 1 °C) for 24 h ± 2h
Day 3 - Subculture
Inoculation of a non-selective agar medium
Incubation at 37 °C ± 1 °C for 24 h ± 2 h
Day 4 - Confirmation and expression of results
Confirmation of Enterobacteriaceae by Oxidase reaction (-) and Fermentation test in Glucose OF medium (+)
Overview of Revised EN ISO 21528-1:2017
- The pre-enrichment step in Buffered Peptone Water (BPW), followed by enrichment in Enterobacteriaceae enrichment (EE) broth has been changed to enrichment in BPW. The selective enrichment step has been deleted.
- Confirmation now takes place in Glucose OF medium instead of using glucose agar.
- Performance testing for the quality assurance of the culture media has been added.
- Performance characteristics for this method have been added to Annex C.
- Most probable number (MPN) method has become an informative Annex A.
Part 2: Colony-count Technique
Day 2 - Plating out step
1 mL of culture + 15 mL molten VRBG agar (47–50 °C) into empty sterile Petri dish
After solidification, cover with a layer of 5-10 mL VRBG agar
24 h ± 2 h | 37 °C
Day 3 - Confirmation and expression of results
Count typical colonies (pink-red or purple) on plates with < 150 of such colonies.
Pick 5 typical colonies. Streak to nutrient agar plates
24 h ± 2 h | 37 °C
1. Perform oxidase test
2. Perform fermentation test:
Inoculate (stab) each oxidase negative colony into tubes with Glucose OF agar medium. Overlay with min. 1 cm of sterile mineral oil 24 h ± 2 h | 37 °C
Oxidase-negative colonies leading to yellow color formation in tubes are confirmed as Enterobacteriaceae
EN ISO 21528-2:2017 Summary
- The confirmation step has been modified by replacing glucose agar with glucose OF medium.
- Precision data based on the results of an interlaboratory study using the method described in the revised edition has been included in annex B (informative).
As a worldwide leading provider of a broad range of dehydrated, granulated, and ready-to-use culture media for food, beverages and water microbiology, we closely track and support the development of relevant standards aimed at increasing consumer confidence and safety.
Culture Media Compliance with Revised ISO Standards
GranuCult® granulated media, Readybag® pre-weighed granulated media in ready-to-use bags, ReadyPlate™, and ReadyTube® ready-to-use media are all compliant with EN ISO 11133:2014. All information regarding compliance with additional reference standards is displayed on the product label and on the product’s technical information available on our website.
We have implemented all the requirements described in both parts of the revised EN ISO 21528:2017. The following culture media and accessories, as described in the revised EN ISO 21528:2017, are available: