Regulatory News 2017 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. In Part 1, a horizontal method for the detection of Enterobacteriaceae is specified, and in Part 2, the enumeration of these organisms, both in food and animal feed samples is stated. The revised standards are also applicable to environme environmental ntal samples in the area of primary production, food production and food handling. Part 1 is applicable when the microorganisms sought are expected to need resuscitation by enrichment, and when the number sought is expected to be below 100 colony forming units (cfu) /mL or gram of test sample. Part 2 is applicable when the number number of colonies sought is expected to be more than 100 cfu / mL or gram of the test sample. The new Part 1 edition cancels and replaces the first edition EN ISO 215281:2004. 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 as minor.
Revised EN ISO 21528-1:2017 at a glance... • 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 enrichtment enrichtment step has been deleted. • Confirmation now takes takes place in glucose OF medium instead of using glucose agar.
Procedure steps for detection of Enterobacteriaceae according to the revised EN ISO 21528-1:2017 Part 1: Detection of Enterobacteriaceae Day
1
Procedure Step
Non-selective Pre-enrichment
2
Plating-out
3
Subculture
4
Confirmation and expression of results
• Performance testing testing for the quality assurance of the culture media has been added. • Performance characteristics characteristics for this this method have been added to Annex C.
Test portion (x g or x mL) + Buffered peptone water (9 x g or 9 x mL) Incubation at 37 °C ± 1 °C for 18 h ± 2 h
by surface inoculation on Violet red bile glucose (VRBG) agar Incubation at 37 °C ± 1 °C (or 30 °C ± 1 °C) for 24 h ± 2 h
Inoculation of a non-selective agar medium Incubation at 37 °C ± 1 °C for 24 h ± 2 h
• Most probable number number (MPN) method has become an informative Annex A.
The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.
Confirmation of Enterobactericeae by Oxidase reaction (-) and Fermentation test in Glucose of medium (+)
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Procedure for detection and enumeration of Enterobacteriaceae in the food chain according to EN ISO 21528-2:2017
Revised EN ISO 21528-2 at a glance...
Part 2: Colony-cou Colony-count nt technique
• The confirmation step has been changed by replacing glucose agar with glucose OF medium.
Day
Procedure Step
Sample Preparation
1
• Precision data based on the results of an interlaboratory study using the method according to this revised edition has been included in annex B (informative).
2
Plating out
x g Sample + 9x mL diluent (e.g. Buffered Peptone Water,, see all parts of ISO 6887) Water
1 mL of culture + 15 mL molten VRBG agar (47-50 °C) into empty sterile Petri dish After solidifcation, cover cover with a layer of 5-10 mL VRBG agar 24 h ± 2 h | 37 °C
As a worldwide leading provider of a broad range of dehydrated granulated and readyto-use culture media for food, beverage beverages s and water microbiology, we closely track and support the developme development nt of relevant standards aimed at increasing consumer confidence and safety. Our 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 are displayed on the product label and in the product’s technical information available on our website.
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
Confirmation and expression of results
3
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
Compliance to revised EN ISO 21528:2017 We have implemented all the requirements described in both parts of the revised EN ISO 21528:2017. For more information, please visit SigmaAldrich.com/Food-Microbiology . The following culture media and accessories described in the revised EN ISO 21528:2017 are available available:: Ordering Information Product
Pack size
GranuCult® Buffered Peptone Water acc. ISO 6579, ISO 21528, ISO 22964, FDA-BAM and EP
500 g
GranuCult® VRBD (Violet Red Bile Dextrose) agar acc. EP, USP, JP and ISO 21528
500 g
VRBD LI agar
20 plates
®
Cat. No.
Other pack sizes available
1.07228.0500
5 kg, 25 kg
1.10275.0500
5 kg
1.46000.0020
120 plates
GranuCult Nutrient agar acc. ISO 6579, ISO 10273 and ISO 21528
500 g
1.05450.0500
GranuCult® Tryptic Soy Agar EP, USP, JP, ISO, FDA-BAM
500 g
1.05458.0500
5 kg
ReadyPlate™ Tryptic Soy Agar ISO, FDA-BAM, EP+USP
20 plates
1.46431.0020
100 plates
Granucult Peptone salt solution (Maximum recovery diluent) acc. ISO 6887 and ISO 8199
500 g
Ringer tablets
100 tablets
Bactident™ Oxidase
50 test strips 1.13300.0010
®
ReadyTube 2 20 00 VRBG ISO 21528
6x200 mL
1.12535.0500
1.15525.0001
1.46435.0006
