🦠 Vibrio spp. in food

Microbiology PN-EN ISO 21872-1

Overview

STATUS (as of 02.11.2026). PN-EN ISO 21872-1:2017-10 — English version, published on 09.10.2017, adopts EN ISO 21872-1:2017 and ISO 21872-1:2017 [IDT]. AMENDMENT PN-EN ISO 21872-1:2017-10/A1:2023-09 (English version, published on 07.09.2023) adopts ISO 21872-1:2017/Amd 1:2023 (20.02.2023) “Inclusion of performance testing of culture media and reagents” — it moves the performance testing table of TCBS agar to the new B.12 and adds criteria for the other media. ISO 21872-1:2017 (first edition, 10.07.2017) replaced ISO/TS 21872-1:2007 with Cor.1:2008: optional molecular identification methods for the major species (V. parahaemolyticus, including potentially enteropathogenic strains, V. vulnificus and V. cholerae) and performance characteristics (Annex E) were introduced; the changes are considered major (ISO 17468).

SCOPE (clause 1). Detection of enteropathogenic Vibrio spp. causing human illness in or via the intestinal tract — V. parahaemolyticus, V. cholerae and V. vulnificus — in products intended for human consumption and for the feeding of animals and in environmental samples in the area of food production and food handling. According to the WHO these are the three major food-borne Vibrio species; the method can also be appropriate for the identification of other Vibrio spp. causing illness in humans. The target species are often present in small numbers accompanied by a much larger number of other bacteria of the Vibrionaceae family and other families. According to chapter 9 of the BAM manual (FDA), V. parahaemolyticus is the leading cause of bacterial diarrhoea associated with seafood consumption in Florida and probably the whole US, and in temperate regions its infections are seasonal, mostly in the warmer months; V. vulnificus is the leading cause of seafood-related deaths in the US, nearly always associated with raw Gulf Coast oysters.

ENRICHMENT TEMPERATURES (4.1–4.3, 9.2, Table 2). Recovery of V. parahaemolyticus and V. cholerae from fresh products is enhanced by enrichment at 41.5 °C, and of V. vulnificus as well as V. parahaemolyticus and V. cholerae from deep-frozen (below −18 °C), dried or salted products — at 37 °C. Primary enrichment: (6 ± 1) h at 41.5 °C and/or 37 °C — for all species in frozen, dried or salted products 37 °C, for V. vulnificus always 37 °C, for V. parahaemolyticus and/or V. cholerae only in fresh products 41.5 °C. Secondary enrichment: 18 h at 41.5 °C for V. parahaemolyticus and V. cholerae, at 37 °C for V. vulnificus. When all three species are to be detected, all temperatures are used; the selection of procedures is stated in the test report.

ISOLATION AND CONFIRMATION (4.4–4.5, 5.2, 5.6.3). From the cultures after the primary and after the secondary enrichment, TCBS agar (37 °C, reading after 24 h) and a second selective medium chosen by the laboratory, e.g. chromogenic (according to the manufacturer), are inoculated. In the performance test of TCBS, V. parahaemolyticus forms green colonies (sucrose negative) and V. furnissii — yellow ones (sucrose positive); E. coli is to be totally inhibited. Presumptive colonies are subcultured and confirmed by biochemical and/or PCR tests; enteropathogenic V. parahaemolyticus (genes of the haemolysins tdh and/or trh) can only be determined by PCR. PCR targets: V. parahaemolyticus — toxR, pathogenic strains — tdh and trh, V. cholerae — the 16S-23S rRNA intergenic spacer region prVC (conventional PCR), V. vulnificus — the haemolysin region vvhA.

SAMPLES (9.1). The standard has been validated for test portions of up to 25 g or 25 ml, homogenized in 225 ml of ASPW; portions larger than 25 g — in ASPW pre-warmed to 37 °C and/or 41.5 °C. Several portions from one batch may be combined (e.g. 10 × 25 g in 2.25 l of ASPW) when it has been shown that this does not modify the result for the product. Cell counts of Vibrio spp. can decline significantly at refrigeration temperatures — storage of samples and suspensions in a refrigerator should be avoided or kept to a minimum.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) contains no criterion for Vibrio spp.

WHAT WE DO NOT GIVE HERE. The available text of the standard ends at 9.3 (secondary enrichment). We do not give the selection and number of colonies to be confirmed, the course of the biochemical tests (oxidase, L-lysine decarboxylase, arginine dihydrolase, β-galactosidase, indole, growth in saline peptone waters) and their interpretation, the PCR conditions (Annexes C and D) or the composition of media and reagents (Annex B).

Method principle

Detection requires four successive phases (Annex A): primary enrichment of the test portion in alkaline saline peptone water (ASPW) at 41.5 °C and/or 37 °C for 6 h, secondary enrichment in fresh ASPW at 41.5 °C and/or 37 °C for 18 h, isolation from both cultures on TCBS agar (37 °C, reading after 24 h) and a second selective medium, and confirmation of presumptive colonies by biochemical and/or PCR tests (4.1–4.5). The temperatures are chosen according to the target species and the state of the product.

Applications

• Products intended for human consumption and for the feeding of animals (clause 1)
• Environmental samples in the area of food production and food handling (clause 1)
• Fresh products and deep-frozen, dried or salted products, e.g. bacalhau, stockfish, katsuobushi — enrichment temperature according to Table 2 (9.2)

Key parameters

Standard

Standard number

PN-EN ISO 21872-1:2017-10 + A1:2023-09

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania Vibrio spp. — Część 1: Wykrywanie potencjalnie enteropatogennych Vibrio parahaemolyticus, Vibrio cholerae i Vibrio vulnificus

Title (EN)

Microbiology of the food chain — Horizontal method for the determination of Vibrio spp. — Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

Step-by-step procedure

1. Test portion

   Homogenize a 25 g or 25 ml portion in 225 ml of ASPW; larger portions — in ASPW pre-warmed to 37 °C and/or 41.5 °C; combining portions from one batch — only when shown not to modify the result (9.1). The sample is prepared according to ISO 6887-1 and ISO 6887-3 (clause 8).

2. Primary enrichment

   Incubate at 41.5 °C and/or 37 °C for (6 ± 1) h according to Table 2: fresh products — 41.5 °C for V. parahaemolyticus and V. cholerae, 37 °C for V. vulnificus; deep-frozen, dried or salted products — 37 °C for all species (9.2).

   🌡 Temperature: 41.5 °C / 37 °C

3. Secondary enrichment

   Transfer 1 ml of the culture taken from the surface (without agitation) into a tube with 10 ml of ASPW; incubate for 18 h at 41.5 °C (V. parahaemolyticus, V. cholerae) and/or at 37 °C (V. vulnificus) (4.3, 9.3).

   🌡 Temperature: 41.5 °C / 37 °C

4. Isolation

   From the cultures after the primary and after the secondary enrichment, inoculate TCBS agar and the second selective medium; incubate TCBS at 37 °C and examine after 24 h, the second medium — according to the manufacturer (4.4). According to chapter 9 of the BAM manual (FDA), V. cholerae forms large (2–3 mm), smooth, yellow colonies on TCBS, and V. parahaemolyticus round, opaque, green or bluish colonies 2–3 mm in diameter.

   🌡 Temperature: 37 °C

5. Confirmation

   Subculture presumptive colonies (e.g. on SNA) and confirm them by biochemical and/or PCR tests; the enteropathogenicity of V. parahaemolyticus (tdh and/or trh genes) is determined only by PCR (4.5, 5.3).

6. PCR screening of broths (optional)

   After the secondary enrichment the laboratory can screen the broth by conventional PCR for Vp-toxR — absence of amplification indicates that V. parahaemolyticus was not detected; this applies only to this species and only if shown to be reliable in the laboratory (4.5).

7. Result

   Presence or absence of V. parahaemolyticus, V. cholerae and V. vulnificus in a determined quantity of product; the selection of procedures for the species is stated in the test report (3.2, 4.1).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Tests for Vibrio spp., and particularly toxigenic V. cholerae, are only conducted in laboratories equipped for this purpose, under the supervision of an experienced microbiologist, with great care in the disposal of contaminated material (warning of the standard)

Frequently asked questions