🔴 Cronobacter spp. in food and feed

Microbiology PN-EN ISO 22964

Overview

STATUS (as of 02.11.2026). PN-EN ISO 22964:2017-06 — English version published on 13.06.2017, Polish version on 15.11.2019; adopts EN ISO 22964:2017 and ISO 22964:2017 [IDT], without a withdrawal note. ISO 22964:2017 (first edition, 04.04.2017) is current and replaced ISO/TS 22964:2006 — the changes are considered major (ISO 17468): the scope was extended to food, feed and environmental samples, mLST broth was replaced by Cronobacter selective broth (CSB), ESIA agar — by chromogenic CCI agar, and several confirmation tests — by the tests of Table 1.

SCOPE (clause 1, 3.1). Detection of Cronobacter spp. in food products and ingredients intended for human consumption and for the feeding of animals and in environmental samples in the area of food production and food handling. Cronobacter spp. are microorganisms forming typical colonies on chromogenic CCI agar and displaying the biochemical characteristics described in the standard; they can be present in small numbers accompanied by other Enterobacteriaceae, such as E. cloacae, that interfere with their detection (note in 5.1). According to chapter 29 of the BAM manual (FDA), the source of infections is unknown, but a growing number of reports point to powdered infant formula as a vehicle; case-fatality rates in the reported infections have varied considerably, with some as high as 80 %.

TEST PORTION AND ENRICHMENT (10.1–10.3). Usually 10 g or 10 ml of sample in 90 ml of buffered peptone water pre-warmed to room temperature; for dry milk — according to ISO 6887-5. The standard has been validated for test portions of 10 g: a smaller portion can be examined in the same ratio without validation, a larger one — after validation or verification (large portions can compromise the recovery of stressed cells when interfering flora, e.g. probiotics, is present); for portions larger than 10 g the peptone water is pre-warmed to 34–38 °C. Pre-enrichment — (18 ± 2) h at 34–38 °C; then 0.1 ml of culture into 10 ml of CSB and (24 ± 2) h at (41.5 ± 1) °C.

ISOLATION AND CONFIRMATION (10.4–10.6). From CSB, CCI agar is streaked with a 10 µl loop and incubated for (24 ± 2) h at 41.5 °C. Typical Cronobacter colonies are small to medium-sized (1–3 mm), blue to blue-green; others are often white or white with a green centre, grey or black, and some naturally pigmented ones — yellow or red. Five typical or suspect colonies (all of them if fewer) are purified on TSA ((21 ± 3) h at 34–38 °C) and tested: oxidase, hydrolysis of 4-nitrophenyl α-D-glucopyranoside, L-lysine and L-ornithine decarboxylase, acid from D-arabitol, D-sorbitol and sucrose, optionally methyl red, Voges-Proskauer and acid from α-methyl-D-glucoside. Cronobacter spp. are oxidase negative, hydrolyse 4-nitrophenyl α-D-glucopyranoside and produce acid from sucrose, do not decarboxylate L-lysine and do not produce acid from D-sorbitol; a positive methyl red and a negative Voges-Proskauer reaction are rare.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026), food safety criterion 1.24: dried infant formulae and dried dietary foods for special medical purposes intended for infants below 6 months of age — Cronobacter spp. (Enterobacter sakazakii) absence in 10 g, n = 30, c = 0, method EN ISO 22964, products placed on the market during their shelf-life. Parallel testing for Enterobacteriaceae and Cronobacter spp. is conducted, unless a correlation between them has been established at the individual plant level; if Enterobacteriaceae are detected in any sample, the batch is tested for Cronobacter spp. (footnote 14). If testing against a food safety criterion gives unsatisfactory results, the product or batch is withdrawn or recalled in accordance with Article 19 of Regulation (EC) No 178/2002 (Article 7(2)).

WHAT WE DO NOT GIVE HERE. The composition of media and reagents (Annex B) and the characteristics distinguishing Cronobacter from related species (Annex C) are not in the available text of the standard — we do not give them.

Method principle

The test portion is added to buffered peptone water and incubated at 34–38 °C for (18 ± 2) h — non-selective pre-enrichment (5.1). The culture is used to inoculate the selective CSB broth, incubated at (41.5 ± 1) °C for (24 ± 2) h (5.2), and then chromogenic CCI agar is streaked and incubated at (41.5 ± 1) °C for (24 ± 2) h (5.3). Typical colonies are purified on a non-selective agar such as TSA and characterized biochemically (5.4).

Applications

• Food products and ingredients 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)
• Dry milk — sample preparation according to ISO 6887-5 (10.1)
• Dried infant formulae and dried dietary foods for special medical purposes for infants below 6 months — food safety criterion (Regulation (EC) No 2073/2005, item 1.24)

Key parameters

Standard

Standard number

PN-EN ISO 22964:2017-06

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania Cronobacter spp.

Title (EN)

Microbiology of the food chain — Horizontal method for the detection of Cronobacter spp.

Step-by-step procedure

1. Test portion

   Usually 10 g or 10 ml of sample to 90 ml of buffered peptone water pre-warmed to room temperature (for portions above 10 g — to 34–38 °C); dry milk according to ISO 6887-5 (10.1).

2. Pre-enrichment

   Incubate at 34–38 °C for (18 ± 2) h (10.2).

   🌡 Temperature: 34–38 °C

3. Selective enrichment

   Mix, transfer 0.1 ml of the culture into 10 ml of CSB, mix and incubate at 41.5 °C for (24 ± 2) h (10.3).

   🌡 Temperature: 41.5 °C

4. Isolation

   Mix the culture and streak CCI agar with a 10 µl loop to obtain well-separated colonies; incubate at 41.5 °C for (24 ± 2) h (10.4).

   🌡 Temperature: 41.5 °C

5. Reading

   Typical colonies: small to medium-sized (1–3 mm), blue to blue-green; others can be white, white with a green centre, grey, black, yellow or red (10.4).

6. Purification

   Streak five typical or suspect colonies (all of them if fewer) on TSA and incubate the plates inverted at 34–38 °C for (21 ± 3) h; the most characteristic colony can be tested first — a positive result ends the test (10.5.1–10.5.2).

   🌡 Temperature: 34–38 °C

7. Biochemical tests

   Oxidase, hydrolysis of 4-nitrophenyl α-D-glucopyranoside (4 h at 37 °C, absorbance at 405 nm of at least 0.3), L-lysine and L-ornithine decarboxylase ((24 ± 2) h at 37 °C), acid from D-arabitol, D-sorbitol and sucrose ((48 ± 2) h at 37 °C), optionally methyl red and Voges-Proskauer; reliable miniaturized galleries for biochemical identification are allowed (10.5.3).

8. Interpretation and result

   Cronobacter spp.: oxidase negative, hydrolysis of 4-nitrophenyl α-D-glucopyranoside and acid from sucrose — positive, L-lysine decarboxylase and acid from D-sorbitol — negative; result: detected or not detected in a test portion of x g or x ml (10.6, clause 11).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard has no clause on safety — for laboratory practice it refers to ISO 7218 (clause 6)

Frequently asked questions