🦠 Coagulase-positive staphylococci (S. aureus)

Microbiology PN-EN ISO 6888-1

Overview

STATUS (as of 02.11.2026). PN-EN ISO 6888-1:2022-03 — English version, published on 04.03.2022, adopts EN ISO 6888-1:2021 and ISO 6888-1:2021 [IDT]; it replaced PN-EN ISO 6888-1:2001 with AMENDMENTS A1:2004 and A2:2018-10. AMENDMENT PN-EN ISO 6888-1:2022-03/A1:2024-02 (English version, published on 28.02.2024) adopts ISO 6888-1:2021/Amd 1:2023: it adds a reference to ISO 19036:2019 (estimation of measurement uncertainty for quantitative determinations), requires the method to be stated in the test report as ISO 6888-1:2021, and in Table B.2 deletes strain WDCM 00090 from the performance test of rabbit plasma with BHI (S. aureus WDCM 00032, 00034, 00035 remain; positive reaction — the clot occupies more than half of the volume of the liquid) and corrects the row for RPFA medium. The 2021 edition changed incubation from “35 °C or 37 °C” to 34–38 °C and the time from “18–24 h” to (24 ± 2) h, introduced pouring of molten medium at 44–47 °C, RPFA medium as an alternative to the coagulase test and performance testing of media according to ISO 11133.

SCOPE (clause 1, introduction). Enumeration of coagulase-positive staphylococci on Baird-Parker medium after aerobic incubation at 34–38 °C and coagulase confirmation — in products intended for human consumption and for the feeding of animals, in environmental samples in the area of food and feed production and handling, and in samples from the primary production stage. Based on the information available at the time of publication, the method is not considered to be (fully) suited to fermented products or other products containing technological flora based on Staphylococcus spp. (e.g. S. xylosus) — such as cheeses made from raw milk and certain raw meat products — when they may contain staphylococci forming atypical colonies on Baird-Parker medium or background flora obscuring the colonies sought; nevertheless ISO 6888-1 and ISO 6888-2 are given equivalent status. The standard is mainly concerned with Staphylococcus aureus, but also with S. intermedius and certain strains of S. hyicus; some strains of S. aureus give weakly positive coagulase reactions — they are distinguished by tests not included in the standard (sensitivity to lysostaphin, haemolysin, thermostable nuclease, acid from mannitol).

COLONIES (9.3.1, 9.2). Typical: black or grey, shining and convex, 1–1.5 mm in diameter after (24 ± 2) h and 1.5–2.5 mm after (48 ± 4) h, surrounded by a clear zone, which can be partially opaque; after at least 24 h an opalescent ring in contact with the colonies can appear in this zone. Atypical, of the same size: shining black with or without a narrow white edge, with no clear zone or a barely visible one, or grey without a clear zone; they are formed mainly by strains contaminating e.g. dairy products, shrimps and giblets. Colonies typical after 24 h can lose their typical appearance after 48 h — counting only at 48 h can give low counts or no counts.

BAIRD-PARKER MEDIUM (ISO 6888-1:1999, 5.3; in the 2021 edition the composition is in Annex B, beyond the available text). Base medium per 1 000 ml: pancreatic digest of casein 10.0 g, yeast extract 1.0 g, meat extract 5.0 g, sodium pyruvate 10.0 g, L-glycine 12.0 g, lithium chloride 5.0 g, agar 12–22 g; pH 7.2 ± 0.2. To 100 ml of base medium are added 1.0 ml of a 1 % potassium tellurite solution and 5.0 ml of egg yolk emulsion (about 20 %), and when Proteus is suspected — 2.5 ml of sulfamethazine solution. The data sheet of the Baird-Parker medium states that lithium chloride and tellurite inhibit most of the accompanying flora, while pyruvate and glycine stimulate the growth of staphylococci.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026) specifies EN/ISO 6888-1 or 2 in the process hygiene criteria for coagulase-positive staphylococci (n = 5, c = 2): cheeses made from milk that has undergone a lower heat treatment than pasteurisation and ripened cheeses made from pasteurised milk or whey — m = 100, M = 1 000 cfu/g (item 2.2.4), unripened soft cheeses (fresh cheeses) made from pasteurised milk or whey — 10 and 100 cfu/g (item 2.2.5), milk powder and whey powder — 10 and 100 cfu/g (item 2.2.7), shelled and shucked products of cooked crustaceans and molluscan shellfish — 100 and 1 000 cfu/g (item 2.4.1). For cheeses made from raw milk (10⁴ and 10⁵ cfu/g, item 2.2.3) the reference method is EN/ISO 6888-2. Where values above 10⁵ cfu/g are detected, the batch is tested for staphylococcal enterotoxins (criterion 1.21: not detected in 25 g, method EN ISO 19020). According to chapter 12 of the BAM manual (FDA), S. aureus is easily destroyed by heat treatment, so its presence in processed foods generally indicates poor sanitation, and post-processing contamination is generally due to human contact or contaminated food-contact surfaces.

WHAT WE DO NOT GIVE HERE. The publicly available text of the 2021 edition ends at 9.3.2 (selection of plates for counting). We do not give the selection of colonies for confirmation, the course of the tube coagulase test and of the test on RPFA medium (9.4), the calculation (clause 10), the composition of media and reagents from Annex B or the precision data (clause 11); the composition of Baird-Parker medium follows the 1999 edition.

Method principle

The surface of a solid selective medium (Baird-Parker) is inoculated with a specified quantity of the liquid sample or the initial suspension and further decimal dilutions; the plates are incubated aerobically at 34–38 °C and examined after 24 h and after 48 h (4.1–4.2). The number of coagulase-positive staphylococci per 1 g, 1 ml, 1 cm² or sampling device is calculated from the number of typical and atypical colonies on plates at dilution levels giving a significant result, confirmed by a positive coagulase test — in a tube or on rabbit plasma fibrinogen agar (RPFA) (3.1, 4.3).

Applications

• Products intended for human consumption and for the feeding of animals (clause 1)
• Environmental samples in the area of food and feed production and handling, and samples from the primary production stage (clause 1)
• Ripened cheeses and cheeses made from milk that has undergone a lower heat treatment than pasteurisation, fresh cheeses, milk powder and whey powder — process hygiene criteria (Regulation (EC) No 2073/2005, items 2.2.4, 2.2.5, 2.2.7)
• Shelled and shucked products of cooked crustaceans and molluscan shellfish (Regulation (EC) No 2073/2005, item 2.4.1)

Key parameters

Standard

Standard number

PN-EN ISO 6888-1:2022-03 + A1:2024-02

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania liczby gronkowców koagulazo-dodatnich (Staphylococcus aureus i innych gatunków) — Część 1: Metoda z zastosowaniem pożywki agarowej Baird-Parkera

Title (EN)

Microbiology of the food chain — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 1: Method using Baird-Parker agar medium

Step-by-step procedure

1. Sample and dilutions

   The test sample, initial suspension and dilutions are prepared according to the relevant part of ISO 6887 and, if necessary, ISO 18593 (clause 8, 9.1). Sampling is not part of the method — recommended standards: ISO/TS 17728, ISO 707, ISO 6887-3, ISO 13307, ISO 17604, ISO 18593 (clause 7).

2. Inoculation

   With a sterile pipette transfer 0.1 ml of the liquid sample or 0.1 ml of the initial suspension (10⁻¹) to a Baird-Parker agar plate; repeat with further decimal dilutions; the number of plates per dilution — according to ISO 7218. For low numbers of staphylococci — 1.0 ml on one 140 mm plate or on three 90 mm plates (9.2).

3. Spreading

   Spread the inoculum with a spreader as quickly as possible over the surface without touching the sides of the dish; allow the plates to dry with their lids on for about 15 min at laboratory temperature (9.2).

4. Incubation

   Invert the plates and incubate for (24 ± 2) h at 34–38 °C, then for a total of (48 ± 4) h (9.2).

   🌡 Temperature: 34–38 °C

5. Reading after 24 h and 48 h

   After (24 ± 2) h mark on the bottom of the plates the positions of typical colonies; after a further (24 ± 2) h mark new typical colonies and atypical colonies (9.3.2).

6. Selection of plates

   For counting retain plates from two successive dilutions with at most 300 colonies in total (typical, atypical and background flora), including at most 150 typical and/or atypical colonies (9.3.2).

7. Confirmation

   Typical and atypical colonies are confirmed by the tube coagulase test or on RPFA medium (3.1, 9.4); a Gram-stained smear before confirmation allows other genera to be distinguished from staphylococci (note in 9.3.1.2). The course of confirmation — 9.4, beyond the available text.

8. Result

   The number of coagulase-positive staphylococci is reported per 1 g, 1 ml, 1 cm² or sampling device (3.2); calculation — clause 10 and ISO 7218; uncertainty — according to ISO 19036:2019, clause 9, when necessary or requested (amendment A1, clause 12).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Tests for staphylococci are only undertaken in properly equipped laboratories, under the control of a skilled microbiologist, with great care in the disposal of all incubated materials (warning of the standard)
• The standard does not address all safety aspects — establishing safety and health practices is the responsibility of the user (warning of the standard)

Frequently asked questions