🦠 Coagulase-positive staphylococci (S. aureus)

Microbiology PN-EN ISO 6888-1

Overview

Staphylococcus aureus is one of the most common bacterial causes of food poisoning worldwide. Coagulase-positive staphylococci produce thermostable enterotoxins (SE A–E) that are not destroyed even during heat treatment of food. Only about 100 ng of enterotoxin is sufficient to cause poisoning symptoms in humans. The source of food contamination is most often production personnel (nasal carriage, infected wounds on hands).

The PN-EN ISO 6888-1 standard describes a horizontal method for enumerating coagulase-positive staphylococci using selective Baird-Parker medium. The medium contains potassium tellurite and lithium chloride as inhibitory agents for accompanying bacteria, while glycine and sodium pyruvate stimulate staphylococci growth. S. aureus colonies on this medium are black or gray, shiny and convex, surrounded by a clear zone (reduction of tellurite to metallic tellurium).

The method is used globally as the gold standard for enumerating staphylococci in food. It allows simultaneous enumeration and preliminary identification based on colony morphology. Confirmation is performed by tube coagulase test or RPFA (Rabbit Plasma Fibrinogen Agar) method.

Testing is mandatory under Regulation (EC) No 2073/2005 for cheeses, milk powder, ready-to-eat products and infant foods. It is a key element of HACCP systems in dairy, confectionery and catering establishments.

Method principle

The method involves surface plating of sample on selective Baird-Parker medium enriched with egg yolk emulsion and potassium tellurite. Potassium tellurite (K₂TeO₃) is reduced by staphylococci to metallic tellurium, giving colonies characteristic black coloration. Lithium chloride and tellurite inhibit growth of most accompanying bacteria. Lecithinase produced by S. aureus causes opalescence (clear zone) around colonies. Lipase degrades egg yolk fats, creating an outer precipitation ring. After 24–48 h incubation at 34–38°C, typical colonies are confirmed by coagulase test.

Applications

• Quality control of cheeses and dairy products
• Testing ready-to-eat (RTE) food — criteria of Regulation (EC) 2073/2005
• Production hygiene monitoring in confectioneries and catering establishments
• Infant and young children food control
• Testing meat and meat products
• Testing personnel hand swabs and work surface swabs (GHP)
• Food analysis in epidemiological investigation (food poisoning)
• Verification of thermal and preservation processes effectiveness

Key parameters

Standard

Standard number

PN-EN ISO 6888-1:2022

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania liczby gronkowców koagulazo-dodatnich (Staphylococcus aureus i inne gatunki) — Część 1: Metoda z zastosowaniem podłoża agarowego 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 preparation

   Weigh 10 g (or 25 g) of sample into stomacher bag. Add 90 mL (or 225 mL) buffered peptone water. Homogenize for 60–120 s.

   ⏱ Time: 5 min

2. Preparation of dilutions

   Prepare decimal dilution series (10⁻², 10⁻³). Use new pipette for each dilution.

   ⏱ Time: 5 min

3. Medium preparation

   Dissolve Baird-Parker Agar Base in distilled water. Sterilize at 121°C for 15 min. Cool to 47–50°C and add egg yolk emulsion with tellurite aseptically. Pour into plates (approx. 18–20 mL). Dry plates.

   ⏱ Time: 30 min • 🌡 Temperature: 47–50°C

4. Surface plating

   Apply 0.1 mL (total up to 0.3 mL on 3 plates from same dilution) to medium surface. Spread evenly over entire surface with Drigalski spatula.

   ⏱ Time: 10 min

5. Inoculum drying

   Leave plates open in laminar flow cabinet until inoculum is absorbed (approx. 15 min).

   ⏱ Time: 15 min

6. Incubation

   Invert plates and incubate at 34–38°C. Read preliminarily after 24 ± 2 h, mark typical colonies and continue incubation up to 48 ± 4 h.

   ⏱ Time: 24–48 h • 🌡 Temperature: 37°C

7. Colony reading

   Count typical colonies (black/gray, shiny, convex, surrounded by clear zone and/or opalescence ring). Also mark atypical colonies (black without zones).

   ⏱ Time: 15 min

8. Coagulase test (confirmation)

   Select min. 5 typical and 5 atypical colonies. Inoculate into 0.3 mL BHI broth, incubate 24 h at 37°C. Add 0.3 mL rabbit plasma. Incubate at 37°C and observe clot formation after 4–6 h (max. 24 h).

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

9. Calculations

   Calculate number of coagulase-positive staphylococci taking into account percentage of confirmed colonies. Express result in CFU/g or CFU/mL.

10. Quality control

    Positive control: S. aureus ATCC 25923 (typical colonies). Negative control: E. coli ATCC 25922 (no growth or atypical colonies). Medium sterility control.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• S. aureus — BSL-2 pathogen, use full personal protective equipment
• Potassium tellurite — toxic, avoid inhalation and skin contact, work in laminar flow cabinet
• Perform all procedures with open sample in class II laminar flow cabinet
• Autoclave used materials before disposal (121°C, 20 min)
• Surface disinfection with 70% ethanol or 0.5% sodium hypochlorite

Frequently asked questions