🦠 Coagulase-positive staphylococci in food — colony-count technique on rabbit plasma fibrinogen agar (RPFA)

Microbiology PN-EN ISO 6888-2

Overview

ISO 6888-1, ISO 6888-2 and ISO 6888-3 describe three horizontal methods for the detection and the enumeration of coagulase-positive staphylococci, among which enterotoxigenic strains occur — above all Staphylococcus aureus, but also S. intermedius and some strains of S. hyicus. The characterisation rests on a positive coagulase reaction; strains with a weakly positive reaction may be confused with other bacteria and are distinguished by additional tests (sensitivity to lysostaphin, haemolysin, thermostable nuclease, acid from mannitol). ISO 6888-2:2021 (the second edition, replacing ISO 6888-2:1999 with Amd 1:2003) specifies a method of counting colonies on rabbit plasma fibrinogen agar after aerobic incubation at 34–38 °C; it is applied to products intended for human consumption and for the feeding of animals, to environmental samples from the production of food and feed and to samples from primary production. The changes with respect to the previous edition are, among others, pouring the medium at 44–47 °C (in accordance with ISO 7218), a temperature range of 34–38 °C instead of "35 °C or 37 °C", a time of 24 h ± 2 h instead of 18–24 h, the requirement to apply ISO 11133 and to carry out performance testing of the RPFA medium; the changes were regarded as minor. The method is not considered fully suitable for fermented products with a technological flora of Staphylococcus spp. (e.g. S. xylosus — cheeses from raw milk, some raw meat products), for products contaminated with staphylococci forming atypical colonies on Baird-Parker or with a flora masking the colonies; ISO 6888-1 and ISO 6888-2 have an equivalent status. Regulation (EC) 2073/2005 indicates EN/ISO 6888-2 as the reference method for the process hygiene criterion for cheeses from raw milk (10⁴–10⁵ cfu/g) and "EN/ISO 6888-1 or 2" for cheeses from pasteurised milk, fresh cheeses, milk powder and whey powder, and products of molluscs and crustaceans (where > 10⁵ cfu/g, an examination for enterotoxins).

Method principle

Pour plates are prepared with a defined amount of the liquid sample or of the initial suspension and of the decimal dilutions (the number of plates according to ISO 7218); the volume of RPFA medium added to the inoculum is a critical point for the coagulase reaction — into each plate 18–20 ml of freshly prepared complete medium (B.2.3) is poured at once, so as to obtain a layer of at least 3 mm. The plates are incubated aerobically at 34–38 °C (incubators set at 35 °C ± 1 °C, 36 °C ± 2 °C or 37 °C ± 1 °C) and assessed after 24 h ± 2 h, the position of the typical colonies being marked on the base; where there are none — reincubation to 48 h ± 4 h in total. The typical colonies are black, grey or even white, small, surrounded by an opaque zone of precipitation indicating coagulase activity; colonies of Proteus may at first look similar, but after 24–48 h they turn brown and swarm. Since the medium rests on the coagulase reaction, confirmation is not needed; despite the trypsin inhibitor in RPFA, typical colonies after 24 h may lose their typical appearance after 48 h (trypsin, overgrowth), so counting only after 48 h may give underestimated results. For counting, the plates with at most 300 colonies, including 100 typical ones, are retained; one plate must have at least 10 colonies; where the numbers are close to the limit of quantification duplicates are recommended (a sum of ≥ 10 colonies, which corresponds to > 5 cfu/ml or > 50 cfu/g). The result is calculated according to ISO 7218 as cfu/g, cfu/ml, cfu/cm² or per sampling device; the composition of the media and the performance testing — annex B / ISO 11133; the precision from the interlaboratory study (cheeses, meat, egg powder, reference materials) — annex C. According to the teaching materials of the UWM, the turbidity around the colonies arises from the reaction of the coagulase with the prothrombin of the plasma — the staphylothrombin that is formed produces, with the fibrinogen, polymers that precipitate around the colonies.

Applications

• The process hygiene criterion for cheeses from raw milk (the reference method EN/ISO 6888-2) and for other cheeses, milk powder and whey powder, products of crustaceans and molluscs (6888-1 or 2) — Regulation (EC) No 2073/2005
• Meat products, delicatessen products, cream cakes, ice cream — assessment of the hygiene of production and of the risk of staphylococcal enterotoxins
• Environmental samples from the production of food and feed and from primary production
• An alternative to Baird-Parker agar (ISO 6888-1) without the stage of confirming the coagulase in a tube
• Feed and raw materials

Key parameters

Standard

Standard number

PN-EN ISO 6888-2:2022-03 (ISO 6888-2:2021)

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania liczby gronkowców koagulazo-dodatnich (Staphylococcus aureus i innych gatunków) — Część 2: Metoda z zastosowaniem pożywki agarowej z plazmą króliczą i fibrynogenem

Title (EN)

Microbiology of the food chain — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 2: Method using rabbit plasma fibrinogen agar medium

Step-by-step procedure

1. Sample

   Prepare the sample, the initial suspension (10⁻¹) and the dilutions according to the ISO 6887 series (surfaces — ISO 18593; sampling: ISO/TS 17728 food, ISO 707 milk, ISO 17604 carcases, ISO 13307 primary production).

2. Pour plating

   With a sterile pipette transfer 1 ml of the liquid sample or of the initial suspension (and of the successive dilutions) into Petri dishes, in the number laid down by ISO 7218.

3. Pouring of the RPFA

   Pour into each plate at once 18–20 ml of freshly prepared complete RPFA medium (44–47 °C), so as to obtain a layer of ≥ 3 mm; mix it carefully and leave it to set on a horizontal surface.

4. Incubation

   Incubate the inverted plates at 34–38 °C for 24 h ± 2 h; mark the typical colonies on the base. If there are no colonies or no typical colonies — reincubate for a further 24 h ± 2 h (48 h ± 4 h in total) and mark the new typical colonies.

5. Counting

   Count the typical colonies (black, grey or white, small, with an opaque zone of precipitation); distinguish Proteus (browning, swarming). Retain the plates with ≤ 300 colonies, including ≤ 100 typical ones, one plate having ≥ 10 colonies; at low numbers use duplicates (sum ≥ 10).

6. Result

   Calculate according to ISO 7218 the number of coagulase-positive staphylococci in cfu/g, ml, cm² or per sampling device; where there are no colonies — the result according to ISO 7218 for special cases; compare it with the criteria of 2073/2005.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Coagulase-positive staphylococci may produce enterotoxins — examinations in a suitably equipped laboratory under the supervision of a microbiologist, careful disposal of the materials after the incubation
• Rabbit plasma and fibrinogen — materials of animal origin, sterilised by filtration, stored in a refrigerator
• Hot agar at 44–47 °C — protection against burns

Frequently asked questions