🦠 Coagulase-positive staphylococci in food — detection and MPN of low numbers (Giolitti-Cantoni broth)
In short
The sample or the dilutions are inoculated into modified Giolitti-Cantoni broth with potassium tellurite and incubated anaerobically at 37 °C for 24 h and 48 h; from the tubes with reduced tellurite (blackening) subcultures are made onto Baird-Parker agar (37 °C, 24–48 h) or RPFA, the coagulase is confirmed and the presence or the MPN is read from tables — a method for dried products, in which the staphylococci are damaged and few in number. The test is performed according to PN-EN ISO 6888-3:2004 (ISO 6888-3:2003); Enrichment: modified Giolitti-Cantoni broth with potassium tellurite; 37 °C anaerobically (a plug of agar/paraffin or a jar); reading after 24 h and 48 h. The procedure comprises 6 steps; it is used for: Dried products and products of low water activity (milk powder, spices, concentrates), in which the staphylococci are damaged and few in number, Detection of the presence of coagulase-positive staphylococci in a defined amount of product (e.g. 1 g, 10 g), Enumeration of low numbers below the limit of the plate methods ISO 6888-1/-2.
At a glance
- Standard: PN-EN ISO 6888-3:2004 (ISO 6888-3:2003)
- Category: Microbiology
- Procedure steps: 6
- Enrichment: modified Giolitti-Cantoni broth with potassium tellurite; 37 °C anaerobically (a plug of agar/paraffin or a jar); reading after 24 h and 48 h
- Indicator in the broth: reduction of the potassium tellurite (blackening) = presumptive staphylococci
- Isolation: Baird-Parker agar at 37 °C, 24 h and 48 h (reduction of the tellurite + reaction with egg yolk) or RPF agar
Overview
ISO 6888-3:2003 specifies a horizontal method for the enumeration and the detection of coagulase-positive staphylococci by the most probable number (MPN) technique in products intended for human consumption and for the feeding of animals and in environmental samples from the production and the distribution of food. It is recommended for products in which the staphylococci are likely to be damaged (stressed) and few in number, for example in dried products. Coagulase-positive staphylococci are chiefly Staphylococcus aureus, but coagulase is also produced by S. intermedius and by some strains of S. hyicus. They are defined as bacteria forming typical and/or atypical colonies on the surface of a selective medium and showing a positive coagulase reaction or a specific reaction with rabbit plasma on RPF agar; the confirmation rests on a clearly positive coagulase reaction, and weakly positive strains are distinguished by additional tests (e.g. thermonuclease, IDF 83). In the teaching materials of the UWM this method figures as the "determination of the presence or of the MPN" of staphylococci: inoculation into Giolitti-Cantoni medium in 3 replicates (double-strength where 10 cm³ of material is used), 37 °C/24–48 h anaerobically, blackening = a positive result, confirmation on RPF or on Baird-Parker. The standard was published with a corrigendum (subclause 9.1.1); in Poland it is in force as PN-EN ISO 6888-3:2004.
Method principle
Detection: a selective liquid medium is inoculated with a defined amount of the liquid sample or of the initial suspension and incubated anaerobically at 37 °C for 24 h and 48 h — the presence of presumptive coagulase-positive staphylococci is indicated by the reduction of the potassium tellurite (anaerobiosis is obtained with a plug of agar or of paraffin in each tube or by incubation in an anaerobic jar/incubator). From the positive tubes after 24 h, and from the remaining ones after 48 h, subcultures are made onto the surface of Baird-Parker agar and incubated at 37 °C for 24 h and 48 h — presumptive staphylococci are indicated by the reduction of the tellurite and the reaction with egg yolk; the typical and atypical colonies are confirmed by the coagulase reaction. Alternatively subcultures are made onto rabbit plasma fibrinogen agar, where colonies with the specific reaction are a confirmation. Result: "present/absent" in x g or x ml. Enumeration: serial dilutions of the product are inoculated into the liquid selective medium, the procedure is as above, and the MPN per g or ml is read from the tables for the confirmed dilutions. Modified Giolitti-Cantoni broth (base medium, per 1000 ml; single-strength / double-strength): casein digest 10/20 g, meat extract 5/10 g, yeast extract 5/10 g, lithium chloride 5/10 g, mannitol 20/40 g, NaCl 5/10 g, glycine 1.2/2.4 g, sodium pyruvate 3/6 g, Tween 80 1/2 g; a solution of potassium tellurite is added to the medium (the further part of clause 5). Diluents according to ISO 6887, ISO 8261 or the standard for the product.
Applications
- Dried products and products of low water activity (milk powder, spices, concentrates), in which the staphylococci are damaged and few in number
- Detection of the presence of coagulase-positive staphylococci in a defined amount of product (e.g. 1 g, 10 g)
- Enumeration of low numbers below the limit of the plate methods ISO 6888-1/-2
- Environmental samples from the production and the distribution of food
- Feed
Key parameters
| Parameter | Value |
|---|---|
| Enrichment | modified Giolitti-Cantoni broth with potassium tellurite; 37 °C anaerobically (a plug of agar/paraffin or a jar); reading after 24 h and 48 h |
| Indicator in the broth | reduction of the potassium tellurite (blackening) = presumptive staphylococci |
| Isolation | Baird-Parker agar at 37 °C, 24 h and 48 h (reduction of the tellurite + reaction with egg yolk) or RPF agar |
| Confirmation | coagulase reaction (typical and atypical colonies) or the specific reaction on RPFA; weakly positive ones — thermonuclease |
| Result | present/absent in x g or ml; MPN per g or ml from the tables for the confirmed dilutions |
| Base medium (per 1 l, single-strength) | casein digest 10 g, meat extract 5 g, yeast extract 5 g, LiCl 5 g, mannitol 20 g, NaCl 5 g, glycine 1.2 g, sodium pyruvate 3 g, Tween 80 1 g |
Standard
- Standard number
- PN-EN ISO 6888-3:2004 (ISO 6888-3:2003)
- Title (PL)
- Mikrobiologia żywności i pasz — Horyzontalna metoda oznaczania liczby gronkowców koagulazo-dodatnich (Staphylococcus aureus i innych gatunków) — Część 3: Wykrywanie obecności i oznaczanie małych liczb metodą NPL
- Title (EN)
- Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 3: Detection and MPN technique for low numbers
Step-by-step procedure
-
Sample
Prepare the initial suspension and the dilutions according to ISO 6887 (the appropriate part), ISO 8261 or the standard for the product.
-
Inoculation of the broth
Detection: a defined amount of the liquid sample or of the initial suspension into a tube of the selective broth (with 10 ml of material — double-strength). Enumeration: serial dilutions into tubes of the broth (in practice three tubes per dilution).
-
Anaerobic incubation
Ensure anaerobiosis (a plug of agar or of paraffin, or an anaerobic jar/incubator) and incubate at 37 °C; assess after 24 h and 48 h — blackening (reduction of the tellurite) means a presumptively positive tube.
-
Plating onto a solid medium
From the positive tubes after 24 h and from all the remaining ones after 48 h plate onto the surface of Baird-Parker agar (37 °C, 24 h and 48 h) — the colonies with reduction of the tellurite and a reaction with egg yolk are presumptive; alternatively onto RPFA, where the specific reaction with the plasma is a confirmation.
-
Confirmation
Confirm the typical and atypical colonies from Baird-Parker by the coagulase reaction (according to the UWM: 0.1 cm³ of the broth culture + 0.3 cm³ of rabbit plasma, 37 °C, reading after 4–6 h and, where it is negative, after a further 24 h); where the reaction is weak — the thermonuclease test.
-
Result
Detection: present/absent in x g or ml. Enumeration: from the number of confirmed tubes in the individual dilutions read the MPN per g or ml from the tables.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator at 37 °C | — | |
| Anaerobic jar or anaerobic incubator (alternatively plugs of agar/paraffin) | anaerobic conditions in the tubes | — |
| Tubes with single-strength and double-strength broth, pipettes | the arrangement of 3 tubes per dilution (UWM) | — |
| Plates with Baird-Parker agar or RPFA | isolation and confirmation | — |
| Tubes for the coagulase test, incubator at 37 °C | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Modified Giolitti-Cantoni broth (base medium + potassium tellurite solution) | — | selective enrichment of the staphylococci under anaerobic conditions |
| Baird-Parker agar (with tellurite and egg yolk) or rabbit plasma fibrinogen agar | — | isolation and preliminary identification |
| Rabbit plasma | — | coagulase test (confirmation) |
| Diluents according to ISO 6887 / ISO 8261 | — | initial suspension and dilutions |
Health and safety (OHS)
- Coagulase-positive staphylococci produce enterotoxins — work in a suitably equipped laboratory under the supervision of a microbiologist, careful disposal of the materials
- Potassium tellurite is toxic — gloves, avoid raising dust; lithium chloride — avoid contact
- Anaerobic jars with gas generators — according to the manufacturer's instructions; paraffin — hot when the plugs are applied
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 6888-3:2004 (ISO 6888-3:2003) — “Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 3: Detection and MPN technique for low numbers”.
How does this method work?
Detection: a selective liquid medium is inoculated with a defined amount of the liquid sample or of the initial suspension and incubated anaerobically at 37 °C for 24 h and 48 h — the presence of presumptive coagulase-positive staphylococci is indicated by the reduction of the potassium tellurite (anaerobiosis is obtained with a plug of agar or of paraffin in each tube or by incubation in an anaerobic jar/incubator). From the positive tubes after 24 h, and from the remaining ones after 48 h, subcultures are made onto the surface of Baird-Parker agar and incubated at 37 °C for 24 h and 48 h — presumptive staphylococci are indicated by the reduction of the tellurite and the reaction with egg yolk; the typical and atypical colonies are confirmed by the coagulase reaction.
What is the measuring range and accuracy?
Enrichment: modified Giolitti-Cantoni broth with potassium tellurite; 37 °C anaerobically (a plug of agar/paraffin or a jar); reading after 24 h and 48 h; Indicator in the broth: reduction of the potassium tellurite (blackening) = presumptive staphylococci; Isolation: Baird-Parker agar at 37 °C, 24 h and 48 h (reduction of the tellurite + reaction with egg yolk) or RPF agar; Confirmation: coagulase reaction (typical and atypical colonies) or the specific reaction on RPFA; weakly positive ones — thermonuclease.
How long does the test take?
The procedure comprises 6 steps. The standard does not give a duration for every stage — the laboratory's own procedure decides.
What equipment is required?
Incubator at 37 °C, Anaerobic jar or anaerobic incubator (alternatively plugs of agar/paraffin), Tubes with single-strength and double-strength broth, pipettes, Plates with Baird-Parker agar or RPFA, Tubes for the coagulase test, incubator at 37 °C.
Where is this test used?
Dried products and products of low water activity (milk powder, spices, concentrates), in which the staphylococci are damaged and few in number; Detection of the presence of coagulase-positive staphylococci in a defined amount of product (e.g. 1 g, 10 g); Enumeration of low numbers below the limit of the plate methods ISO 6888-1/-2; Environmental samples from the production and the distribution of food; Feed.
What safety precautions apply?
Coagulase-positive staphylococci produce enterotoxins — work in a suitably equipped laboratory under the supervision of a microbiologist, careful disposal of the materials; Potassium tellurite is toxic — gloves, avoid raising dust; lithium chloride — avoid contact; Anaerobic jars with gas generators — according to the manufacturer's instructions; paraffin — hot when the plugs are applied.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-EN ISO 6888-3.