🦠 Presumptive Pseudomonas spp. in meat and meat products — surface plating on CFC agar (25 °C, 44 h)
In short
0. 1 ml portions of the initial suspension and of the decimal dilutions are spread with a spreader on CFC agar (base medium with cephalothin 50 µg/ml, sodium fusidate 10 µg/ml and cetrimide 10 µg/ml), incubated at 25 °C ± 1 °C for 44 h ± 4 h, and the colonies are confirmed by the oxidase test (positive); the result is given as the number of presumptive Pseudomonas spp. The test is performed according to PN-EN ISO 13720:2010 (ISO 13720:2010); Incubation: 25 °C ± 1 °C, 44 h ± 4 h. The procedure comprises 6 steps; it is used for: Meat and meat products, including poultry products — enumeration of presumptive Pseudomonas spp. (the scope of the standard), The CFC medium according to the manufacturer: isolation of Pseudomonas spp. from meat and meat products, dairy products, water and environmental samples, In-house and official laboratories examining raw meat — commercial medium (base + CFC supplement).
At a glance
- Standard: PN-EN ISO 13720:2010 (ISO 13720:2010)
- Category: Microbiology
- Procedure steps: 6
- Incubation: 25 °C ± 1 °C, 44 h ± 4 h
- Inoculation: 0.1 ml of the initial suspension and of the decimal dilutions, spread over the surface with a spreader; 1 plate/dilution, ≥ 2 successive dilutions
- Inhibitors in CFC: cephalothin 50 µg/ml, sodium fusidate 10 µg/ml, cetrimide 10 µg/ml (5 + 1 + 1 ml of the solutions per 100 ml of medium)
Overview
ISO 13720:2010 specifies a method for the enumeration of presumptive Pseudomonas spp. present in meat and meat products, including poultry products. According to the standard, presumptive Pseudomonas spp. are bacteria which at 25 °C form colonies on agar with cephalothin, sodium fusidate and cetrimide (CFC) and give a positive oxidase test. In Poland it is in force as PN-EN ISO 13720:2010 (it replaced PN-ISO 13720:1999).
The CFC medium is commercially available as a base (Pseudomonas Agar Base) with a CFC supplement; the manufacturer of the medium describes it as a selective medium for the detection and enumeration of Pseudomonas spp. in accordance with ISO 13720 (meat and meat products), with different supplements for milk (ISO/TS 11059, supplement PP) and for water (ISO 16266, supplement CN). Typical composition of the base: gelatin peptone 16.0 g/l, casein hydrolysate 10.0 g/l, anhydrous potassium sulfate 10.0 g/l, anhydrous magnesium chloride 1.4 g/l, agar 15.0 g/l, pH 7.1 ± 0.2; the potassium sulfate and the magnesium chloride favour the production of pyocyanin, and the glycerol is the source of carbon and energy. The CFC supplement contains cetrimide, fusidic acid and cephaloridine; all the colonies that grow on CFC are regarded as suspect Pseudomonas spp., and the colonies that are oxidase-positive and do not ferment glucose are confirmed.
Method principle
An initial suspension and decimal dilutions are prepared from the sample. The solid selective medium — CFC agar — is inoculated with a defined amount of the initial suspension, and further plates are prepared from the decimal dilutions; in accordance with ISO 7218 one plate per dilution is used, with at least two successive dilutions. With a pipette 0.1 ml of the initial suspension is transferred onto a CFC plate and 0.1 ml of the first decimal dilution onto the next one (with a new sterile pipette), after which the liquid is spread with a sterile spreader over the whole surface of the agar. The plates are incubated at 25 °C ± 1 °C for 44 h ± 4 h. The colonies of presumptive Pseudomonas spp. are confirmed by the oxidase test (positive), and the number per millilitre or gram is calculated from the number of confirmed colonies per plate. The complete medium is prepared from the base medium (pH after sterilisation 7.2 ± 0.2 at 25 °C; sterilisation for 15 min at 121 °C), to which, after it has been cooled in a bath to 47 °C ± 2 °C, the solutions of the inhibitors are added: per 100 ml of medium 5 ml of cephalothin solution (final concentration 50 µg/ml), 1 ml of sodium fusidate solution (10 µg/ml) and 1 ml of cetrimide solution (10 µg/ml); the solutions of the inhibitors are not kept for longer than 7 days at 5 °C ± 3 °C. About 15 ml of the complete medium is poured into sterile plates; undried plates may be stored for up to 4 weeks at 5 °C ± 3 °C.
Applications
- Meat and meat products, including poultry products — enumeration of presumptive Pseudomonas spp. (the scope of the standard)
- The CFC medium according to the manufacturer: isolation of Pseudomonas spp. from meat and meat products, dairy products, water and environmental samples
- In-house and official laboratories examining raw meat — commercial medium (base + CFC supplement)
Key parameters
| Parameter | Value |
|---|---|
| Incubation | 25 °C ± 1 °C, 44 h ± 4 h |
| Inoculation | 0.1 ml of the initial suspension and of the decimal dilutions, spread over the surface with a spreader; 1 plate/dilution, ≥ 2 successive dilutions |
| Inhibitors in CFC | cephalothin 50 µg/ml, sodium fusidate 10 µg/ml, cetrimide 10 µg/ml (5 + 1 + 1 ml of the solutions per 100 ml of medium) |
| Base medium | pH 7.2 ± 0.2 after sterilisation (121 °C, 15 min); addition of the inhibitors at 47 °C ± 2 °C; about 15 ml/plate |
| Confirmation | positive oxidase test |
| Storage | solutions of the inhibitors ≤ 7 days at 5 °C ± 3 °C; undried plates ≤ 4 weeks at 5 °C ± 3 °C |
Standard
- Standard number
- PN-EN ISO 13720:2010 (ISO 13720:2010)
- Title (PL)
- Mięso i przetwory mięsne — Oznaczanie liczby przypuszczalnych Pseudomonas sp.
- Title (EN)
- Meat and meat products — Enumeration of presumptive Pseudomonas spp.
Step-by-step procedure
-
Preparation of the CFC agar
Prepare the base medium (pH 7.2 ± 0.2 after sterilisation), sterilise it for 15 min at 121 °C, cool it in a bath to 47 °C ± 2 °C and to every 100 ml add 5 ml of cephalothin solution, 1 ml of sodium fusidate solution and 1 ml of cetrimide solution (the solutions ≤ 7 days at 5 °C ± 3 °C); mix carefully and pour about 15 ml into sterile plates (undried plates ≤ 4 weeks at 5 °C ± 3 °C).
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Initial suspension and dilutions
From the meat sample prepare an initial suspension and decimal dilutions.
-
Surface plating
With a pipette transfer 0.1 ml of the initial suspension onto a CFC plate; with a new sterile pipette transfer 0.1 ml of the first decimal dilution onto the next plate (one plate per dilution, at least two successive dilutions). Spread the liquid with a sterile spreader over the whole surface of the agar.
-
Incubation
Incubate the plates at 25 °C ± 1 °C for 44 h ± 4 h.
-
Confirmation
Confirm the colonies of presumptive Pseudomonas spp. by the oxidase test (positive).
-
Calculation and result
From the number of confirmed colonies per plate calculate the number of presumptive Pseudomonas spp. per gram or millilitre of sample.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 25 °C ± 1 °C | incubation for 44 h ± 4 h | — |
| Autoclave 121 °C | sterilisation of the base medium for 15 min | — |
| Water bath 47 °C ± 2 °C | cooling of the medium before the inhibitors are added | — |
| Sterile 1 ml pipettes graduated in 0.1 ml (ISO 835) | dispensing of 0.1 ml | — |
| Sterile spreaders and Petri dishes | surface plating | — |
| Homogeniser (e.g. of the Stomacher type), pH meter | initial suspension, control of the pH | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| CFC base medium (Pseudomonas Agar Base) | — | base of the selective agar; pH 7.2 ± 0.2 |
| Cephalothin solution | — | inhibitor — 50 µg/ml in the complete medium |
| Sodium fusidate solution | — | inhibitor — 10 µg/ml |
| Cetrimide solution | — | inhibitor — 10 µg/ml |
| Oxidase reagent | — | confirmation of the colonies (Pseudomonas are oxidase-positive) |
| Diluent for the initial suspension | — | decimal dilutions |
Health and safety (OHS)
- Raw meat may contain pathogens (Salmonella, Campylobacter) — aseptic working, disinfection, autoclaving of the cultures
- Antibiotics (cephalothin, fusidic acid) and cetrimide — avoid inhaling the powders and contact with the skin, gloves
- Medium at 47 °C and after autoclaving — risk of burns
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-EN ISO 13720:2010 (ISO 13720:2010) — “Meat and meat products — Enumeration of presumptive Pseudomonas spp.”.
How does this method work?
An initial suspension and decimal dilutions are prepared from the sample. The solid selective medium — CFC agar — is inoculated with a defined amount of the initial suspension, and further plates are prepared from the decimal dilutions; in accordance with ISO 7218 one plate per dilution is used, with at least two successive dilutions.
What is the measuring range and accuracy?
Incubation: 25 °C ± 1 °C, 44 h ± 4 h; Inoculation: 0.1 ml of the initial suspension and of the decimal dilutions, spread over the surface with a spreader; 1 plate/dilution, ≥ 2 successive dilutions; Inhibitors in CFC: cephalothin 50 µg/ml, sodium fusidate 10 µg/ml, cetrimide 10 µg/ml (5 + 1 + 1 ml of the solutions per 100 ml of medium); Base medium: pH 7.2 ± 0.2 after sterilisation (121 °C, 15 min); addition of the inhibitors at 47 °C ± 2 °C; about 15 ml/plate.
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 25 °C ± 1 °C, Autoclave 121 °C, Water bath 47 °C ± 2 °C, Sterile 1 ml pipettes graduated in 0.1 ml (ISO 835), Sterile spreaders and Petri dishes, Homogeniser (e.g. of the Stomacher type), pH meter.
Where is this test used?
Meat and meat products, including poultry products — enumeration of presumptive Pseudomonas spp. (the scope of the standard); The CFC medium according to the manufacturer: isolation of Pseudomonas spp. from meat and meat products, dairy products, water and environmental samples; In-house and official laboratories examining raw meat — commercial medium (base + CFC supplement).
What safety precautions apply?
Raw meat may contain pathogens (Salmonella, Campylobacter) — aseptic working, disinfection, autoclaving of the cultures; Antibiotics (cephalothin, fusidic acid) and cetrimide — avoid inhaling the powders and contact with the skin, gloves; Medium at 47 °C and after autoclaving — risk of burns.
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 13720.