🦠 Detection of Salmonella spp.

Microbiology PN-EN ISO 6579-1

Overview

Salmonella is a genus of Gram-negative rods belonging to the Enterobacteriaceae family, and one of the most important foodborne pathogens worldwide. It causes salmonellosis — a disease manifested by diarrhoea, fever, and abdominal pain. The most common species are S. Enteritidis (eggs, poultry) and S. Typhimurium (meat, vegetables).

Standard PN-EN ISO 6579-1 is the "gold standard" for Salmonella detection in food, feed, and environmental samples from food production facilities. The method is horizontal — applicable to all types of food and feed. It is required by Regulation (EC) 2073/2005 (microbiological criteria) and national monitoring programmes.

Criterion: Salmonella — absent in 25 g (NOT DETECTED/ABSENT) for most ready-to-eat products, meat, eggs, and infant foods. Detection of Salmonella in food results in batch withdrawal from the market.

Method principle

Four-stage method based on classical culture:

1. **Pre-enrichment** — sample in buffered peptone water (BPW), 18 h/37°C. Resuscitation of injured cells. 2. **Selective enrichment** — inoculation into RVS (Rappaport-Vassiliadis) 41.5°C/24 h and MKTTn (Müller-Kauffmann with tetrathionate) 37°C/24 h. Selective inhibition of competing flora. 3. **Plating on selective-differential media** — XLD agar + second medium (BGA, chromogenic). Typical colonies: black centre on XLD (H₂S production). 4. **Biochemical and serological confirmation** — TSI, LDC, urea, indole, VP, agglutination with antisera.

Applications

• Ready-to-eat food control (Regulation 2073/2005)
• Poultry, pork, and beef testing
• Eggs and egg product control
• Vegetables, fruits, and sprouts testing
• Environmental monitoring of food production facilities (swabs)
• Feed and feed ingredient testing
• Water and environmental testing (method adaptation)

Key parameters

Standard

Standard number

PN-EN ISO 6579-1:2017

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania, oznaczania liczby i serotypowania Salmonella — Część 1: Wykrywanie Salmonella spp.

Title (EN)

Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1: Detection of Salmonella spp.

Step-by-step procedure

1. Sample preparation

   Weigh 25 g of sample into a stomacher bag. Add 225 mL of buffered peptone water (BPW). Ratio 1:10.

   ⏱ Time: 5 min

2. Homogenization

   Homogenize in a stomacher for 1–2 minutes (intensity depends on matrix: meat — longer, milk — shorter).

   ⏱ Time: 2 min

3. Pre-enrichment

   Incubate the bag with sample in BPW at 37±1°C for 18±2 hours. Objective: resuscitation of injured Salmonella cells.

   ⏱ Time: 18 h • 🌡 Temperature: 37°C

4. Selective enrichment — RVS

   Transfer 0.1 mL of pre-enrichment to 10 mL of RVS broth. Incubate at 41.5±1°C for 24±3 hours.

   ⏱ Time: 24 h • 🌡 Temperature: 41.5°C

5. Selective enrichment — MKTTn

   Transfer 1 mL of pre-enrichment to 10 mL of MKTTn broth. Incubate at 37±1°C for 24±3 hours.

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

6. Plating on solid media

   From both broths, streak with a loop onto XLD agar and a second medium (BGA or chromogenic). Incubate 24±3 h/37°C.

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

7. Colony reading

   On XLD: typical colonies — pink-red with black centre (H₂S+) or without centre (H₂S–). On BGA: pink-red surrounded by a red zone.

8. Pure culture isolation

   Select min. 5 suspect colonies from each medium. Subculture onto nutrient agar. Incubate 24 h/37°C.

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

9. Biochemical confirmation

   TSI: alkaline slant (K)/acid butt (A), gas +, H₂S +/–. Urea: negative. LDC: positive. Indole: negative (usually). VP: negative.

10. Serological confirmation

    Slide agglutination with polyvalent antisera O and H. Positive reaction = clumping within 1 min.

11. Result interpretation

    Colony confirmed biochemically and serologically = Salmonella DETECTED in 25 g. No typical colonies or negative confirmation = NOT DETECTED.

12. Reporting

    Result: "Salmonella spp. detected / not detected in 25 g". Optionally: serotyping (S. Enteritidis, S. Typhimurium, etc.).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Salmonella — BSL-2 pathogen! Work exclusively in a Class II biosafety cabinet
• Nitrile gloves, laboratory coat, and safety glasses mandatory
• All waste must be autoclaved (121°C/20 min) before disposal
• Disinfect work surfaces after completion (70% ethanol or chlorine solution)
• Positive results must be reported to public health authorities (Sanitary Inspectorate) — legal obligation

Training and video materials

• Salmonella Isolation: XLD & BGA Agar — JoVE (Source: JoVE)

Frequently asked questions