🦠 Detection of Salmonella spp.
Classical culture method for Salmonella detection in food and feed. Four-stage process: pre-enrichment → selective enrichment → plating on selective media → confirmation.
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
| Parameter | Value |
|---|---|
| Result | Detected / Not detected in 25 g (presence/absence) |
| Sample size | 25 g (or as specified by the standard) |
| Time to result | 4–5 working days |
| Limit of detection | 1 cell / 25 g (theoretical) |
| Incubation temperature | 37°C (BPW, MKTTn), 41.5°C (RVS) |
| Specificity | >98% for genus Salmonella |
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.
2. Homogenization
Homogenize in a stomacher for 1–2 minutes (intensity depends on matrix: meat — longer, milk — shorter).
3. Pre-enrichment
Incubate the bag with sample in BPW at 37±1°C for 18±2 hours. Objective: resuscitation of injured Salmonella cells.
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.
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.
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.
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.
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
| Equipment | Example | Indicative price |
|---|---|---|
| Incubator 37±1°C | Memmert IN110, Binder BD 115, POL-EKO ST 2 | 5 000–15 000 PLN |
| Incubator / water bath 41.5±1°C | Memmert WNB 14, GFL 1003 water bath | 3 000–10 000 PLN |
| Stomacher (homogenizer) | Seward Stomacher 400 Circulator, BagMixer 400 | 12 000–25 000 PLN |
| Autoclave | Tuttnauer 3870ELV, Systec VX-75 | 30 000–80 000 PLN |
| Class II biosafety cabinet | ESCO Airstream, Thermo MSC-Advantage | 25 000–60 000 PLN |
| Automatic pipettes | Eppendorf Research Plus 1 mL + 10 mL | 800–2 000 PLN/pc. |
| Colony counter | Interscience Scan 500, BZG 30 | 3 000–15 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Buffered peptone water (BPW) | mixture | Peptone 10 g + NaCl 5 g + Na₂HPO₄ 3.5 g + KH₂PO₄ 1.5 g per litre. Merck 1.07228, Oxoid CM0509 |
| RVS broth (Rappaport-Vassiliadis) | — | Selective with malachite green and MgCl₂. Merck 1.07700, Oxoid CM0669. Incubation 41.5°C. |
| MKTTn broth (Müller-Kauffmann) | — | With tetrathionate, ox bile, and novobiocin. Merck 1.05878. Incubation 37°C. |
| XLD agar (Xylose Lysine Deoxycholate) | — | Salmonella colonies: red with black centre (H₂S+). Merck 1.05287, Oxoid CM0469. |
| BGA agar (Brilliant Green Agar) | — | Second selective medium. Salmonella colonies: pink-red. Oxoid CM0329. |
| Nutrient agar | — | For isolation of pure cultures, non-selective medium. |
| TSI medium (Triple Sugar Iron) | — | Confirmation: K/A with H₂S (alkaline slant, acid butt, black precipitate) |
| Urea medium (Christensen) | — | Salmonella — negative (no urea hydrolysis, no colour change) |
| Lysine decarboxylase broth (LDC) | — | Salmonella — positive (purple coloration) |
| Polyvalent Salmonella antisera O and H | — | bioMérieux, Denka Seiken — for slide agglutination |
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