Reviewed by: Darwin Benedict, BSc Microb, MPH, Co-Editor. March 10, 2026

Introduction

Salmonella are Gram-negative bacteria belonging to the family Enterobacteriaceae. They are among the most important foodborne pathogens worldwide, causing illnesses such as gastroenteritis and typhoid fever.

Foods commonly associated with Salmonella contamination include poultry, eggs, meat, dairy products, vegetables, and ready-to-eat foods. Because even low numbers of these bacteria can lead to outbreaks, routine monitoring in food laboratories is essential.

The ISO 6579‑1 standard provides a globally accepted laboratory procedure for detecting Salmonella in food, animal feed, and environmental samples.

This guide presents the ISO 6579-1 detection method in a clear step-by-step format, explaining the principle, laboratory procedure, confirmation tests, and interpretation of results.

Why Salmonella Testing Matters

Public health protection
Salmonella infections cause millions of cases of foodborne illness globally each year.

Regulatory requirement
Many food safety regulations require absence of Salmonella in 25 g of food.

Hygiene monitoring
Detection of Salmonella indicates serious contamination or sanitation failure.

Outbreak prevention
Routine testing helps identify contaminated food before it reaches consumers.

Principle of Test method

The method is a qualitative detection method designed to determine whether Salmonella is present or absent in a food sample.

The method involves several stages:

1. Non-selective pre-enrichment to revive injured bacteria.
2. Selective enrichment to promote growth of Salmonella while suppressing competing microbes.
3. Isolation on selective agar plates.
4. Biochemical confirmation of suspect colonies.
5. Serological confirmation using Salmonella antisera.

Materials & Equipment

Consumables

• Sterile sample containers or stomacher bags
• Sterile pipettes and pipette tips
• Buffered Peptone Water (BPW)
• Selective enrichment broths:
  • Rappaport-Vassiliadis Soya broth (RVS)
  • Müller-Kauffmann Tetrathionate-Novobiocin broth (MKTTn)
• Selective agar plates:
  • XLD agar
  • Hektoen Enteric agar
  • Brilliant Green agar
• Biochemical test media (TSI agar, urease medium, lysine decarboxylase medium)
• Sterile inoculating loops

Equipment

• Incubator capable of 37 °C and 41.5 °C
• Stomacher or homogenizer
• Biosafety cabinet (recommended)
• Colony counter
• Autoclave

Personal Protective Equipment (PPE)

• Lab coat
• Gloves
• Eye protection

Step-by-Step Test Procedure

1. Sample Preparation

1. Weigh 25 g of solid food or measure 25 mL of liquid sample.
2. Place the sample into a sterile stomacher bag.
3. Add 225 mL Buffered Peptone Water (BPW).
4. Homogenize using a stomacher for 1–2 minutes.

This produces the initial suspension used for pre-enrichment.

2. Pre-Enrichment

1. Incubate the suspension at 37 °C for 18 ±2 hours.
2. This step allows stressed or injured Salmonella cells to recover before selective enrichment.

Pre-enrichment improves the sensitivity of the detection method.

3. Selective Enrichment

Two selective enrichment broths are typically used:

RVS Broth

1. Transfer 0.1 mL of pre-enrichment culture into 10 mL RVS broth.
2. Incubate at 41.5 °C for 24 hours.

MKTTn Broth

1. Transfer 1 mL of pre-enrichment culture into 10 mL MKTTn broth.
2. Incubate at 37 °C for 24 hours.

Selective enrichment encourages growth of Salmonella while inhibiting competing bacteria.

4. Isolation on Selective Agar

Using a sterile loop:

1. Streak cultures from the enrichment broths onto selective agar plates:
   • XLD agar
   • Hektoen Enteric agar
   • Brilliant Green agar
2. Incubate plates at 37 °C for 24 hours.

5. Colony Identification

Typical Salmonella colony appearance:

XLD agar

• Red colonies with black centers (H₂S production)

Hektoen Enteric agar

• Green or blue-green colonies with black precipitate

Brilliant Green agar

• Pink or white colonies surrounded by a red medium

Suspected colonies should be selected for confirmation.

Biochemical Confirmation Tests

Common biochemical tests include:

Test	Expected Result for Salmonella
TSI agar	Red slant / Yellow butt
Gas production	Often positive
H₂S production	Positive (black precipitate)
Urease	Negative
Lysine decarboxylase	Positive
Motility	Positive

These characteristics help distinguish Salmonella from other enteric bacteria.

Serological Confirmation

Serological confirmation is performed using slide agglutination tests with Salmonella antisera.

Procedure

1. Place a drop of saline on a slide.
2. Mix a suspected colony into the drop.
3. Add specific Salmonella antisera.
4. Observe for agglutination (clumping).

Interpretation

Agglutination indicates that the isolate belongs to the genus Salmonella.

Further serotyping can identify specific strains such as S. Typhimurium or S. Enteritidis.

Result Interpretation

Result	Interpretation	Action
No Salmonella detected	Sample compliant	Acceptable for consumption
Suspected colonies unconfirmed	Additional testing needed	Repeat confirmation
Confirmed Salmonella detected	Serious contamination	Investigate source and take corrective action

Most food safety regulations require:

Absence of Salmonella in 25 g of food product.

Recording & Reporting

A laboratory report should include:

• Sample identification
• Date of analysis
• Media and enrichment broths used
• Colony morphology observations
• Biochemical test results
• Serological confirmation results
• Final result: Presence/absence of Salmonella in 25g