Testing for Salmonella in Food and Associated Samples

The genus Salmonella belongs to the family Enterobacteriaceae. Salmonella are Gram-negative, non-spore forming rods. There are over 2,500 serovars of Salmonella, which are characterized according to somatic (O) and flagellar (H) antigens.

Salmonellosis is the most frequently reported cause of foodborne illness. An estimated one million cases occur annually in the United States; of these, approximately 35,000 are laboratory-confirmed cases reported to Centers for Disease Control and Prevention.

The ingestion of 1-10 cells can constitute a human infectious dose. Low cell numbers can be highly infectious, especially in high fat foods where the microorganism can escape the gastric acidity and be released in the intestine through bile mediated dispersion of the lipids.

Despite the general perception that chicken and egg products are the primary source of Salmonella infections, many outbreaks in recent years have been associated with tomatoes, peanut butter, and vegetable sprouts. Other affected foods include raw meat, powdered infant formula, milk and dairy products, fish, shrimp, salad dressing, cake mixes and chocolate.

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Salmonella test options

ISO 6579-1:2017 Standard Method

A culture media-based standard reference method cited in Commission Regulation (EC) No 2073/2005 and other national regulations and guidelines for the detection of Salmonella species in a range of different foods, animal feeds, environmental samples and samples from the primary production environment.

This is a summary of the ISO 6579 Part 1: Detection of Salmonella spp. in food (including milk and milk products), in animal feed, in animal faeces, and in environmental samples from the primary production stage.

Workflow overview for ISO 6579:2017 Part 1

Day 0

25 g + 225 mL Buffered Peptone Water (BPW)
1 swab + 10 mL BPW
1 sponge + 100 mL BPW
1 wipe + 225 mL BPW

Incubate for 18±2 h at 36±2°C

Day 1

Add 100 μL of primary enrichment to 10 mL of Rappaport-Vassiliadis Peptone Soya broth (RVS) or Modified Semi-solid Rappaport Vassiliadis (MRSV) agar.

Incubate for 24±3 h at 41.5±1°C

     

Add 100 μL of primary enrichment broth to 10 mL of Muller-Kauffman Tetrathionate Broth (MKTTn) supplemented with Novobiocin and Iodine-iodide.

Incubate for 24±3 h at 36±2°C

Day 2

Plate 10 μL of secondary enrichment broth onto Xylose Lysine Deoxycholate Agar (XLD) and a secondary isolation medium
and
Plate 0.1 μL from MSRV or RVS onto XLD and a secondary isolation medium

Incubate XLD plates for 24±3 h at 36±2°C 
(incubate the secondary isolation medium according to the manufacturer's instructions)

Day 3

Confirm up to five presumptive-positive colonies by selecting either well isolated colonies where there is sufficient growth, or by first sub-culturing on to a non-selective agar such as Nutrient Agar.

Incubate for 24±3 h at 36±2°C

Day 4-5


Biochemical confirmation
Refer to ISO standard for method details.

Acid and gas production: Triple Sugar Iron Agar (TSI)
Detection of urease: Urea Agar
Detection of β-galactosidase: L-Lysine Decarboxylation
Indole reaction: Tryptone Water, Kovac’s Reagent

  

Serological confirmation
Refer to ISO standard for method details.

Detection of O, Vi and H antigens:
Salmonella O Polyvalent Agglutinating Sera
Salmonella Vi Polyvalent Agglutinating Sera
Salmonella H Polyvalent Agglutinating Sera


Thermo Scientific™ SureTect Salmonella species PCR Assay Method

A rapid real-time PCR method for the enrichment, detection and confirmation of Salmonella species in food, feed products and environmental samples:

  • Validated according to ISO 16140-part 2:2016 standard
  • Streamlined and rapid workflows – 96-well runs can be prepared with just a few simple steps
  • Single enrichment protocols for faster time-to-result and streamlined workflow
  • Pre-dispensed reagents, reducing handling steps and risk of error
  • Universal PCR conditions for detecting multiple targets in the same run
  • Intuitive, user-friendly software, avoiding subjective interpretation
  • ‘Plug and play’ ready-to-use instruments out of the box
  • Reduced time to result: 1 day compared with up to 5 days for the ISO reference method
Thermo Scientific™ SureTect Salmonella species PCR Assay Method

Workflow overview for SureTect Salmonella Species PCR Assay validated according to ISO 16140-part 2:2016 method

Day 0

Environmental Samples

 
 

• 25 g + 225 mL BPW

• 1 swab + 10 mL BPW

• 1 sponge + 100 mL BPW

• 1 wipe + 225 mL BPW

20–24 h at 37±1°C

Cocoa and chocolate products

 

• Enrichment broth according to the ISO 6887-4:2017 standard

20-28 h at 36±2°C

 

• Pre-warmed BPW

22-30 h at 36±2°C

Raw beef meats
 

 

25 g + 225 mL pre-warmed BPW

9–24 h at 41.5±1°C


Broad range of foods*
 
 

• 25 g + 225 mL BPW + 12 mg/L Novobiocin

20–24 h at 37±1°C

 

• 25 g + 225 mL ONE Broth Salmonella + 12 mg/L Novobiocin

20–24 h at 37±1°C

 

*including meat, fish, dairy, seafood, fruit & vegetables

Powdered infant formula (10g)
 

25 g + 225 mL BPW

16–20 h at 37±1°C

Powdered milk products

 

Up to 375 g diluted 1:10 with BPW + 6 mg/L Vancomycin

18–22 h at 37±1°C

Day 1

 
Add 10 μL of SureTect Proteinase K to each required SureTect Lysis Tube (supplied pre-filled with Lysis Reagent 1).
 
Add 10 μL enriched sample to the SureTect Lysis Tube. For the 7500 Fast; add 10 μL sterile nuclease-free water to a SureTect Lysis Tube as a negative control.
 
Incubate SureTect Lysis Tubes in the Applied Biosystems SimpliAmp Thermal Cycler at 37±1°C for 10 minutes followed by 95±1°C for 5 minutes, and 2 minutes at 10±1°C.
 
Dilute 1:5 the lysate of cocoa and chocolate products in BPW to avoid PCR inhibition and transfer 20 μL of lysate to SureTect PCR Tubes.

Load SureTect PCR Tubes to the Applied Biosystems 7500 Fast or the Applied Biosystems QuantStudio 5 PCR Instrument. Start PCR and review results at end of run.

(Report negative results)

Day 2

Confirm PCR positive results by plating 10 μL of enrichment broth onto Brilliance ™ Salmonella Agar or XLD for cocoa or chocolate product samples.
For samples with high background microflora first subculture in RVS Broth (incubate for 24 h±3 h at 41.5°C±1°C).
 
Confirm presumptive-positive colonies with the Oxoid™ Salmonella Latex Kit, MicroBact™GNB24E or confirmatory tests described in ISO 6579-1:2017 method or with an ISO 16140-part 6:2019 validated confirmatory test.

Thermo Scientific RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Assay method

A rapid method for the enrichment, detection and confirmation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis in raw pork and poultry, ready-to-eat and ready-to-reheat pork and poultry, production environment samples and primary production samples (PPS):

  • Validated according to ISO 16140-part 2:2016 standard
  • First validated multiplex PCR assay for simultaneous detection of Salmonella species, and Salmonella serovars; Typhimurium and Enteritidis
  • Designed as a tool specifically for Salmonella control programs in pork and poultry production
  • Streamlines testing workflow and reduces waiting time for product release or intervention
  • Reduced time to results as little as 16 hours compared with up to 5 days for standard culture method
Thermo Scientific RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Assay method

Workflow overview for RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Assay validated according to ISO 16140-part 2:2016 standard.

Day 0

Raw and ready-to-eat pork and poultry samples:

Add 25 g sample to 225 ml BPW (ISO-meat peptone) supplemented with 12 mg/L Novobiocin at room temperature.

Incubate at 41.5±1°C for 16±2 hours

 

Production environment samples:

Add 25 g of production environment sample to 225 ml of BPW (ISO-meat peptone). Add 1 swab to 10 ml of BPW (ISO-meat peptone). Add 1 sponge to 100 ml of BPW (ISO-meat peptone). Add 1 wipe to 225 ml of BPW (ISO-meat peptone).

Incubate at 41.5±1°C for 16±2 hours

Day 1

Remove ~1.5 mL of enrichment to a sterile sealable tube.

First, add 10 μL of Proteinase K to each required Lysis Tube.

Second, add 10 μL of enrichment to the Lysis Tube.

Incubate SureTect Lysis Tubes in the Applied Biosystems SimpliAmp Thermal Cycler at 37±1°C for 10 minutes followed by 95±1°C for 5 minutes, and 2 minutes at 10±1°C.

Transfer 20 μL to RapidFinder PCR Tubes.

 

7500 Fast PCR Instrument

 

QuantStudio 5 PCR Instrument

Load samples to the Applied Biosystems 7500 Fast Food Safety PCR Instrument, with the addition of a negative control tube (prefilled with Lysis Reagent 1) containing nuclease free sterile water and start PCR, review results at end of run (approx. 45 min)

OR

Load into the Applied Biosystems QuantStudio 5 Food Safety PCR Instrument and start PCR, review results at end of run (approx. 50 min)
 

Report PCR-negative results and confirm PCR-positive results by culturing onto Brilliance™ Salmonella Agar

Day 2

Confirm colonies grown on the Brilliance Salmonella Agar using biochemical and serological procedures described in ISO 6579-1:2017 or with an ISO 16140-part 6:2019 validated confirmatory test.

 

 

 

Workflow overview for Thermo Scientific RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex Flex PCR Assay according to ISO 16140-part 2:2016 standard

Day 0

Primary production samples (PPS):
Add 25 g sample to 225 mL Tetrathionate (Hajna) Broth (TT Broth) supplemented with Iodine-iodide

Incubate at 37±1°C for 18±2 hours

Day 1

1 mL of enriched sample is transferred in 9 mL BPW

Incubate at 37±1°C for 5±1 hour

KingFisher Flex Instrument

500 μL of secondary enrichment is loaded onto the KingFisher Flex Instrument

Allow lysates to cool at room temperature for at least 2 min, then transfer 20 μL to RapidFinder Salmonella PCR Tubes

 

7500 Fast PCR Instrument

OR

QuantStudio 5 PCR Instrument

Load PCR Tubes into the Applied Biosystems 7500 Fast Food Safety Instrument, with the addition of a negative control tube containing nuclease free sterile water and start PCR, review results at end of run (approx. 45 min)   Load PCR Tubes into Applied Biosystems QuantStudio 5 Food Safety Instrument and start PCR, review results at end of run (approx. 50 min)
 

Report PCR-negative results and confirm PCR-positive results by culturing onto Brilliance™ Salmonella Agar.

Day 2

Confirm colonies grown on the Brilliance Salmonella Agar using Oxoid Salmonella Latex Test (Salmonella species), or biochemical and serological procedures (Salmonella Enteritidis and Salmonella Typhimurium) described in ISO 6579-1:2017 or with an ISO 16140-part 6:2019 validated confirmatory test.

 


Thermo Scientific™ Salmonella Precis™ Method

A quick and easy method for the enrichment, detection and confirmation of Salmonella species from food, animal feed and environmental samples:

  • Validated according to ISO 16140-part 2:2016 standard
  • Simple procedure—no specialised equipment required
  • Single overnight enrichment
  • Single sample transfer
  • Single 24-hour plate incubation
  • Quick and convenient confirmation: Oxoid Salmonella Latex Test or ISO 6579:2017 standard tests
  • Reduced time to result: 2 days compared with up to 5 days for standard culture methods
  • Brilliance Salmonella Agar contains novel Inhibigen technology, giving targeted specificity and reduced background flora
Thermo Scientific™ Salmonella Precis™ Method

Workflow overview for Thermo Scientific Salmonella Precis Method validated according to NF VALIDATION for AFNOR Certification

Day 0

Food and feed

Environmental samples

Up to 375 g of milk powder, infant formula* and infant cereals*

Up to 375 g of cocoa and chocolate products

Up to 150g animal feed

25 g + 225 mL BPW (ISO)
+ 12 mg/L 
Novobiocin

16-20 h at 37±1 °C

or

25 g + 225 mL
ONE Broth Salmonella

16-20 h at 41±1 °C

 

• 25 g + 225 mL

• 1 wipe + 225 mL

• 1 swab + 10 mL

• 1 sponge + 100 mL


BPW (ISO)

16-20 h at 37±1 °C

 

or

ONE Broth Salmonella

16-20 h at 41±1 °C

Up to 375 g
diluted 1:10 with BPW (ISO)
+ 6 mg/L Vancomycin

19-23 h at 37±1 °C

Up to 375 g
diluted 1:10 with prewarmed BPW (ISO)

22-28 h at 36±2°C

or

Recommendations of the ISO 6887-4:2017 method
20-26 h at 36±2 °C

 

Up to 150 g diluted 1:10 with BPW (ISO)  + 12 mg/L Novobiocin

20-26 h at 36±2 °C

Day 1

Inoculate onto Brilliance Salmonella Agar

Incubate for 24±2 h at 37±1 °C

Day 2

Confirm typical colonies as Salmonella using Oxoid Salmonella Latex Test

or

ISO 6579-1:2017 confirmatory tests tests or with an ISO 16140-part 6:2019 validated confirmatory test.

 

*with or without probiotics


Food Microbiology Testing Methods: Salmonella species

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