Salmonella species
The genus Salmonella belongs to the family Enterobacteriaceae. Salmonella are Gram-negative, nonspore forming rods. There are over 2,500 serovars of Salmonella, which are characterized according to somatic (O) and flagellar (H) antigens.
Salmonellae are chemoorganotropic, with an ability to metabolize nutrients by both respiratory and fermentative pathways. The bacteria are oxidase and catalase negative, grow optimally at 37°C, and catabolize D-glucose and other carbohydrates with the production of acid and gas. However, they readily adapt to extreme environmental conditions and can grow at both elevated and refrigerator temperatures.
Incidence
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.
Infective dose
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.
The detection of Salmonella in foods before they are consumed is vital for safeguarding public health, and essential for preserving the financial health and reputation of food businesses.
Methods Overview
The following methods illustrated for Salmonella species include reference and alternative validated and certified methods as follows:
- Reference method: ISO 6579-1:2017 Microbiology of the food chain – Horizontal method for the detection, enumeration and serotyping of Salmonella – Part 1: Detection of Salmonella spp.
- Alternative method: Thermo Scientific™ SureTect™ Salmonella species PCR Assay workflow
- Alternative method: Thermo Scientific™ RapidFinder™ Salmonella species, Typhimurium and Enteritidis Multiplex PCR Assay methods
- Alternative method: Thermo Scientific™ Salmonella Precis™ method
Reference Method: ISO 6579:2017 Microbiology of the food chain – Horizontal method for the detection, enumeration and serotyping of Salmonella – Part 1: Detection of Salmonella spp.
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
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 16-20 h at 34-38 °C
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 21-27 h at 40.5-42.5 °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 21-27 h at 34-38 °C
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 21-27 h at 34-38 °C
(incubate the secondary isolation medium according to the manufacturer’s instructions)
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 21-27 h at 34-38 °C
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
Product order information for testing according to ISO 6579:2017 Part 1: Detection of Salmonella spp.
Please note that a range of alternative formats of culture media such as Bagged Enrichment Media and Prepared Plate
Media are available. Please contact your local representative or technical services to find out more.
Alternative Method: ISO 6579:2017 Microbiology of the food chain – Horizontal method for the detection, enumeration and serotyping of Salmonella – Part 1: Detection of Salmonella spp.
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
Enrichment
Single enrichment step
Lysis
Direct lysis automatically carried out in Applied Biosystems SimpliAmp
Run & Read
Results automatically
interpreted
Report/Confirm
Report PCR negative results and confirm PCR positive results after overnight incubation
Validation
The Thermo Scientific™ SureTect™ Salmonella Assay workflow has been validated and approved by NF VALIDATION for AFNOR Certification according to ISO 16140-2:2016 standard against the reference method ISO 6579-1:2017 Detection of Salmonella spp.
AFNOR Certification validation certificate No. UNI 03/07-11/13 is available in PDF format from the AFNOR
website https://nf-validation.afnor.org/en/food-industry/salmonella-spp/.
Workflow overview for SureTect Salmonella Species PCR Assay validated according to ISO 16140-part 2:2016 method
Environmental samples:
1 wipe + 225 mL BPW
1 swab + 10 mL BPW
1 sponge + 100 mL BPW
20–24 h at 36-38 °C
Meat products, seafood and vegetables:
25 g + 225 mL
BPW + 12 mg/L
Novobiocin
20–24 h at 36-38 °C
Raw beef meats:
25 g + 225 mL
pre-warmed BPW
9–24 h at 40.5-42.5°C
Dairy products:
25 g + 225 mL
BPW + 12 mg/L
Novobiocin
20–24 h at 36-38 °C
25 g + 225 mL
ONE Broth Salmonella
12 mg/L
Novobiocin
20–24 h at 36-38 °C
Powdered infant
formula (10 g):
25 g + 225 mL
BPW
16–20 h at 36-38 °C
Powdered milk
products:
375 g + 3375 mL
BPW + 6 mg/L
Vancomycin
18–22 h at 36-38 °C
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 36-38 °C for 10 minutes followed by 94-96 °C for 5 minutes, and 2 minutes at 9-11 °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.
Report negative results
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.
Confirm PCR positive results by plating 10 µL of enrichment broth onto Brilliance™ Salmonella or XLD.
For samples with high background microflora first subculture in RVS Broth (incubate for 21-27 h at 41.5 °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.
Product order information for SureTect Salmonella Species PCR Assay workflow
Please note that a range of alternative formats of culture media such as Bagged Enrichment Media and Prepared Plate Media are available. Please contact your local representative or technical services to find out more.
Alternative Method: Thermo Scientific RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Assays
.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 methods
Enrichment
Single enrichment step for meat and production environment samples
Lysis
Direct lysis and DNA extraction automatically carried out in SimpliAmp Thermal Cycler or KingFisher Flex Purification System
Run & Read
Results automatically
interpreted
Report/Confirm
Report PCR-negative results and confirm Salmonella spp., SE/ST results with single plate and serology
Validation
The RapidFinder™ Salmonella Multiplex Assay and RapidFinder™ Salmonella Multiplex Flex Assay methods have been
validated and approved by NF VALIDATION for AFNOR Certification according to ISO 16140-2:2016 standard against the
reference method ISO 6579-1 2017 Detection and serotyping of Salmonella spp.
NF VALIDATION certificate No. UNI 03/12-01/18 is available in PDF format from the AFNOR
website https://nf-validation.afnor.org/en/food-industry/salmonella-spp/.
Workflow overview for RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Assay validated according to ISO 16140-part 2:2016 standard
Raw and ready-to-eat pork and poultry samples:
25 g + 225 mL BPW (ISO-meat peptone) supplemented with 12 mg/L Novobiocin.
40.5-42.5 °C for 14-18 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).
40.5-42.5 °C for 14-18 hours
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 36-38°C for 10 minutes followed by 94-96 °C for 5 minutes, and 2 minutes at 9-11 °C.
Transfer 20 µL to RapidFinder PCR Tubes
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
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
Primary production samples (PPS): Add 25 g sample to 225 mL Tetrathionate (Hajna) Broth (TT Broth) supplemented with Iodine-iodide
Incubate for 16-20 hours at 36-38 °C
1 mL of enriched sample is transferred in 9 mL BPW
Incubate for 4-6 hours at 36-38 °C
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
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
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) procedures described in ISO 6579-1:2017 standard or with an ISO 16140-part 6:2019 validated confirmatory test.
Product order information for SureTect Salmonella Species PCR Assay workflow
Please note that a range of alternative formats of culture media such as Bagged Enrichment Media and Prepared Plate Media are available. Please contact your local representative or technical services to find out more.
Alternative Method: 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 against ISO 6579-1:2017
- 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
Single overnight enrichment
Single 24-hour plate incubation
Rapid and easy confirmation with Salmonella Latex Test or ISO 579-1 confirmation steps
Reactions on Thermo Scientific™ Oxoid™ Brilliance™ Salmonella Agar
E. coli and other bacteria and yeasts are inhibited by the combination of the Inhibigen and other selective agents in the medium.
Validation
The Salmonella Precis™ method has been validated and approved by NF VALIDATION for AFNOR Certification according to ISO 16140-2:2016 standard against the reference method ISO 6579-1:2017 Detection of Salmonella spp.
For flexibility, confirmation was validated using both the Oxoid Salmonella Latex Test and the tests outlined in ISO 6579:2017.
Alternatively, biochemical panels such as Thermo Scientific™ Microbact™ GNB 24E or Thermo Scientific™
RapID ONE™ Panel, may be used.
AFNOR Certification validation certificate No. UNI 03/06-12/07 is available in PDF format from the AFNOR website https://nf-validation.afnor.org/en/food-industry/salmonella-spp/.
Workflow overview for Thermo Scientific Salmonella Precis Method validated according to NF VALIDATION for AFNOR Certification
Food and feed:
25 g + 225 mL
BPW (ISO)
12 mg/L
Novobiocin
20-26 h at 34-38 °C
or
25 g + 225 mL
ONE Broth
Salmonella
16-22 h at
40.5-42.5 °C
Environmental samples:
25 g + 225 mL
1 wipe + 225 mL
1 swab + 10 mL
1 sponge + 100 mL
BPW (ISO)
20-26 h at 34-38 °C
or
ONE Broth
Salmonella
16-22 h at
40.5-42.5 °C
Up to 375 g
Milk powder, Infant
formula* and Infant
cereals*:
Up to 375 g
diluted 1:10 with
BPW (ISO)
+ 6 mg/L
Vancomycin
18-24 h at 34-38 °C
Up to 375 g Cocoa
and chocolate
products:
Up to 375 g
diluted 1:10 with
prewarmed
BPW (ISO)
22-28 h at 34-38 °C
Recommendations of
the ISO 6887-4:2017 method
20-26 h at 34-38 °C
Up to 150 g
Animal feed:
Up to 150 g
diluted 1:10 with
BPW (ISO)
+ 12 mg/L
Novobiocin
20-26 h at 34-38 °C
Inoculate onto Brilliance Salmonella Agar
Incubate for 22-26 h at 36-38 °C
Confirm typical colonies as Salmonella using Oxoid Salmonella Latex Test
or
ISO 6579-1:2017 confirmatory tests or with an ISO 16140-part 6:2019 validated confirmatory test.
*with or without probiotics
Product order information for Thermo Scientific Salmonella Precis Method
Please note that a range of alternative formats of culture media such as Bagged Enrichment Media and Prepared Plate Media are available. Please contact your local representative or technical services to find out more.
Find out more at thermofisher.com/salmonella-testing-food