May 12, 2026
Screening Canine Feces for Salmonella species with V-CLASP culture method
- Sarah Nemser1,
- Shelley Rankin2,
- Timothy Frana3,
- Beverly Byrum4,
- Shuping Zhang5,
- Amy Swinford6,
- Sara Lawhon7,
- Jing Cui8,
- Yan Zhang9,
- Shannon Kiener10,
- Megan Miller1,
- Renate Reimschuessel11
- 1FDA CVM;
- 2UPenn (Retired);
- 3Iowa State University (Retired);
- 4Ohio Department of Agriculture (Retired);
- 5University of Missouri;
- 6Texas A&M Veterinary Medical Diagnostic Laboratory;
- 7Texas A&M University - College Station;
- 8Ohio Department of Agriculture;
- 9University of Arizona;
- 10FDA HFP Moffett PT;
- 11FDA CVM (Retired)
- Vet LIRN
External link: https://doi.org/10.1128/jcm.02137-16
Protocol Citation: Sarah Nemser, Shelley Rankin, Timothy Frana, Beverly Byrum, Shuping Zhang, Amy Swinford, Sara Lawhon, Jing Cui, Yan Zhang, Shannon Kiener, Megan Miller, Renate Reimschuessel 2026. Screening Canine Feces for Salmonella species with V-CLASP culture method . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnodxgk5/v1
Manuscript citation:
Reimschuessel R, Grabenstein M, Guag J, Nemser SM, Song K, Qiu J, Clothier KA, Byrne BA, Marks SL, Cadmus K, Pabilonia K, Sanchez S, Rajeev S, Ensley S, Frana TS, Jergens AE, Chappell KH, Thakur S, Byrum B, Cui J, Zhang Y, Erdman MM, Rankin SC, Daly R, Das S, Ruesch L, Lawhon SD, Zhang S, Baszler T, Diaz-Campos D, Hartmann F, Okwumabua O. Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014. J Clin Microbiol. 2017 May;55(5):1350-1368. doi: 10.1128/JCM.02137-16. Epub 2017 Feb 15. PMID: 28202802; PMCID: PMC5405253.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2024
Last Modified: May 12, 2026
Protocol Integer ID: 102742
Keywords: Microbiology, Culture, Salmonella, Animal Diagnostics, canine feces for salmonella species, screening canine fece, detection of salmonella spp, salmonella spp, animal fece, grant evaluation of salmonella, salmonella, salmonella species, study for the vet, participating vet, culture method, vet, clasp culture method, asymptomatic pet
Funders Acknowledgements:
Vet-LIRN
Grant ID: RFA-FD-11-010
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
The culture method was developed and validated by 11 participating Vet-LIRN laboratories as a collaborative effort under the grant Evaluation of Salmonella in Symptomatic and Asymptomatic Pets: Study for the Vet-LIRN Program (https://grants.nih.gov/grants/guide/rfa-files/rfa-fd-11-010.html).
This method is based on ISO 6579:2002 Annex D- “Detection of Salmonella spp. in animal feces and in samples of the primary production stage” with some modifications
Guidelines
Sample submission
Feces should be received fresh or on ice in individually marked containers. At least 1 g of feces is required and 10-25 g of feces is preferred. Frozen samples or fecal swabs are not acceptable. Samples should be set-up in pre-enrichment within 4 hours of receipt. If a sample is to be used for additional testing, then the sample should be split as needed and the results reported separately.
Materials
Critical media and sources include:
| A | B | C | D | |
| Catalog No. | Vendor | Description | Powder or Prepared | |
| *R452672 | Remel | Buffered Peptone Water 500g each | Powder | |
| *R117660 | Remel | RV Broth (10ml) 100/PK | Prepared | |
| *R110463 | Remel | XLT4 Agar 10/PK | Prepared | |
| R01247 | Remel | Bril Green Agar w/Novobiocin 10/PK | Prepared | |
| R061300 | Remel | Lysine Iron Agar (LIA) (slant) 100/PK | Prepared | |
| CM0381B | Remel | LYSINE IRON AGAR 500g | Powder | |
| R064850 | Remel | Triple Sugar Iron Agar (slant) 100/PK | Prepared | |
| R01550 | Remel | MacConkey Agar 10/PK | Prepared | |
| R453802 | Remel | MacConkey Agar 500g each | Powder | |
| R01202 | Remel | Blood Agar,5% Sheep Blood 100/PK | Prepared | |
| *222641 | BD | Salmonella Poly O Antisera | Prepared |
*Items are critical*
The general QC requirements for these media are:
- Verify delivery of the amount ordered, check lot numbers and for impending expiration dates
- Record lot numbers, expiry date, and received date
- Store media as specified by manufacturer
- Perform a visual inspection of each lot for obvious problems such as: cracked damaged plates, detached agar, frozen or melted agar, etc.
- Because contamination testing is routinely performed by the manufacturer, sterility testing is not required. Perform visual inspection prior to inoculation.
Reference strains for controls:
S. Typhimurium ATCC 14028 (used for Specificity)
E. coli ATCC 25922 or ATCC 8739 (used for Selectivity)
Pre-enrichment in non-selective liquid medium
Using a tongue depressor, aseptically weight 10 g of fecal sample into a Whirl-Pak‱ bag
Add 90 mL of ambient temperature buffered peptone water (BPW)
Mix the fecal sample in the bag. Use the necessary quantity of pre-enrichment medium to yield a 1/10 dilution with feces
Incubated at 37 °C ± 1 °C for 18 h ± 2 h.
Enrichment in selective liquid media
Transfer 0.1 mL of the enriched BPW sample to a tube containing 10 ml of Rappaport-Vassiliadis (RV) broth.
Incubate the inoculated RV broth at 41.5 °C ± 1 °C for 24 h ± 3 h.
Note
Care should be taken that the maximum allowed incubation temperature (42.5 °C) is not exceeded.
Plating and Presumptive Identification
Inoculate a XLT4 agar and Brillliant Green with Novobiocin (BGN) agar standard-sized Petri dish
Take a10 µL loop of the enriched RV broth in the RV broth,
Spread the loop on the surface of the XLT4 and Brilliant Green with Novobiocin (BGN) agar plates so that well-isolated colonies will be obtained
Invert the dishes so that the bottom is uppermost, and place them in the incubator set at 37 °C.
After incubation for 24 h ± 3 h examine the plates for the presence of typical colonies of Salmonella.
Mark their position on the bottom of the dish.
Note
Typical colonies of Salmonella grown on XLT4 agar have a black center and a lightly transparent zone of reddish color due to the color change of the indicator. On BGN typical Salmonella colonies are pink.
Confirmation of identity
The recognition of colonies of Salmonella is to a large extent a matter of experience, and their appearance may vary somewhat. Colonies of presumptive Salmonella are subcultured, and then plated out as described in steps 7.1-7.5, and their identity is confirmed by means of appropriate biochemical and serological tests.
Selection of colonies for confirmation
Take from each dish of each selective medium at least five colonies considered to be typical or suspect
Note
If on one dish there are fewer than five typical or suspect colonies, take for confirmation all the typical or suspect colonies.
Streak the selected colonies onto the surface of MacConkey agar plates in a manner which will allow well-isolated colonies to develop
Incubate the inoculated plates at 37 °C ± 1 °C for 24 h ± 3 h.
Use pure cultures for biochemical and serological confirmation.
Biochemical confirmation
Inoculate triple sugar/iron agar (TSI) and Lysine iron agar (LIA) with each of the cultures obtained from the colonies selected.
Using an inoculation needle, inoculate TSI slant by streaking slant and stabbing butt.
Note
Typical Salmonella are alkaline (red) on the slant and acid (yellow) with gas and hydrogen sulfide, H2S (blackening) production in the butt. H2S production may obscure the butt color reaction.
Inoculate LIA slant by stabbing butt twice and then streak slant.
Note
Typical Salmonella are alkaline on the slant and butt with H2S production.
Incubate at 37 +/- 1C for 24 +/- 3h
Salmonella confirmation and serotyping
The detection of the presence of Salmonella O- antigens is tested by slide agglutination with polyvalent anti-O sera from pure colonies from TSI/LIA slants or other appropriate media.
Use the antisera according to the producer's instructions.
Suspect Salmonella colonies can be sub-cultured to blood agar plates for purification, incubated at 37°C ± 1°C for 24 ± 3 h, and identified using matrix-assisted laser desorption and ionization
time of flight mass spectrometry (MALDI-TOF MS) according to the manufacturer’s
protocol.
Freezing Isolates
The preferred method of storing microorganisms is to suspend the organism in a stabilizer such as:
• sterile 10%-20% glycerol in Brucella broth,
• defibrinated whole sheep blood,
• 50% calf serum, or
• commercial cryopreservation tubes.
Incubate the organisms overnight on a blood agar plate under appropriate incubation conditions,
37°C ± 1°C for 24 ± 3 h.
Observe the colonies on the agar surface to ensure the purity and identity of the culture. A gram stain may provide additional information as to the purity and identity of the culture.
Using a very heavy inoculum, suspend the colonies from the agar in the stabilizing fluid to be used. Make a turbid suspension.
For each isolate, label the side of two or more freezer vials with the isolate number and the date the isolate is frozen. A round label is to be placed on the top of each vial with the isolate number.
To ensure quick freezing of the organisms, place the vials in the refrigerator (4°C) for approximately 30 minutes
Remove the vials from the refrigerator, place them in a freezer box and store at < -70°C.
Stocks prepared in this manner can remain at < -70°C indefinitely without significant risk to viability or genetic alterations.
Protocol references
ISO 6579:2002 Annex D- “Detection of Salmonella spp in animal feces and in samples of the primary production stage
CLSI document M22-A3, "Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard - Third Edition (2004)