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Salmonella Rapid Culture Method

ONE Broth-Salmonella & Brilliance Salmonella Agar (OSCM II)

Summary

The Salmonella Rapid Culture Method provides a simple protocol for the recovery and presumptive identification of Salmonella species in as little as 38 hours. ONE Broth-Salmonella is a highly nutritious and selective medium which allows sufficient recovery to be achieved within 16-20 hours with no need for a secondary enrichment broth. Simply inoculate the incubated broth onto Brilliance™ Salmonella Agar.

Brilliance Salmonella Agar is a unique agar that combines the benefits of chromogenic media and new Inhibigen™ technology with impressive results. Inhibigens are a new class of selective agents, which can be used to target specific groups of organisms and prevent them from growing. In Brilliance Salmonella Agar, a specifically tailored Inhibigen is combined with other selective agents to significantly reduce the growth of nontarget micro-organisms. Carefully selected chromogens give excellent differentiation, making Salmonella easier to identify and reducing the number of false positive colonies requiring confirmation.

ONE Broth-Salmonella

Code: CM1091

ONE Broth- Salmonella is a selective medium for the recovery of Salmonella spp. in a single enrichment step.

Typical Formula*	gm/ liter
Peptone	5
Yeast extract	5
Salt buffer mix	10
Growth promoter mix	5
pH 7.0† ± 0.2 at 25ºC

* Adjusted as required to meet performance standards
† pH of supplemented medium

ONE Broth-Salmonella SELECTIVE SUPPLEMENT

Code: SR0242

Vial Contents (each vial is sufficient for 500ml of medium)	per vial	per litre
Novobiocin	2.7mg	12mg

BRILLIANCE SALMONELLA AGAR BASE

Code: CM1092

Brilliance Salmonella Agar (formerly OSCM II) is used to produce a selective medium for the presumptive identification and differentiation of Salmonella spp.

Typical Formula*	gm/ liter
Inhibigen™ mix	14
Chromogenic Mix	25
Agar	15
pH 7.3† ± 0.1 @ 25ºC

* Adjusted as required to meet performance standards
† pH of supplemented medium

SALMONELLA SELECTIVE SUPPLEMENT

Code: SR0194

Vial Contents (each vial is sufficient for 500ml of medium)	per vial	per litre
Novobiocin	5mg	10mg
Cefsulodin	12mg	24mg

Materials required but not supplied
Incubators, balances, pipettes, micropipettes, sterile disposable tips, heated stir plate, autoclave, sterile containers, tubes and plates, inoculating loops, stomacher/blender, quality control organisms, and demineralized water.

Directions

ONE Broth-Salmonella
Suspend 5.6g of ONE Broth-Salmonella Base in 225ml of distilled water. Sterilize by autoclaving at 121°C for 15 minutes. Cool to below 50°C and add the contents of 1 vial of ONE Broth-Salmonella Supplement (SR0242E), re-suspended as directed.

Brilliance Salmonella Agar
Suspend 27g of Brilliance Salmonella Agar Base in 500ml of distilled water. Add the contents of 1 vial of Salmonella Selective Supplement (SR0194E), resuspended as directed. Mix well and sterilize by bringing to the boil with frequent agitation. Cool to around 50ºC, mix well and pour into sterile Petri dishes.
NOTE: It is critical that the selective supplement is added prior to heating. When preparing volumes greater than 1 litre contact Technical Support for directions.

Description
An Inhibigen compound is comprised of two components, combined together by a bond that can only be cleaved by a specific enzyme. When bound together, the inhibitor compound is not toxic and therefore can exist in a medium without harming micro-organisms. Once inside the cell, the bond will be cleaved if the target enzyme is present. When the bond is cleaved, the inhibitor molecule is released and disrupts cell wall synthesis, causing death of the organism. As cells die and lyse, free inhibitor is released but cannot be taken up by other cells, resulting in targeted inhibition. The Inhibigen in Brilliance Salmonella Agar targets Escherichia coli. Novobiocin and cefsulodin, supplied as a freeze-dried supplement (SR0194), are added to the medium to inhibit the growth of other competing flora such as Proteus spp. and Pseudomonas spp.

Differentiation of Salmonella from the other organisms that grow on Brilliance Salmonella Agar is achieved through the inclusion of two chromogens that also target specific enzymes: caprylate esterase and ß-glucosidase. Caprylate esterase is an enzyme present in all samonellae as well as some species of Klebsiella, Enterobacter and Proteus1. Organisms possessing caprylate esterase cleave the chromogen to release an insoluble purple chromophore. As the cells grow, the chromophore builds up and produces a purple-coloured colony. Some Enterobacteriaceae, including Klebsiella and Enterobacter, but not Salmonella, possess ß-glucosidase2. If these organisms grow, they will form blue or dark blue colonies, even if they are esterase positive, which make them easy to differentiate from purple Salmonella colonies.

Technique
The following method is a summary of the AOAC-RI Salmonella Rapid Culture method, using ONE-Broth Salmonella for enrichment and Brilliance Salmonella Agar for identification.  Food matrices that have AOAC-RI approval include: ground beef, ground chicken, lettuce, shrimp and shell eggs.

1. Add 25g or 25ml of food sample to 225ml of ONE Broth-Salmonella.  For solid samples, stomach for a minimum of 30 seconds to mix the sample.
2. Incubate the broth at 42ºC ± 1ºC for 18h ± 2h
3. Gently agitate the bag then, using a 10μl microbiological loop, inoculate the broth onto a Brilliance Salmonella (OSCM II) plate using a diminishing sweep technique to produce single colonies.
4. Using a microbiological loop, inoculate each broth onto one XLD Agar plate and one Brilliance Salmonella Agar plate, to give a total of 4 plates.
5. Incubate the plates at 37ºC for 24h ± 2h
6. Confirm purple colonies as Salmonella spp by appropriate biochemical and serological methods (as indicated in the FDA BAM and USDA MLG).4,5

The following Oxoid products may be useful for confirmation: Oxoid Salmonella Latex Test Kit (DR1108), Microbact  (see www.remel.com or www.oxoid.com for codes) and Agglutinating Sera.

Please confirm prepared media codes with your local supplier.

Storage conditions and Shelf life
ONE Broth-Salmonella should be stored tightly capped in the original container at 10-30ºC. The ONE Broth-Salmonella Selective Supplement should be stored in the dark at 2-8ºC. When stored as directed, the unopened products will remain stable until the expiry date printed on the packaging.

Once prepared, ONE Broth-Salmonella can be stored for up to 2 weeks, when made from CM1091 and SR0242 according to the manufacturer’s instructions and stored at 2-8ºC, in the dark. A longer shelf life may be attainable, but should be validated under the relevant, local manufacturing and storage conditions.

Brilliance Salmonella Agar should be stored in the tightly capped original container at 10-30ºC. Salmonella Selective Supplement should be stored in the dark at 2-8ºC. When stored as directed, the unopened products will remain stable until the expiry date printed on the packaging.

Once prepared, Brilliance Salmonella Agar plates can be stored for up to 2 weeks when made from CM1092 and SR0242 according to the manufacturer’s instructions and stored at 2-8ºC, out of direct sunlight. A longer shelf life may be attainable, but should be validated under the relevant, local manufacturing and storage conditions.

Appearance

ONE Broth-Salmonella
Dehydrated broth base: straw colored, free-flowing powder
Selective supplement: white, freeze-dried pellet
Prepared medium: straw colored clear liquid

Brilliance Salmonella Agar
Dehydrated medium: White/cream colored, free-flowing powder
Selective supplement: White, freeze-dried pellet
Prepared medium: White/cream colored opaque gel

Quality control

All lot numbers are tested using the following quality control organisms and have been found to be acceptable.  Testing of control organisms should be performed in accordance with established laboratory quality control procedures.

ONE Broth-Salmonella

Positive controls:	Expected results
Salmonella Typhimurium ATCC®10428*	Good growth; turbid broth
Negative controls
Staphylococcus aureus ATCC® 25923*	Inhibited

Brilliance Salmonella Agar

Positive controls:	Expected results
Salmonella Typhimurium ATCC®10428*	Good growth; purple colonies
Klebsiella pneumoniae ATCC®13883*	Good growth; blue colonies
Negative controls:
Enterococcus faecalis ATCC®29212*	Inhibited
Escherichia coli ATCC®25922*	Inhibited

*This organism is available as a Culti-Loop®

Precautions
ONE Broth-Salmonella medium and Brilliance Salmonella Agar are for in vitro diagnostic use only, by experienced microbiologists. They must not be used beyond the stated expiry date, or if the product shows any sign of deterioration. Sterilize specimens, equipment and media properly after use; dispose waste in accordance with local rules and regulations.  Directions should be read and followed carefully.

Limitations
It should be noted that, as with all Salmonella media, organisms with atypical enzyme patterns may give anomalous reactions in ONE Broth-Salmonella or on Brilliance Salmonella Agar. A small number of atypical strains that may give a weak reaction or fail to grow, especially when low numbers are present in the sample.

Note: For AOAC-RI approved methods, a number of S. typhii and S. paratyphii spp. may fail to grow in the ONE Broth-Salmonella enrichment.4,5. If testing for S. typhii or S. paratyphii, confirm absence of the organism using a secondary method for enrichment, followed by plating on Brilliance Salmonella Agar.  For additional information, please contact technical service at 1-800-255-6730.

Refs
1. Cooke. V.M. et al, (1999) A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae. Appl. Environ. Microbiol. 65(2), 807-812
2. Garrity, G., Brenner, D.J., Krieg, N.R., Staley, J.R. (Eds.), Bergey’s Manual of Systematic Bacteriology. Vol 2, Part B. 2nd ed., 2005. US, Springer.
3. ISO 6579:2002, Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp.
4. Isolation and Identification of Salmonella from Meat, Poultry, and Egg Products. USDA Microbiology Laboratory Guidebook 4.03. USDA/FSIS, Laboratory QA/QC Division, Athens, GA. (http://www.fsis.usda.gov/PDF/MLG_4_04.pdf)
5. Andrews, W.H., Hammack, T.S. (1998) US FDA/CFSAN Bacteriological Analytical Manual (FDA-BAM), 8th edition, Chapter 5: Salmonella, U.S. Food and Drug Administration, Washington, DC. (online)(http://www.cfsan.fda.gov/~ebam/bam-5.html)

ATCC® is a registered trademark of American Type Culture Collection