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Material Safety Data Sheet

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Dehydrated Culture Media

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MODIFIED SEMI-SOLID RAPPAPORT VASSILIADIS AGAR (ISO)

Code: CM1112

Modified Semi-solid Rappaport-Vassiliadis (MSRV) Agar (ISO) is used for the selective enrichment of Salmonella spp. from food, animal faeces and environmental samples from the primary stage of animal production. This medium meets the specifications for formulation and performance described in ISO 6579:2002 +A1:2007Annex D1.

Typical Formula*

gm/ litre

Enzymatic digest of animal and plant tissue

4.6

Acid hydrolysate of casein

4.6

Sodium chloride

7.3

Potassium dihydrogenphosphate (KH2PO4)

1.5

Magnesium chloride anhydrous (MgCl2)

10.9

Malachite green oxalate

0.04

Agar

2.7

pH 5.2 (5.1 to 5.4) @ 25°C 
*Adjusted as required to meet performance standards
† pH of supplemented medium

NOVOBIOCIN SUPPLEMENT

Code: SR0181

Vial Contents (each vial sufficient for 1 litre of medium)

per vial

per litre

Novobiocin

10mg

10mg


Directions

Suspend 31.6g of MSRV (ISO) Agar base in 1 litre of distilled water. Bring to the boil with frequent agitation. DO NOT AUTOCLAVE. Cool to 50°C, and aseptically add the contents of 1 vial of Novobiocin Supplement (SR0181), reconstituted as directed. Mix well, and pour into sterile Petri dishes. This medium is semi-solid, therefore do not invert plates.

Description
Modified Semi-solid Rappaport Vassiliadis Agar (ISO) is based on the formulation described by De Smedt et al., which has been shown to detect more Salmonella-positive samples than traditional enrichment procedures2,3. Further collaborative studies have confirmed these findings4,5.

Motility enrichment on MSRV Agar has been designed as a simple and sensitive method for the isolation of salmonellae from food and environmental samples. It has been incorporated into ISO 6579:2002 +A1:2007 Annex D1, which applies to the isolation of Salmonella spp. from animal faeces (such as poultry, pigs and cattle) and environmental samples associated with the primary stage of animal production (such as dust). In Annex D, MSRV Agar replaces Rappaport-Vassiliadis Broth, which is still used in ISO 6579:2002 +A1:2007 for the selective enrichment of Salmonella spp. in food.

The efficiency of the medium is based on the ability of salmonellae to migrate through the selective medium ahead of competing Gram-negative, motile organisms, thus producing opaque halos of growth. Novobiocin, low pH, high magnesium chloride concentration and malachite green inhibit the growth of Gram-positive flora.

Subculture should be carried out from the migrated culture, with the inoculum being taken from the furthest edge of the growth. Presumptive identification is achieved by subculture onto XLD Agar and a second Salmonella agar of choice. Characteristic presumptive Salmonella colonies should be confirmed according to directions in ISO 6579:2002 +A1:2007 Annex D1. Alternatively, Oxoid Salmonella Latex Test FT0203 or DR1108 may be used for serological confirmation of Salmonella spp. MSRV (ISO) Agar is not suitable for the detection of non-motile strains of Salmonella (clinical incidence <0.1%)6.

Technique
Refer to relevant standard method for detailed instructions. The following method is a summary of ISO 6579:2002 +A1:2007 Annex D1.

  1. Prepare samples in accordance with the appropriate standard.
  2. Add 25g of sample to 225ml of Buffered Peptone Water (ISO) (CM1049) and homogenise for 30 seconds.
  3. Incubate at 37°C for 18h ± 2h.
  4. Inoculate MSRV plates with 3 drops of the pre-incubated BPW culture totalling 0.1ml, equally spaced on the medium.
  5. Incubate plates at 41.5°C for 24h ± 3h.
  6. Motile Salmonella colonies are characterised by white/grey, turbid zones radiating from the point of inoculation. Zones are surrounded by a white halo with a sharply defined border.
  7. If plates are negative after 24h, they should be re-incubated for a further 24h ± 3h.
  8. Select area of zone furthest from the point of inoculation and sample with a 1µl sterile loop.
  9. Streak sample onto Xylose Lysine Desoxycholate Agar (CM0469), and a second selective medium such as Brilliance™ Salmonella Agar (CM1092).
  10. Incubate at 37°C for 18h ± 3h.
  11. Colonies can be subcultured onto Nutrient Agar (CM0003) to confirm purity and perform further biochemical and serological tests.

Storage conditions and Shelf life
MSRV (ISO) should be stored in the tightly capped original container at 10-30°C. Novobiocin Supplement should be stored in the dark at 2-8°C. When stored as directed, the un-opened products will remain stable until the expiry date printed on the packaging.

Locally prepared MSRV (ISO) plates can be stored for up to 2 weeks when made from CM1112 and SR0181 according to the manufacturer’s instructions and stored at 2-8°C, out of direct sunlight. A longer shelf life may be attainable, but should be validated under the relevant, local manufacturing and storage instructions.

Appearance
Dehydrated culture medium: Blue/green, free-flowing powder
Selective Supplement: White, freeze-dried pellet
Prepared medium: Blue, semi-solid gel

Quality control

Positive controls: Expected results
Salmonella Typhimurium ATCC®14028*Straw colonies at site of inoculation surrounded by white/grey halo of growth
Salmonella Enteritidis ATCC®13076*Straw colonies at site of inoculation surrounded by white/grey halo of growth
Negative controls: 
Escherichia coli ATCC®8739*Restricted or no growth
Enterococcus faecalis ATCC®19433*Restricted or no growth

* This organism is available as a Culti-Loop®

Precautions
MSRV (ISO) Agar is for laboratory use only. Do not use the dehydrated medium beyond the stated expiry date or if the product shows any sign of deterioration.
Basal medium is very hygroscopic.
When handling powder a face mask and gloves must be worn. Refer to material safety data sheet for details.

Refs
1. ISO 6579:2002 +A1:2007 Annex D.
2. De Smedt J. M., Bolderdijk R., Rappold H. and Lautenschlaeger D. (1986) J. Food. Prot. 49. 510-514.
3. De Smedt J. M., Bolderdijk R. (1987) J. Food. Prot. 50. 658-661.
4. De Zutter L. et al. (1991) Int. J. Food Micro. 13. 11-20.
5. De Smedt J. M. et al. (1991) Int. J. Food Micro. 13. 301-308.
6. Holbrook R., Anderson J. M., Baird-Parker A. C., Dodds L. M., Sawhney D., Struchbury S. H. and Swaine D. (1989) Lett. Appl. Microbiol. 8. 139- 142.

ATCC® is a registered trademark of American Type Culture Collection

 
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