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MODIFIED TRYPTONE SOYA BROTH (mTSB)

CODE: CM0989

two products designed specifically for the selective enrichment of Escherichia coli O157 from food or faecal samples

* Adjusted as required to meet performance standards

VCC SELECTIVE SUPPLEMENT

CODE: SR0190

Directions
Suspend 33g of Modified Tryptone Soya Broth (mTSB) in 1 litre of distilled water. Mix well, dispense into final containers and sterilise by autoclaving at 121°C for 15 minutes. Allow the medium to cool to 50°C before adding a selective supplement as appropriate.

Description
Many selective broths have been formulated for the enrichment of Escherichia coli O157. mTSB is a nutritious, selective base, which was devised by Doyle and Shoeni¹. The broth was incubated for at 37°C for 24 hours in shaken flasks. Szabo and co-workers² also used mTSB and found incubation at 43°C with agitation gave better recovery rates from inoculated food samples.

Buffered Peptone Water (BPW) (CM0509), supplemented with VCC Selective Supplement (SR0190) was used for the isolation of Escherichia coli O157 from beef carcasses, bovine faeces and from milk and minced beef³.

In EHEC enrichment broth (EEB), VCC Selective Supplement is added to mTSB, which is used instead of BPW to overcome some of the inhibitory effects of the antibiotics on Escherichia coli O157⁴.

Both BPW-VCC (CM0509+SR0190) and EEB (CM0989+SR0190) are incubated for 6hr at 37°C³.

Tryptone Soya Broth (CM0129) has been modified by increasing the concentration of the buffer, dipotassium hydrogen phosphate, to 4g/l and the addition of 1.5g/l Bile Salts. The bile salts inhibit the growth of spore formers and enterococci, but allow the growth of Escherichia coli O157.
VCC Selective Supplement contains three antibiotics vancomycin for inhibition of Gram-positive organisms. Cefixime for the inhibition of Proteus spp. cefsulodin suppresses Pseudomonas spp.

Technique
mTSB can be used with either Novobiocin Selective Supplement (SR0181) or VCC Selective Supplement (SR0190) depending on which protocol is followed. VCC Selective Supplement is a highly selective supplement, which can be used either with mTSB, to give EHEC Enrichment Broth (EEB), or with Buffered Peptone Water (CM0509) depending on which protocol is followed.

1. ISO⁵/ PHLS⁶/ FDA/BAM⁷/NMKL⁹
mTSB+n
500ml of Modified Tryptone Soya Broth (CM0989) and 1 vial of Novobiocin Selective Supplement (SR0181). Incubate at 41.5°C for 6 and 22 hours.
2. FDA/BAM⁷
EEB
225ml of Modified Tryptone Soya Broth (CM0989) and 1 vial of VCC Selective Supplement (SR0190). Incubate at 37°C with agitation for 6 and 24 hours.
3. CCFRA⁸
BPW-VCC
225ml of Buffered Peptone Water (CM0509) and 1 vial of VCC Selective Supplement (SR0190). Incubate at 37°C for 6 and 24 hours.

Storage conditions and Shelf Life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Prepared medium should only be used fresh.

Appearance
Dehydrated medium: Straw coloured, free flowing powder
Prepared medium: Straw coloured solution

Quality control

* This organism is available as a Culti-Loop®

References
1.Doyle, M. P. and J. L Schoeni (1987) Applied Environmental Microbiology 53: 2394-2396
2.Szabo R. E. et al. (1990) Applied Environmental Microbiology 56: 3546-3549
3.Desmarchelier, P. M. and Grau, F. H. (1997) Escherichia coli. In: Foodborne Microorganisms of Public Health Significance. 5th Edition. pp.231-264.A. D. Hocking (Ed.). AIFST (NSW Branch) Food Microbiology Group, Australia
4.Weagant S. D. et al. (1995) Journal of Food Protection 58: 7-12
5. ISO:16654:2001 Microbiology of food and animal feed stuffs. Horizontal method for the detection of E. coli O157 (Pub March 22, 1999, expired June 30 1999)
6.PHLS Standard Methods F17 & W16: Detection of E. coli O157
7.FDA/BAM 8^th Edition (Revision A) (1998) Chapter 4: 4.20-4.26
8.Campden and Chorleywood Food Research Association Method 19.1: 1997. Detection of E. coli O157
9.Nordic Committee on Food Analysis No 164 1999 Escherichia coli O157. Detection in food and feeding stuffs