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Material Safety Data Sheet

Organisms

Organisms this product works with:

Dehydrated Culture Media

EC BROTH (Reduced Bile Salts)

Code: CM0990

A selective enrichment broth for the growth of E. coli O157, from food and environmental samples.

Typical Formula*

gm/litre

Tryptone

20.0

Lactose

5.0

Bile Salts No. 3

1.12

Di-potassium phosphate

4.0

Mono-potassium phosphate

1.5

Sodium chloride

5.0

pH 6.9 ± 0.2 @ 25°C

 

* Adjusted as required to meet performance standards 

Novobiocin Supplement

Code: SR0181

Vial contents (each vial is sufficient for 500ml of medium)
per vial
per litre

Novobiocin

10mg

20mg

Directions
Dissolve 18.3g in 500ml of distilled water. Sterilise by autoclaving at 121°C for 15 minutes. Cool the EC Broth (Reduced Bile Salts) to 50°C and aseptically add the contents of 1 vial of Novobiocin Supplement SR0181, reconsituted as directed. Dispense into final sterile containers.

Description
Novobiocin is an antibiotic which is active against Gram-positive bacteria such as Staphylococcus aureus (including methicillin-resistant strains) and other staphylococci. Some Gram-negative organisms including Haemophilus influenzae, Neisseria spp. and some strains of Proteus spp. are also susceptible; however most Enterobacteriaceae are resistant.

Novobiocin is primarily bacteriostatic, but in higher concentrations it may be bactericidal against more sensitive species. It principally inhibits synthesis of DNA, although RNA synthesis is also inhibited to a lesser extent. Its action on DNA is specifically related to the selective and competitive inhibition of the binding of ATP to gyrase B substrates. The gyrase enzyme forms negative supercoils from closed circular double-stranded DNA, and is inhibited by novobiocin at low concentrations. However, novobiocin-resistant Escherichia coli cells contain a DNA gyrase which is unaffected by the drug. Novobiocin also inhibits protein and cell wall synthesis.

EC Broth (Reduced Bile Salts) can be used for the enrichment of Escherichia coli O157 directly from food and environmental samples, especially for use with frozen food samples, where the bacterium may be sub-lethally damaged1. Novobiocin may be added to provide a more selective primary enrichment medium.

The formulation includes a buffered lactose broth and reduced levels of Bile Salts No. 3 based on a recommendation of Szabo et al.2. The presence of the lower level of bile salts inhibits the growth of spore formers and enterococci, but allows the growth of E. coli O157 at 37°C.
When using the reduced temperature of 37°C suggested by Okrend, supplementation with Novobiocin Supplement SR0181 at a final concentration of 20mg per litre improves reduction in background flora with no reduction in growth of Escherichia coli O1573.

Technique
One method of use is as follows (based on Okrend3 ) :
Prepare a 1 in 10 dilution of the sample in EC Broth (Reduced Bile Salts) CM0990, supplemented with novobiocin as directed. Blend in a StomacherÔ for 2 minutes. Incubate for either 6 hours at 37°C on a rotary shaker at 100rpm or static for 24 hours at 35°C.

After incubation, dilute the shaken culture to 10-2 and the overnight broth culture to 10-6. Streak on to appropriate selective agar e.g. Sorbitol MacConkey Agar (SMAC) CM0813 or SMAC with BCIG CM0981. Cefixime Tellurite Selective Supplement SR0172 may be added to increase selectivity of either medium.

Incubate at 37°C for 24 hours. Escherichia coli O157 colonies should look clear and colourless. Confirm suspected Escherichia coli O157 with the E. coli O157 Latex Test DR0620 or Dryspot E. coli O157 DR0120. Please refer to the reference for full method.

EC Broth (Reduced Bile Salts) and Novobiocin Supplement SR0181 is a USDA/FSIS4 and AIFST1 recommended enrichment medium for the detection of Escherichia coli O157:H7 and O157:NM (NonMotile) from meat and poultry products.

Storage conditions and Shelf life 
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Novobiocin Supplement SR0181 should be stored in the dark at 2-8°C. When stored as directed, the supplement remains stable until the expiry date printed on the label.

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

Quality control

Positive control:

Expected results

Escherichia coli 0157 (Non-toxigenic strain) NCTC 12900 *Turbid growth

Negative control:

 
Staphylococcus aureus ATCC®25923*No growth
* This organism is available as a Culti-Loop®

 References
1. 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.
2. Szabo, R. A., Todd, E. C. D. and Jean, A. (1986) J. Food Prot. 49: 768-772
3.Okrend, A. J. G., Rose, B. E. and Bennett, B. (1990) J. Food Prot. 53: 249-252
4. UDSA/FSIS Microbiology Laboratory Guidebook 3rd Ed. (1998) Chapter 5. Detection, Isolation and Identification of Escherichia coli O157:H7 and O157:NM (NonMotile) from Meat and poultry Products.

 
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