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EE BROTH

Code: CM0317

An enrichment medium for Enterobacteriaceae in the bacteriological examination of foods.

Directions
Add 43.5g to 1 litre of distilled water. Distribute 100ml quantities in 250ml flasks and heat at 100°C for 30 minutes only. Cool rapidly in cold running water. This medium is heat sensitive. DO NOT AUTOCLAVE.

Description
EE Broth (Buffered glucose - Brilliant Green-bile broth) is recommended as an enrichment medium for Enterobacteriaceae in the bacteriological examination of foods¹ and animal feed stuffs². This medium is more inhibitory to non- Enterobacteriaceae than other non-selective media e.g. Mannitol broth³ or Lactose broth⁴ by virtue of the presence of brilliant green and bile salts in the preparation.

The enumeration of Enterobacteriaceae is of great importance in monitoring the sanitary quality of food and drugs but the reliability of the methods used depends upon resuscitation of damaged cells. Such weakened cells may arise from exposure to dehydration, low pH and other unfavourable conditions⁵.

Incubation for 2 hours in well-aerated Tryptone Soya Broth CM0129 at 25°C should precede enrichment in EE Broth. This procedure is recommended for dried foods⁶, animal feeds⁷ and semi-preserved foods⁸. Occasionally, with a particular dry product, a longer incubation period is necessary but never over eight hours of resuscitation.

Oxoid EE Broth was formulated to overcome the unsatisfactory effects of inhibition on small numbers of Enterobacteriaceae cells due to bile salt variations. The inclusion of purified ox bile eliminated these problems and a preliminary assay can be used to check growth by inoculating approximately one viable cell per medium unit^9,10.

For the bacteriological evaluation of processed foods the entire Enterobacteriaceae group can be used as indicator organisms¹⁰. This will overcome the discrepancies that can arise when lactose-negative, anaerogenic lactose-positive or late lactose fermenting Enterobacteria are present but are missed by the standard `coli-aerogenes’ tests. To overcome these problems lactose media have been replaced by those containing glucose. Mossel et al¹ cited several examples in the literature which referred to various foods contaminated with salmonellae, although results for coliforms were negative. A later example quoted by Mossel⁹ involved an outbreak of diarrhoea caused by French mould-fermented soft cheese contaminated by Escherichia coli serotype O124. This organism is lactose-negative and therefore was not detected in coliform tests but only recognised when the commodity was tested for Enterobacteriaceae since it fermented glucose rapidly.

EE Broth should be used as an enrichment broth in conjunction with Violet Red Bile Glucose Agar CM0485. When specific organisms, rather than Enterobacteriaceae in general, are required subcultures must be made onto lactose differential media e.g. Desoxycholate Citrate Agar CM0035, Brilliant Green Agar CM0263, or MacConkey Agar CM0007 for the detection of lactose-negative or delayed organisms.
Sample size should not be less than 10g to yield the organisms being sought.

Technique
1. Resuscitate debilitated cells by incubating 1:10 dilutions of the food samples under investigation in Tryptone Soya Broth CM0129 at 25°C for 2-8 hours. The fluid layer should not be much deeper than one centimetre. Shake the flask to disperse the contents alternately in clockwise and anti-clockwise directions for 30 seconds on three successive occasions.
2. After the period of time necessary for resuscitation, ten-fold volumes of EE Broth are added to the resuscitated suspensions.
3. Shake to disperse as above. For large samples it is desirable to add the resuscitation medium containing the product under examination, to equal volumes of double strength EE Broth.
4. Incubate at 44°C for 18 hours for thermotrophic bacteria; 32°C for 24/48 hours for mesophilic bacteria; 4°C for 10 days for psychrotrophic bacteria depending on the groups of Enterobacteriaceae sought.
5. Examine the tubes of broth and look for turbidity with some change of colour towards yellowish-green for presumptive evidence of Enterobacteriaceae.
6. Subcultures can be made on to Violet Red Bile Glucose Agar CM0485 or on to lactose-containing media for confirmation of LF or NLF status. Further tests must be made to confirm the identity of the isolate.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Pale green coloured, free-flowing powder
Prepared medium: Green coloured solution

Quality control

* This organism is available as a Culti-Loop®

Precautions
Avoid overheating the medium, especially the double-strength broth.

References
1. Mossel. D. A. A., Vissar M. and Cornellisen A. M. R. (1963) J. Appl. Bact. 26(3). 444-452.
2. Van Schothurst M., Mossel D. A. A., Kampelmacher E. H. and Drion E. F. (1966) Vet. Med. 13(3) 273-285.
3. Taylor W. I. (1961) Appl. Microbiol. 9. 487-490.
4. North W. R. (1961) Appl. Microbiol. 9. 188-195.
5. Mossel D. A. A. and Harrewijn G. A. (1972) Alimenta 11. 29-30.
6. Mossel D. A. A. and Ratto M. A. (1970) Appl. Microbiol. 20. 273-275.
7. Mossel D. A. A., Shennan Jean L. and Vega Clare (1973) J. Sci. Fd. Agric. 24. 499-508.
8. Mossel D. A. A. and Ratto M. A. (1973) J. Fd. Technol. 8. 97-103.
9. Mossel D. A. A., Harrewijn G. A. and Nesselrooy-van Zadelhoff C. F. M. (1974) Health Lab. Sci. 11. 260-267.
10. Richard N. (1982) in Quality Assurance and quality control of microbiological culture media. Ed. J.E.L. Corry. G.I.T. - Verlag Darmstadt. pp 51-57.
11. Mossel D. A. A. (1973) Food R. A. Technical Circular no 526, February 1973.