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Dehydrated Culture Media

BRILLIANT GREEN AGAR (MODIFIED)

Code: CM0329

A selective and diagnostic agar for salmonellae (other than Salmonella typhi) from food and feeds.

Typical Formula*

gm/litre

`Lab-Lemco’ powder

5.0

Peptone

10.0

Yeast extract

3.0

Disodium hydrogen phosphate

1.0

Sodium dihydrogen phosphate

0.6

Lactose

10.0

Sucrose

10.0

Phenol red

0.09

Brilliant green

0.0047

Agar

12.0

pH 6.9 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards 

Directions
Suspend 52g in 1 litre of distilled water. Heat gently with occasional agitation and bring just to the boil to dissolve the medium completely. DO NOT AUTOCLAVE. Cool to 50°C, mix well and pour plates.

SULPHAMANDELATE SUPPLEMENT

Code: SR0087

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

per vial
per litre

Sodium sulphacetamide

500mg

1000mg

Sodium mandelate

125mg

250mg

Directions
Reconstitute one vial as directed, aseptically add the contents to 500ml of sterile Oxoid Brilliant Green Agar (Modified) cooled to 50-55°C. Mix gently and pour into sterile Petri dishes.

Description
Brilliant Green Agar (Modified) was developed from a formula supplied by the Rijks Instituut voor de Volksgezondheid (National Institute for Public Health), Utrecht1,2.

The medium has been widely assessed in Europe and has been used in ISO standards3,4,5.

The advantages claimed for the medium are the greater inhibition of Escherichia coli and Proteus species than other formulations: the restriction of growth of Pseudomonas species, whose colonies may resemble salmonellae on Brilliant Green Agar and cause confusion or much extra work to confirm their identity: the absence of inhibitory properties towards small numbers of salmonellae6.

SELECTIVE BRILLIANT GREEN AGAR (MODIFIED)
Watson and Walker7 incorporated a combination of sulphacetamide (1.0 mg/ml) and mandelic acid (at 0.25 mg/ml) into Oxoid Brilliant Green Agar (Modified) to obtain maximum recovery of salmonellae from Muller-Kauffmann Tetrathionate Broth whilst giving maximum suppression of contaminating organisms.

Oxoid Salmonella Sulpha-Mandelate Supplement, SR0087 used for the isolation and enumeration of salmonellae from sewage and sewage sludge, is based on the formulation of Watson and Walker7. These authors showed that the use of Brilliant Green Agar (Modified) incorporating a combination of sulphacetamide and mandelic acid incubated at 43°C resulted in maximum recovery of salmonellae from Muller-Kauffmann Tetrathionate Broth.

The method described7 has been shown to be a quick and reliable technique for the isolation of sub-lethally damaged salmonellae from treated sewage and sewage sludge.


Use of antibiotic supplemented Brilliant Green Agar is necessary because the pre-enrichment of the sewage in phosphate buffered peptone (PBP) water will encourage not only the growth of stressed salmonellae but many competing organisms.

The inhibitory properties of Muller-Kauffmann Tetrathionate Broth are not sufficient by themselves to suppress the growth of the latter. The advantage claimed for Selective Brilliant Green Agar is greater inhibition of contaminating organisms and a lower incidence of false positives.
This advantage was confirmed by Fricker and his co-workers when using Brilliant Green Agar (Modified) containing sodium sulphacetamide and sodium mandelate for plating enrichment cultures in Rappaport Broth, from sewage and sewage polluted water8,11, seagull faeces9 and chicken10,12.

Vassilliadis et al.13 added 2.5g of sodium desoxycholate to one litre of Brilliant Green Agar (Modified) to prevent swarming by Proteus hauseri, during examination of sewage effluents. They found desoxycholate to be superior to sulphonamides in suppressing swarming without affecting the growth of a wide range of salmonellae serotypes.

Colonial characteristics

Salmonellae

Red colonies surrounded by bright red medium

Lactose/Sucrose fermenters

Inhibited to a certain extent, but producing yellow green colonies when growth is evident

Proteus Almost completely inhibited, those colonies that grow produce red colonies without swarming
Pseudomonas Inhibited growth of small, crenated red colonies

Techniques
Technique for food and feeds
An outline of the method used by Edel and Kampelmacher2 in their trials is as follows:
1. One part of the food sample was added to 20 parts of Muller-Kauffmann Tetrathionate Medium CM0343.
2. After agitation, the flask of broth was placed into a 45°C waterbath for 15 minutes only.
3. The flask was then transferred to a 43°C incubator.
4. The broth was subcultured to Brilliant Green Agar (Modified) after 18 and 48 hours.
A single loopful of broth was used to streak inoculate either two 9 cm diameter plates (without recharging the loop between plates) or one 14cm diameter plate.
5. The plates were incubated at 35°C for 18-24 hours.
6. Red colonies, resembling salmonellae, were picked off the plates and subcultured to Lysine Decarboxylase Broth CM0308 and Triple Sugar Iron Agar CM0277. These media were incubated at 35°C for 18-24 hours.

If the reactions on these media were positive for salmonellae then slide agglutination tests were carried out on the surface growth of the Triple Sugar Iron Agar.

Technique for sewage7
1. Take a representative sample of sewage or sludge for examination.
2. Homogenise a suitable volume in a macerator or stomacher.
3. Inoculate five 10ml samples into 35ml of Buffered Peptone Water CM0509, five 1ml samples and five 0.1ml samples into 10ml of Buffered Peptone Water. Incubate at 35°C overnight.
4. Transfer 10ml portions into 35ml of Muller-Kauffmann Tetrathionate Broth and incubate at 43°C.
5. Subculture the broths on to Brilliant Green Agar (Modified) containing Sulphamandelate Selective Supplement SR0087 after 24 and 48 hours incubation.
6. Incubate the Brilliant Green Agar plates overnight at 43°C.
7. Identify suspicious (red) colonies using further diagnostic tests.

The Sulphamandelate Selective Supplement SR0087 inhibits competing organisms which multiply during the resuscitation and recovery stages in Buffered Peptone Water.

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 plates of medium at 2-8°C.

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

Quality control

Positive control:

Expected results

Salmonella typhimurium ATCC® 14028 *Good growth; red coloured colonies and media
Negative controls:  
Escherichia coli ATCC® 25922 *Inhibited or no growth; yellow-green coloured colonies   
Proteus mirabilis ATCC® 29906 * Inhibited or no growth; pink coloured colonies
* This organism is available as a Culti-Loop®

Precautions
Lactose-fermenting salmonellae may be present in foods.
Salmonella typhi and Shigella species may not grow on this medium.
Proteus, Citrobacter and Pseudomonas species may mimic enteric pathogens by producing small red colonies.

References
1. Edel W. and Kampelmacher E. H. (1968) Bull. Wld Hlth. Org. 39. 487-491.
2. Edel W. and Kampelmacher E. H. (1969) Bull. Wld Hlth. Org. 41. 297-306.
3. Anon. (1975) International Organization for Standardization. Meat and Meat products - detection of Salmonella. Ref. method ISO 3565-1975(E).
4. Anon. (1981) International Organization for Standardization. Microbiology - General guidance on methods for the detection of Salmonella. Ref. method ISO 6579-1981(E).
5. Anon. (2001) International Organization for Standardization. Milk and Milk products - detection of Salmonella species. ISO 6785.
6. Read R. B. and Reyes A. L. (1968) Appl. Microbiol. 16. 746-748.
7. Watson U. C. and Walker A. P. (1978) J. Appl. Bact. 45. 195-204.
8. Fricker C.R. (1984) Zbl. Bakt. Hyg. Abt. I. Orig.B. 179. 170-178.
9. Fricker C.R. (1984) lnt. J. Food Microbiol. 1. 171-177.
10. Fricker C.R. and Girdwood R.W.A. (1985) J. Appl. Bact. 58. 343-346.
11. Fricker C.R., Quail E., McGibbon L. and Girdwood R.W.A. (1985) J. Hyg. 95. 337-344.
12. Vassilliadis P., Trichopoulos J., Papadakis V. K. and Ch. Serie. (1979) Ann. Soc. belge. Med. trop. 59. 117-120.
13. Harvey R. W. S., Price T. H. and Hall L. M. (1973) J. Hyg. Camb. 71. 481-486.

 
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