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VIOLET RED BILE LACTOSE AGAR (ISO)

CODE: CM0968

Violet Red Bile Lactose (VRBL) Agar (ISO) is for the detection and enumeration of coliforms in food, animal feed and environmental samples. This medium meets the formulation and performance criteria laid down in ISO 4832:20061 and ISO 11133:2014.

Typical Formula*

gm/ litre

Peptone

7.0

Yeast extract

3.0

Bile salts No. 3

1.5

Sodium chloride

5.0

Neutral red

0.03

Crystal violet

0.002

Lactose

10.0

Agar

12.0

pH 7.4 ± 0.2 @ 25°C  
*Adjusted as required to meet performance standards

Directions
Suspend 38.5g of VRBL Agar (ISO) dehydrated medium in 1 litre of distilled water. Bring to the boil while mixing. Continue to boil for up to 2 minutes or for the minimum time necessary to dissolve completely. DO NOT AUTOCLAVE. Cool to 44-47°C and use within 4 hours. Mix well before pouring.

Description
Methods for indicator or index organisms provide a simple approach for routine screening of foods. Besides safety, indicator groups provide a measure of food quality and spoilage potential. One of the most common groups of bacteria employed as indicator organisms by the food industry are the coliforms, which can be regarded as a subgroup within the Enterobacteriaceae.

Coliforms are commonly defined by their ability to ferment lactose rapidly, producing acid and gas, typically within 24 hours. The genera that would be commonly expected to belong to this group include Enterobacter, Klebsiella, Citrobacter and Escherichia, particularly Escherichia coli. However, species belonging to other genera, for example Erwinia and Serratia, can also ferment lactose, albeit slowly, and some strains of Citrobacter and Klebsiella, as well as Salmonella arizonae and Hafnia alvei, show delayed or variable fermentation ability.

Coliforms rapidly ferment the lactose in VRBL Agar (ISO) and so reduce the pH of the medium, producing red-purple colonies, due to the inclusion of neutral red and crystal violet. These colonies are usually surrounded by red-purple halos of precipitated bile salts.

Non-lactose or late lactose fermenters produce straw or colourless colonies with greenish halos. Other related Gram-negative bacteria may grow but can be suppressed by incubation at >42°C or by anaerobic incubation. Crystal violet and bile salts inhibit the growth of Gram-positive flora.

Technique
According to ISO 11133 :2014, VRBL Agar (ISO) should deliver a productivity ratio of ≥0.5 for E. coli when compared to the same inoculum grown on a reference medium (Tryptone Soya Agar). VRBL Agar (ISO) is tested in accordance with the requirements of ISO 11133:20142. Quality certificates indicating organisms tested and productivity are available for every batch.

Refer to the appropriate standard method for full instructions. The following is an overview of ISO 4832:20061.

  1. Prepare the sample to be tested according to the instructions in ISO 68873 or the specific standard as appropriate.
  2. Take two Petri dishes and transfer to each 1.0ml of the test sample or test suspension. Repeat the process with decimal dilutions of the sample if required.
  3. Within 15 minutes add approximately 15ml of molten VRBL Agar (ISO) at 44-47°C. Carefully mix the medium with the inoculum by horizontal movements and allow the medium to cool.
  4. Once the agar has solidified, pour an additional 4ml of medium onto the surface of the inoculated plate to prevent spreading growth and achieve semi-aerobic conditions.
  5. Incubate for 24 ± 2 hours at 30°C or 37°C as appropriate. (The temperature is subject to agreement between parties concerned. In the case of milk and milk products, the incubation temperature is 30°C.)
  6. Count the total number of characteristic colonies. Coliforms form dark red to purple coloured colonies larger than 0.5mm in diameter, usually surrounded by a purple zone but sometimes without. These are considered as typical colonies of coliforms and do not require further confirmation.
  7. Also count and confirm atypical colonies (e.g. of smaller size) and all colonies derived from milk products that contain sugars other than lactose.
  8. Inoculate five colonies of each atypical type, if available, into tubes of Brilliant Green Lactose Bile Broth (CM0031) containing a Durham tube.
  9. Incubate the tubes at 30°C or 37°C (as appropriate) for 24 ± 2 hours.
  10. Colonies that show gas formation in the Durham tube should be considered coliforms.

Storage conditions and Shelf life
VRBL Agar (ISO) should be stored in the tightly capped original container at 10-30°C. When stored as directed, the un-opened product will remain stable until the expiry date printed on the packaging.

Locally prepared VRBL Agar (ISO) plates can be stored for up to 2 weeks when made from CM0968 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 conditions.

Appearance
Dehydrated medium: Straw-pink, free-flowing powder
Prepared medium: Dark purple gel

Quality Control

Quality control includes testing in acordance with ISO 11133:20142

Positive controls: Expected results
Escherichia coli ATCC® 25922*
WDCM00013
Good growth;-purple-pink colonies with or purple haloes
Escherichia coli ATCC® 8739 *
WDCM00012
Good growth;-purple-pink colonies with or purple haloes
Negative controls:  
Pseudomonas aeruginosa ATCC® 27853*
WDCM00025
Good growth straw colonies
Enterococcus faecalis ATCC® 29212*
WDCM00087
No growth
Enterococcus faecalis ATCC® 19433*
WDCM00009
No growth

* This organism is also available as a Culti-Loop®

Precautions
VRBL Agar (ISO) should be prepared, cooled to 44-47°C and used within 4 hours.

VRBL Agar (ISO) 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.

References

1. ISO 4832:2006 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of coliforms - Colony-count technique
2. ISO 11133:2014 Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media
3. ISO 6887 series Microbiology of food and animal feed - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination.

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