CRITERION™ VIOLET RED BILE GLUCOSE AGAR (VRBGA)

Cat. no. C7270 CRITERION™ Violet Red Bile Glucose Agar 71gm
Cat. no. C7271 CRITERION™ Violet Red Bile Glucose Agar 500gm
Cat. no. C7272 CRITERION™ Violet Red Bile Glucose Agar 2kg
Cat. no. C7273 CRITERION™ Violet Red Bile Glucose Agar 10kg

INTENDED USE

Hardy Diagnostics CRITERION™ Violet Red Bile Glucose Agar is recommended for the detection and enumeration of Enterobacteriaceae in food and dairy products.

SUMMARY

Enterobacteriaceae include lactose-fermenting coliform bacteria, non-lactose-fermenting strains of Escherichia coli, and other non-lactose-fermenting species of Salmonella and Shigella involved in food spoilage. Because of their potential contamination of food and dairy products, it is important to detect members of the Enterobacteriaceae, rather than traditional coliform bacteria.(1-4)

All species in the Enterobacteriaceae family ferment glucose. Mossel et al. modified traditional Violet Red Bile Agar, adding glucose, resulting in the formulation now known as Violet Red Bile Glucose Agar.(5-7)

CRITERION™ Violet Red Bile Glucose Agar contains peptones and yeast extract to supply carbon, nitrogen, essential minerals, and B-complex vitamins to stimulate the growth of bacteria. Glucose supplies energy for growth and metabolism. Bile salts and crystal violet inhibit the growth of gram-positive bacteria. Neutral red is added as a pH indicator. Agar is the solidifying agent. Organisms that ferment glucose will produce red to purple colonies with red-purple halos, demonstrating bile precipitation in the presence of neutral red.

FORMULA*

Gram weight per liter: 35.6gm/L
Glucose 10.0gm
Enzymatic Digest of Gelatin 7.0gm
Sodium Chloride 5.0gm
Yeast Extract 3.0gm
Bile Salts 1.5gm
Neutral Red 0.03gm
Crystal Violet 2.0mg
Agar 9.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-3ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original reddish-beige.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 35.6gm of the dehydrated culture media in one liter of distilled or deionized water. Stir to mix thoroughly.

2. Heat with frequent agitation and boil for one minute to dissolve completely. Do not autoclave.

3. Cool to 45-50ºC. and dispense desired amount into sterile containers.

PROCEDURE

Consult listed references for information on sample collection and procedures for use.(1-8)

The medium is appropriate for use with spread plate or pour plate methods, with or without an agar overlay. The medium can also be used as an agar overlay for spread plates to prevent colony swarming and to provide semi-anaerobic conditions to suppress the growth of nonfermentative, gram-negative microorganisms. Additionally, stab inoculation procedures using agar deeps can be used with Violet Red Bile Glucose Agar.

Spread Plate Method:

1. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

2. Aseptically inoculate agar surface with 0.1ml of well mixed diluted sample.

3. Spread the dilution evenly over the surface of the medium.

4. Using a sterile spreader device, distribute the inoculum evenly over the agar surface.

5. Incubate plates aerobically for 48 +/- 2.0 hours at 35ºC.

Pour Plate Method:

1. Melt agar by placing in a boiling waterbath until liquified.

2. Cool media to 45-50ºC. Maintain in a 45-50ºC waterbath until ready to pour.

3. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

4. Place a 1ml inoculation into a sterile petri plate.

5. Aseptically pour approximately 18ml of the cooled media (45-50ºC) over the inoculum. Carefully swirl the plate to mix the inoculum evenly.

Note: Do not keep media longer than 3 hours at 45-50ºC. Sterile solidified medium can only be remelted once.

6. Allow to solidify.

7. Incubate plates aerobically for 48 +/- 2.0 hours at 35ºC.

INTERPRETATION OF RESULTS

Enterobacteriaceae ferment glucose, thereby producing acid by-products, and form red to dark purple colonies surrounded by a reddish zone, or halo, of bile precipitate.

LIMITATIONS

If using the pour plate technique, the medium should be prepared fresh, tempered to 45ºC, and used within 3 hours.

Due to a variation in nutritional requirements, some strains encountered may grow poorly or fail to grow at all on this medium.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC ® 25922
B 18-24hr 35°C Aerobic Growth; red to purple colonies, with reddish precipitate
Salmonella enterica
ATCC ® 14028
B 18-24hr 35°C Aerobic Growth; red to purple colonies, with reddish precipitate
Enterobacter aerogenes
ATCC ® 13048
A 18-24hr 35°C Aerobic Growth; red colonies, may have a slight precipitate
Staphylococcus aureus
ATCC ® 25923
A 18-24hr 35°C Aerobic Partial to complete inhibition; colorless to red colonies, no bile precipitate

User Quality Control

Physical Appearance

CRITERION™ Violet Red Bile Glucose Agar powder should appear homogeneous, free-flowing, and reddish-beige in color. The prepared medium should appear clear, slightly opalescent, and reddish-purple in color.

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Draft Standard Methods for Microbiological Examination of Meat Products. 1977. Part 3: Detection and enumeration of Enterobacteriaceae. BS5393: Part 3, ISO/DIS 5552.

4. Mossel, D.A.A. 1985. Media for Enterobacteriaceae. Int. J. Food. Microbiol.; 2:27.

5. Mossel, D.A.A., W.H.J. Mengerink, and H.H. Scholts. 1962. Use of a modified MacConkey agar medium for the selective growth and enumeration of Enterobacteriaceae. J. Bacteriol.; 84:381.

6. Mossel, D.A.A., I. Eelderink, M. Koopmans, and F. van Rossem. 1978. Lab Practice; 27:1049-1050.

7. Mossel, D.A.A., I. Eelderink, M. Koopmans, and F. van Rossem. 1979. Influence of carbon source, bile salts and incubation temperature on recovery of Enterobacteriaceae from food using MacConkey-type agars. J. Food Protect.; 42:470.

8. The Official Compendia of Standards. 2008. USP27-NF22. United States Pharmacopeial Convention, Rockville, MD.


ATCC is a registered trademark of the American Type Culture Collection.

062716gr