VIOLET RED BILE (VRB) AGAR

Cat. no. G78 Violet Red Bile Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. U96 Violet Red Bile Agar, 125ml Polycarbonate Bottle, 100ml Deep 20 bottles/box
Cat. no. U296 Violet Red Bile Agar, 8oz. Glass Bottle, 200ml 12 bottles/box

INTENDED USE

Hardy Diagnostics Violet Red Bile Agar is recommended for the detection of coliforms in food or dairy products.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Violet Red Bile Agar is a selective medium used to detect and enumerate lactose-fermenting coliform microorganisms. The medium is recommended for use in the microbiological analysis of milk and other dairy products, and for use in the examination of water. (1,2)

The medium contains bile salts and crystal violet which serve as inhibitory agents toward some gram-positive microorganisms, especially staphylococci. Neutral red is employed as the pH indicator.

Lactose-fermenting microorganisms produce pink to red colonies that are generally surrounded by a reddish zone of precipitated bile. Non-lactose-fermenting microorganisms result in colorless colonies.

FORMULA

Ingredients per liter of deionized water:*

Lactose 10.0gm
Pancreatic Digest of Gelatin 7.0gm
Sodium Chloride 5.0gm
Yeast Extract 3.0gm
Bile Salts No. 3 1.5gm
Neutral Red 30.0mg
Crystal Violet 2.0mg
Agar 15.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample Collection: Consult listed references for information on sample collection. (1,2)

Method of Use: Allow medium to warm to room temperature prior to inoculation. Consult listed references for information concerning inoculation procedures. (1,2)

For melting bottled media: Autoclave at 121ºC. for 1-3 minutes or until melted. Alternatively, a covered, boiling waterbath (100 degrees C.) can be used. There should be enough water in the waterbath to reach the media line. A covered waterbath will help to reach and maintain the temperature. Heat in waterbath until melted.

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 45-50º 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: After autoclaving, do not heat 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

Lactose-fermenting microorganisms, including coliforms, produce pink to red colonies that are generally surrounded by a reddish zone of precipitated bile.

Surface colonies of Escherichia coli appear as entire-edged colonies, while deep colonies appear lens-shaped.

Colonies of Enterobacter aerogenes often appear mucoid and pinkish in color.

Enterococcus spp. may grow, and if so, usually appear pinpoint in size and rose colored.

Non-lactose-fermenting microorganisms produce colorless colonies.

LIMITATIONS

Violet Red Bile Agar contains a low concentration of bile salts, and therefore is not completely specific for enterics.

Enterococci may grow, and if so, usually appear pinpoint in size and rose colored.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, staining supplies, Lauryl Sulfate Broth, other culture media, microscopes, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Enterobacter aerogenes
ATCC ® 13048
A 18-24hr 35°C Aerobic Growth; pink to red colonies, may have a slight precipitate around colonies colonies
Escherichia coli
ATCC ® 25922
A 18-24hr 35°C Aerobic Growth; pink to red colonies, with a red precipitate around colonies
Salmonella enteritidis
ATCC ® 13076
A 18-24hr 35°C Aerobic Growth; colorless colonies
Staphylococcus aureus
ATCC ® 25923
B 18-24hr 35°C Aerobic Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

Violet Red Bile Agar should appear slightly opalescent, and reddish-purple in color.

E. aerogenes growing on Violet Red Bile Agar

Enterobacter aerogenes (ATCC ® 13048) colonies growing on Violet Red Bile Agar (Cat no. G78). Incubated aerobically for 24 hours at 35ºC.

E. coli growing on Violet Red Bile Agar

Escherichia coli (ATCC ® 25922) colonies growing on Violet Red Bile Agar (Cat no. G78). Incubated aerobically for 24 hours at 35ºC.



S. enteritidis growing on Violet Red Bile Agar

Salmonella enteritidis (ATCC ® 13076) colonies growing on Violet Red Bile Agar (Cat no. G78). Incubated aerobically for 24 hours at 35ºC.

S. aureus inhibited on Violet Red Bile Agar

Staphylococcus aureus (ATCC ® 25923) growth inhibited on Violet Red Bile Agar (Cat no. G78). Incubated aerobically for 24 hours at 35ºC.



REFERENCES

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

2. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

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

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

041816gr