Violet Red Bile Lactose Agar: Selective Medium for the Detection of Lactose-Fermenting Bacteria

Table of Contents

• Introduction
• Composition of VRBL Agar
• Principle of VRBL Agar
• Macroscopic Observation on VRBL Agar
• Preparation of VRBL Media
• Procedure
• Interpretation
• Uses of VRBL Media
• Limitations of VRBL Media
• Precautions of VRBL Media

Introduction:

• VRBL Agar (Violet Red Bile Lactose Agar):
• Selective medium for isolating, detecting, and enumerating lactose-fermenting bacteria.
• Differentiates coliforms or Coli aerogenes group from non-lactose fermenting organisms.
• Used in water, milk, dairy products, dairy equipment, and other foodstuffs.
• Coliforms are gram-negative bacilli, non-sporulated, oxidase negative, aerobic or anaerobic, able to multiply in the presence of bile salts or other surface agents which can ferment lactose rapidly, producing acid and gas, usually within 24 hours or 48 hours at the temperature of 44 ± 0.5°C.
• According to the International Organization for Standardization (ISO), fecal coliforms are also known as thermotolerant coliforms.
• It belongs to several bacterial species:
• Enterobacter aerogenes
• Enterobacter cloacae
• Klebsiella (Klebsiella pneumoniae, Klebsiella oxytoca)
• Citrobacter freundii
• Citrobacter diversus
• Citrobacter amalonaticus
• Escherichia coli
• Other organisms that can also ferment lactose are:
• Erwinia and Serratia (slowly)
• Moellerella wisconsensis
• Salmonella enterica subsp. III (Salmonella arizonae)
• Yersinia enterocolitica
• Hafnia alvei

Composition of VRBL Agar

Ingredients in grams/liter:

• Peptone: 7.0 gm
• Yeast Extract: 3.0 gm
• Sodium chloride: 5.0 gm
• Lactose: 10.0 gm
• Bile Salts No. 3: 1.5 gm
• Crystal Violet: 0.002 gm
• Neutral Red: 0.03 gm
• Agar: 15.0 gm
• Distilled or deionized water: 1000.00 ml
• Final pH: 7.4 +/- 0.2 at 25°C

Storage instructions:

• Store the prepared media at 2°C-8°C and protect from direct sunlight.
• Store the dehydrated powder in a cool, dry place in tightly sealed containers or bottles at 15°C-25°C to protect from moisture and light.
• After opening and replacing the cap, store the container in a low-humidity area at the same storage temperature.

Expiration and disposal:

• The expiration date is on the package or container.
• Discard if the dehydrated medium is not freely flowing or its appearance has altered from its original color.

Principle of VRBL Agar

• VRBL Agar, containing bile salts and crystal violet, is based on MacConkey Agar.
• Not required for use in the diagnosis of disease or other conditions in humans.
• Peptone: Source of nitrogen, vitamins, minerals, and amino acids essential for growth.
• Yeast Extract: Source of vitamins, especially those in the B group.
• Lactose: Fermentable sugar or carbohydrate providing carbon and energy.
• Bile Salts and Crystal Violet: Inhibit non-target Gram-positive and some Gram-negative bacteria.
• Neutral Red: pH indicator that changes to a red-purple color due to acid production during fermentation.
• Sodium Chloride: Provides essential electrolytes for transport and osmotic balance.
• Bacteriological Agar: Solidifying agent.

Microorganisms or Coliforms: 

• Rapid lactose fermenters produce purple or red-purple colonies with bile salt precipitation, often surrounded by a halo of the same color.
• Non-lactose fermenters and late lactose fermenters produce pale colonies with greenish areas.
• Occasionally, intestinal tract cocci can form small, punctiform red colonies.
• Red-purple colonies typically have a diameter > 0.5mm.

Selectivity:

• Increased by anaerobic incubation or elevated temperatures above 42°C for 18 hours to suppress unwanted microorganisms or other Gram-positive bacteria.

Macroscopic Observation on VRBL Agar

After incubation at 37°C for 18-24 hours under anaerobic conditions, the color and growth of colonies are as follows:

Preparation of VRBL Media

• Weigh 38.5 grams of VRBL agar and suspend it in 1 liter of purified or distilled water.
• Allow it to absorb for 5 minutes.
• Heat gently with frequent agitation.
• Mix the medium properly until a homogeneous suspension is obtained by boiling it for a few seconds.
• Note: Do not autoclave, sterilize, or overheat.
• Cool the mixture to 45°C.
• If necessary, adjust the pH to 7.4 ± 0.2.
• Pour the medium into sterilized Petri dishes or tubes.
• The medium should appear light purple-violet and transparent.
• The medium can be used up to 4 hours after preparation.

Procedure

• Perform the pour or spread plate procedure with or without overlay.
• For the pour plate method:
• Freshly prepare the medium, tempered to 47°C, and use within 3 hours.
• Incubation guidelines:
• Coliforms: Incubate at 37°C for 18-24 hours.
• Psychrotrophs: Incubate at 4°C for ten days.
• Mesotrophs: Incubate at 32°C for 24-48 hours.
• Thermotrophs: Incubate at 42°C for 18 hours.
• Escherichia coli: Incubate at 44 ± 0.1°C.
• Thermotolerant coliforms: Incubate at 44 ± 1°C for 24 ± 2 hours.
• Examine for purple or red/purple colonies with red/purple haloes with a diameter ≤ 0.5 mm.
• Continue confirmatory testing of typical organism colonies according to the applicable standards.

Interpretation

• After incubation of coli-aerogens groups at 35 or 37°C for 24 hours, count coliform colonies on Petri plates.
• Coliform characteristics:
• Round, purple, or red-purple colonies with red-purple haloes (lactose positive types).
• Pale colonies with greenish zones (lactose negative types).
• Colonies have a diameter ≤ 0.5 mm.

Uses of VRBL Medium

• VRBL agar is used to examine food, animal feed, and environmental samples for the presence of coliforms, which are included as a subgroup within the Enterobacteriaceae.
• VRBL agar is used to enumerate fecal coliforms and thermotolerant coliforms in foods.

Limitations of VRBL Media

• Some strains may grow poorly or not at all on VRBL agar due to variations in nutritional requirements.
• During the pour-plating procedure, elevated temperatures can stress coliforms, affecting their growth.
• Therefore, fecal coliform agar (FCA) can also be used to recover and enumerate stressed fecal coliforms.

Precautions of VRBL Media

• Always wear a proper lab coat and gloves when working in the laboratory.
• Handle chemicals and live infectious microorganisms (bacteria) with care.
• According to local rules and regulations, media that come into contact with food samples are considered contaminated and should be disposed of as tainted.
• Ensure the time interval between preparing the stock solution (or the 10-1 dilution for solid products) and the dilutions coming into contact with the culture medium does not exceed 15 minutes.