In 1979, Dr. A. Rambach invented and patented the first chromogenic medium for E.coli detection. This technology uses a color-based differentiation technique. It uses soluble colorless molecules, also known as chromogens. They are composed of a substrate that targets a specific enzyme activity and a chromophore. The chromophore can be released when the enzyme of the target organism cleaves the colorless, chromogenic conjugate.
The unconjugated chromophore has a distinctive color, and forms precipitates due to its reduced solubility. Under normal lighting conditions, the result is extremely specific and easily distinguishable by the naked eye. This technique allows you to identify specific microorganism colonies by their color at a glance. Rambach Agar eliminates many false positives, allowing technicians to concentrate on the actual contaminated samples.
Composition of CHROMagar
pH 7.4 ± 0.2 at 25°C
Principle of CHROMagar
Enterobacteriaceae can multiply easily because of the Rambach Agar’s nutritive substrates. The accompanying Gram-positive flora is inhibited by sodium deoxycholate. Rambach Agar allows Salmonella species to be distinguished clearly from other bacteria by adding propylene glycol in the culture medium. Salmonellae produce acid with propylenegl, which when combined with a pH indicator gives rise to the distinctive red color of the colonies. Peptone provides nitrogen and vitamins to the organism’s growth. The integrity of cells is maintained by sodium chloride, which maintains an osmotic equilibrium.
The medium must contain a chromogen that indicates the presence of b–galactosidase split, which is a characteristic of coliforms. Blue-violet colonies are produced by lactose-fermenting bacteria (ss-galactosidase-producing). Salmonellae make acid from propylene glycol, which when combined with the pH indicator results in typical pink-red colonies. Other enteric bacteria gram-negative form colorless-yellowish colonies. Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Typhimurium produce pink-to-red colonies.
Preparation of Medium of CHROMagar
- You can add components to deionized/distilled water to increase volume to 1.0 L.
- Mix well.
- Place in a boiling water bath, or in a stream of steam. Stir occasionally.
- If there are no visible particles sticking to the glass wall, then the medium is suspended completely.
- The medium shouldn’t be heated further.
- It takes approximately 35-40 minutes to complete dissolution by shaking the mixture in 5-min intervals.
- Autoclave is not recommended. Avoid overheating.
- Keep at 45°C to 50°C, while gently shaking every now and again.
- Pour into sterile Petri dishes.
- Petri dishes should be placed on cool surfaces during pouring to prevent precipitate and clotting of the chromogenic mixture. 25degC) surface.
- These plates are pink and opaque.
Result on CHROMagar
Salmonella → red/intense mauve
Many Coliforms → blue-green or blue-violet
Proteus, Pseudomonas → colorless-yellowish colonies
Applications of CHROMagar
- To distinguish organisms based on their specific color colonies.
- It is a differential-diagnostic culture medium for identifying types of organisms in foodstuffs and clinical samples.
- Rambach agar can be used as a supplementary agar for Salmonella testing foods
- Additional applications include rapid detections of Clostridium perfringens and Listeria monocytogenes as well as Bacillus cereus and Staphylococcus Aureus using enzyme detection methods in food microbiology and water microbiology with chromogenic yeast medium.