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Dubos Oleic Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

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Dubos Oleic Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Dubos Oleic Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Dubos Oleic Agar

Dubos Oleic Agar is commonly used for isolation and antimicrobial susceptibility testing (AST) of Mycobacterium tuberculosis . M. tuberculosis, the etiological agent of tuberculosis. The organism may present in airborne particles(droplet nuclei)that are generated when patients with pulmonary tuberculosis cough.  The disease can happen when a susceptible person inhales the droplet nuclei containing M. tuberculosis. It is one of the common agar-based media for the isolation of mycobacteria.

Principle of Dubos Oleic Agar

Casein enzymic hydrolysate and L-aspargine as sources of nitrogen in Dubos Oleic Agar for  Mycobacterium species. The phosphates together with calcium chloride act as buffers for the media as well as serve as sources of phosphates. The combination of magnesium sulfate, zinc sulfate, copper sulfate, and ferric ammonium citrate provides trace metals and sulfates. Dubos Oleic Agar is lacking glycerol or dextrose to stop the growth of commensals. Agar acts as the solidifying agent.  Water is an essential ingredient for the growth and reproduction of organisms and also serves as a transport medium for the agar’s various substances. The medium becomes selective with the addition of antibiotics while the Oleic albumin supplement provides an important role in the metabolism of mycobacteria and binding to free fatty acids.

Composition of Dubos Oleic Agar

Ingredients  Gms / Litre

Extra Ingredients for 180 ml medium

  1. Oleic Albumin Supplement: 20 ml
  2. Penicillin:  5,0000 -10,000U

 Preparation of Dubos Oleic Agar

  1. Suspend 4.0 grams in 1 80 ml of purified/distilled or deionized water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  4. After autoclaving,  leave for cooling to 45-50°C.
  5. Aseptically add sterile Oleic Albumin Supplement and antibiotics (Penicillin)  volume as mentioned in the composition section.
  6. Mix thoroughly.
  7. Pour in sterile test tubes or Petri plates and leave tubes/plates on the sterile surface until the agar has solidified.
  8. Store the tubes/plates in a refrigerator at 2-8°C.

Storage and Shelf life of Nutrient agar

Test Requirements

Procedure of Dubos Oleic Agar

  1. Allow the tubes/ plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
  2. Perform inoculating in a biosafety cabinet since M. tuberculosis is the risk group 3rd organism.
  3. Inoculate and streak the specimen or bacterium.
  4. If the culturing specimen is on a swab, roll the swab over a small area of the agar surface.
  5. Streak for isolation with a sterile loop.
  6. Incubate at 35-37°C  until growth is observed or discarded as negative after 8  weeks.
  7. Examine culture weekly if possible otherwise on at least three occasions. After a week to detect rapidly growing mycobacteria which may be mistaken for M. tuberculosis. After 3-4 weeks to detect positive cultures of Mycobacterium tuberculosis as well as other slow-growing mycobacteria which may be either harmless saprophytes or potential pathogens. Afterwards 8 weeks to detect very slow-growing mycobacteria, including Mycobacterium tuberculosis, before judging the culture to be negative.

Colony Morphology of Dubos Oleic Agar

Organism- Growth- Colony Morphology

Result Interpretation of Dubos Oleic Agar

Uses of Dubos Oleic Agar

Keynotes on Dubos Oleic Agar

Further Readings on Dubos Oleic Agar