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

  • Casein enzymic hydrolysate: 0.5
  • L-Asparagine: 1.0
  • Monopotassium phosphate:1.0
  • Disodium phosphate: 2.5
  • Ferric ammonium citrate: 0.05
  • Magnesium sulfate: 0.01
  • Calcium chloride: 0.0005
  • Zinc sulfate: 0.0001
  • Copper sulfate: 0.0001
  • Agar: 15.0
  • Final pH ( at 25°C) 6.6±0.2

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

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
  • The product is light and temperature sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements

  • Test specimens ( samples or growth of bacteria)
  • Inoculating loop
  • Bunsen burner
  • Incubator
  • Biosafety cabinet (BSC)
  • Control strain (Mycobacterium smegmatis ATCC 14468)

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

  • Mycobacterium smegmatis– luxuriant- rough or smooth, white dome-shaped colonies
  • M. tuberculosis–  luxuriant- flat, rough, dry, and usually non-pigmented
  • Mycobacterium avium -luxuriant smooth, thin, non-pigmented
    colonies
  • Mycobacterium gordonae-luxuriant -Smooth, yellow to orange
    colonies which are occasionally rough.
  • Mycobacterium kansasii-luxuriant -photochromogenic with flat, smooth, or somewhat granular surface and slightly undulating margins

Result Interpretation of Dubos Oleic Agar

  • No growth after 8 weeks: Negative
  • Growth: Positive (mention colony characteristics)
  • Control strain (Mycobacterium smegmatis ATCC 14468): rough or smooth, white dome-shaped colonies

Uses of Dubos Oleic Agar

  • Dubos Oleic Agar is used for the cultivation of Mycobacterium species.
  • It is also useful in antimicrobial susceptibility testing of Mycobacterium tuberculosis.
  • This medium is superior to others for the isolation of Mycobacterium tuberculosis from non-sterile clinical specimens.

Keynotes on Dubos Oleic Agar

  • Middlebrook and Dubos media are agar-based media while Lowenstein-Jensen medium is egg based.
  • Agar-based media for mycobacteria are normally recommended for testing clinical samples obtained from non-sterile sites.
  • Dubos Oleic Broth Base is Dubos and Middlebrook’s recommended medium for the primary isolation and subsequent cultivation of the tubercle bacilli.
  • On comparative analysis of various mycobacteria cultivating media, Dubos Oleic Agar was found to be superior to other media for the primary isolation of the Mycobacterium species.

Further Readings on Dubos Oleic Agar

  • https://www.himedialabs.com/TD/M198.pdf
  • Dubos R. J., and Middlebrook G., 1947, Am. Rev. Tuberc., 56:334
  • https://www.himedialabs.com/TD/M179.pdf
  • Bailey & Scott’s Diagnostic Microbiology. Editors: Betty A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  • Kent and Kubica, 1985, Public Health Mycobacteriology: A Guide For the Level III Laboratory, USDHHS, Center for Disease Control, Atlanta c.a.
  • Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC
  • Byham, 1950, Am. J. Clin. Pathol., 20:678
  • Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken, 7th ed 2005, Publisher ASM, USA.
  • Roberts A. H., Wallace R. J. and Erlich P., 1950, Am. Rev. Tuberc., 61:563.
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