Dubos Oleic Agar Base

Intended Use

Dubos Oleic Agar Base is used with Dubos Oleic Albumin Complex and penicillin for isolating and determining the susceptibility of M. tuberculosis.

Summary and Explanation

Mycobacterial infections, particularly tuberculosis, are a worldwide health problem. Almost three million people worldwide die of tuberculosis each year.¹ During the mid 1980s, the number of tuberculosis (TB) cases in the U.S. began increasing. Prior to this time, the number of cases in the U.S. had been decreasing, reaching a low in 1984.² Non-tuberculous mycobacterial infections have also increased since the mid 1980s.³
Dubos Broth is prepared according to the Dubos, Fenner and Pierce⁴ modification of the medium originally described by Dubos and Davis⁵ and Dubos and Middlebrook.⁶
Dubos and Middlebrook⁶ described Dubos Oleic Medium Albumin as suitable for primary isolation and cultivation of the tubercle bacillus and for studying colony morphology. In comparative studies, Dubos Oleic Albumin Agar Medium was superior to other media studied for primary isolation.^7,8 There are two types of solid culture media for the primary isolation of mycobacteria, those that have coagulated egg as a base and those that have agar. Lowenstein formulations are examples of media that contain egg; Middlebrook and Dubos formulations contain agar.
Agar based media are not liquefied by contaminating proteolytic organisms but overgrowth may occur. These media are recommended for specimens from nonsterile sites.⁹ The medium is clear so colonies of mycobacteria can be viewed through a stereo microscope even if contaminating organisms are present. Colonies can be observed in 10-12 days.
Drugs may be added to Dubos media in exact concentrations because the medium is solidified with agar rather than by inspissation. Also, there is less drug inactivation when egg ingredients are not present.
Mycobacteria grow more rapidly in broth media. Primary culture of all specimens in broth media is recommended.¹⁰ Polysorbate 80 in the medium acts as a surfactant, dispersing the bacilli, which increases growth.
Dubos Broth, Enriched is a modified medium based on the formulation of Dubos et al.⁴ This formulation differs from the original in that it has a strong buffering system and an acid pH.¹¹ The particular value of Dubos Broth, Enriched is that it provides dispersed growth, free of excessive clumps, which can be used to prepare a relatively uniform suspension of mycobacteria for use in bacterial studies. It is also used as a subculture and enrichment medium for the rapid cultivation of M. tuberculosis and other mycobacterial species from treated clinical specimens and from direct inoculation of specimens that may yield pure cultures; e.g., cerebrospinal fluid.¹²

Principles of the Procedure

Peptone and asparagine are sources of nitrogen. Disodium phosphate and monopotassium phosphate are sources of phosphates and, along with calcium chloride, help maintain the pH of the medium. Magnesium sulfate, ferric ammonium sulfate, zinc sulfate and copper sulfate are sources of trace metals and sulfates. Polysorbate 80, an oleic acid ester, supplies essential fatty acids for the replication of mycobacteria. Bovine albumin acts as a protective agent by binding free fatty acids that may be toxic to mycobacteria. The albumin is heat-treated to inactivate lipase, which may release fatty acids from the polysorbate 80. Phosphate buffers maintain the pH of the medium. Agar is the solidifying agent.

User Quality Control

Identity Specifications
Dubos Oleic Agar Base
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       2.0% solution, soluble in purified water upon
                                   boiling. Solution is light amber, slightly opalescent
                                   to opalescent with fine precipitate.
Prepared Appearance:    Light amber, slightly opalescent to opalescent
                                   with fine precipitate.
Reaction of 2.0%
Solution at 25°C:           pH 6.6 ± 0.2
Cultural Response
Dubos Oleic Agar Base
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C with 5-10% CO2 for up to 3 weeks.

Formula

Dubos Oleic Agar Base
Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 0.5 g
Asparagine................................................................ 1.0 g
Monopotassium Phosphate.......................................... 1.0 g
Disodium Phosphate (anhydrous)................................. 2.5 g
Agar........................................................................ 15.0 g
Ferric Ammonium Citrate......................................... 50.0 mg
Magnesium Sulfate................................................. 10.0 mg
Calcium Chloride...................................................... 0.5 mg
Zinc Sulfate............................................................. 0.1 mg
Copper Sulfate......................................................... 0.1 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Precautions^13,14

1. Biosafety Level 2 practices, containment equipment and facilities are required for non-aerosol-producing manipulations of clinical specimens such as preparation of acid-fast    smears. All aerosol-generating activities must be conducted in a Class I or II biological safety cabinet.
2. Biosafety Level 3 practices, containment equipment and facilities are required for laboratory activites in the propagation and manipulation of cultures of M. tuberculosis and M.     bovis. Animal studies also require special procedures.

Directions for Preparation from Dehydrated Product

1. Suspend 4 g of the powder in 180 mL of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. Cool to below 50-55°C.
4. Aseptically add 20 mL Dubos Oleic Albumin Complex and 5,000-10,000 units of penicillin (25-50 units per mL of medium). Mix thoroughly.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

The test procedures are recommended by the Centers for Disease Control and Prevention (CDC) for primary isolation from specimens containing mycobacteria.¹³ N-acetyl-L-cysteinesodium hydroxide (NALC-NaOH) solution is recommended as a gentle, but effective digesting and decontaminating agent. These reagents are provided in the BBL™ MycoPrep™ Specimen Digestion/Decontamination Kit. For detailed decontamination and culturing instructions, consult an appropriate text.^3,9,12,13,15
Specimens that are less likely to be contaminated with other microorganisms (cerebrospinal fluid, pleural fluid, tissue biopsy, etc.) may be inoculated directly into the medium. Consult appropriate texts for recommended procedures.^3,9,12,13,15 Incubate the tubes at 35 ± 2°C in a CO₂-enriched atmosphere. Keep the tube caps loosened for at least one week to permit circulation of CO₂, but tighten the caps thereafter to prevent dehydration. Loosen briefly once a week to replenish CO₂. Six to eight weeks of incubation may be necessary for evidence of growth of many mycobacteria.

Expected Results

Growth of mycobacterial colonies on the agar medium or in broth media, as indicated by turbidity compared to an uninoculated control.

Limitations of the Procedure

1. Negative culture results do not rule-out active infection by mycobacteria. Some factors that are responsible for unsuccessful cultures are:
• The specimen was not representative of the infectious material; i.e., saliva instead of sputum.
• The mycobacteria were destroyed during digestion and decontamination of the specimen.
• Gross contamination interfered with the growth of the mycobacteria.
• Proper aerobic conditions and increased CO₂ tension were not provided during incubation.
2. Mycobacteria are strict aerobes and growth is stimulated by increased levels of CO₂. Screw caps on tubes or bottles should be handled as directed for exchange of CO₂.

*Store at 2-8° C.

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