Columbia CNA Agar

Intended Use

Columbia CNA Agar, Columbia CNA Agar, Modified, and Columbia PNA Agar, all supplemented with 5% sheep blood, are selective and differential media used for the isolation and differentiation of gram-positive microorganisms from clinical and nonclinical materials.

Summary and Explanation

Ellner et. al., in 1966, reported the development of a blood agar formulation, which has been designated as Columbia Agar.¹ The Columbia Agar base, which achieves rapid and luxuriant growth and sharply defined hemolytic reactions, is utilized as the base for media containing blood and for selective formulations in which various combinations of antimicrobial agents are used as additives.
Ellner and his colleagues found that a medium consisting of 10 mg of colistin and 15 mg of nalidixic acid per liter in a Columbia Agar Base enriched with 5% sheep blood would support the growth of staphylococci, hemolytic streptococci and enterococci while inhibiting the growth of Proteus, Klebsiella and Pseudomonas species. In BBL™ Columbia CNA Agar with 5% Sheep Blood, the concentration of nalidixic acid has been reduced to 10 mg/L to increase the recovery of gram-positive cocci from clinical specimens. The concentration of nalidixic acid has been further reduced in Columbia CNA Agar, Modified to 5 mg/L.
In the Columbia PNA version of Ellner’s medium, polymyxin B has been substituted for colistin (10 mg). Although the antimicrobial properties of the two agents are nearly the same, some species of gram-negative bacteria are more sensitive to polymyxin B than colistin.²

Principles of the Procedure

These media derive their superior growth-supporting properties from the combination of peptones prepared from pancreatic digest of casein, peptic digest of animal tissue and beef extract. Yeast extract and corn starch are also included in the formulation and serve as energy sources, with yeast extract being a supplier of the B-complex vitamins.
Sheep blood supports the growth of fastidious organisms and allows detection of hemolytic reactions. It should be noted that this medium has a relatively high carbohydrate content and, therefore, beta-hemolytic streptococci may produce a greenish hemolytic reaction that may be mistaken for alpha hemolysis.
The addition of the antimicrobial agents, colistin (or polymyxin B) and nalidixic acid, renders the medium selective for grampositive microorganisms.³ Colistin and polymyxin B disrupt the cell membrane of gram-negative organisms, whereas the nalidixic acid blocks DNA replication in susceptible gramnegative bacteria.⁴

User Quality Control

Identity Specifications
Columbia CNA Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                        4.25% solution, soluble in purified water upon
                                     boiling. Solution is medium, tan to yellow, hazy.
Prepared Appearance:    Tan to yellow, hazy.
Reaction of 4.25%
Solution at 25°C:            pH 7.3 ± 0.2
Cultural Response
Columbia CNA Agar
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C with 3-5% CO2 for 18-24 hours.

Formula

Columbia CNA Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 12.0 g
Peptic Digest of Animal Tissue...................................... 5.0 g
Yeast Extract.............................................................. 3.0 g
Beef Extract................................................................ 3.0 g
Corn Starch................................................................ 1.0 g
Sodium Chloride......................................................... 5.0 g
Agar......................................................................... 13.5 g
Colistin................................................................... 10.0 mg
Nalidixic Acid........................................................... 10.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 42.5 g of the powder in 1 L 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 12 minutes. Cool to 45-50°C.
4. Add 5% sterile, defibrinated sheep blood.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. Incubate plates at 35 ± 2°C for 24-48 hours in an aerobic atmosphere supplemented with carbon dioxide.

Expected Results

Typical colonial morphology on Colombia CNA Agar with 5% Sheep Blood is as follows:
Streptococci (non-group) D.........Small, white to grayish. Beta or alpha hemolysis.
Enterococci (group D) ............... Small, but larger than group A streptococci, blue-gray. Beta or alpha hemolysis.
Staphylococci ........................... Large, white to gray or cream to yellow, with or without hemolysis.
Micrococci ................................ Large, white to gray or yellow to orange, with or without hemolysis.
Corynebacteria ........................ Small to large, white to gray or yellow, with or without hemolysis.
Candida .................................. Small, white.
Listeria monocytogenes ............ Small to large, blue-gray, with beta hemolysis.
Gram-negative bacteria ............ No growth to trace growth.

*Store at 2-8° C.

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