Columbia CNA Agar – Composition, Preparation, Uses, and Results

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Columbia CNA Agar

  • Columbia CNA Agar is used to isolate gram-positive cocci from clinical and non-clinical samples.
  • Ellner et al. created Columbia Agar in 1966 as a nutrient-rich blood medium that promoted healthy growth and clearly defined hemolysis reactions.
  • Ellner and company also described a selective formulation using colistin and nalidixic acid as antibacterial agents.
  • Colistin, a polypeptide antibiotic belonging to the polymyxin group, and nalidixic acid, a first-generation quinolone, are largely active against gram-negative bacteria; hence, Columbia CNA Agar is an excellent medium for the selective isolation of gram-positive cocci.
  • Enterobacteriaceae and Pseudomonas species are inhibited while yeast, staphylococci, streptococci, and enterococci are permitted to flourish.

Columbia CNA Agar with 5% Sheep Blood

  • The selective medium Columbia CNA Agar with 5% Sheep Blood is used to isolate gram-positive bacteria (particularly staphylococci and streptococcus) from clinical specimens.
  • Ellner et al. announced in 1966 the invention of a blood agar formulation, now known as Columbia Agar.
  • This medium, which produces larger colonies and lusher growth than equivalent blood agar bases, is used for media containing blood and for specific formulations.
  • Ellner et al. discovered that a medium containing 10 mg of colistin and 15 mg of nalidixic acid per litre in a Columbia agar base enriched with 5% sheep blood promotes the growth of staphylococci, hemolytic streptococci, and enterococci while inhibiting the growth of Proteus, Klebsiella, and Pseudomonas species.
  • Columbia Agar provides a nutrient-rich medium basis. Colistin and nalidixic acid make the medium selective for gram-positive bacteria, particularly streptococci and staphylococci.
  • The concentration of nalidixic acid in BD Columbia CNA Agar with 5% Sheep Blood has been decreased to 10 mg/l in order to increase the recovery of gram-positive cocci from clinical specimens.
  • Sheep blood permits the detection of hemolytic responses, which are crucial in the presumptive diagnosis of streptococci.

Composition of Columbia CNA Agar per Liter of Medium

Casein-Meat Peptone10.0 g
Casein-Yeast Peptone10.0 g
Heart Peptone3.0 g
Sodium Chloride5.0 g
Corn Starch1.0 g
Agar13.5 g
Defibrinated Sheep Blood50.0 mL
Colistin Sulfate10 mg
Nalidixic Acid15 mg


  1. Permit the medium to reach room temperature before inoculation.
  2. Perform a four-quadrant streak using a direct inoculum from the specimen to obtain well-isolated colonies. If desired, punctures can also be formed in the medium to enhance and better observe hemolytic reactions.
  3. Depending on the organism of interest, incubate plates at 35°C in an aerobic, anaerobic, or CO2-enriched atmosphere.
  4. Examine following 24 and 48 hours.

Interpretation of Results

After the incubation period, check for growth and hemolysis on the plates. Hemolysis is a helpful differential characteristic that is best observed when a bright light is passed through the plate from behind. There are four different forms of hemolysis:

  • Alpha-hemolysis (α) — Partial hemolysis characterised by a greenish tint surrounding the colony.
  • Beta-hemolysis (β) – Complete lysis of red blood cells, resulting in a clean zone surrounding the colony.
  • Gamma-hemolysis (γ) — Absence of hemolysis resulting in no medium change.
  • Alpha-prime-hemolysis (α′) – A small region of full hydrolysis surrounded by a region of incomplete hemolysis.

To complete identification, additional biochemical or serological tests must be conducted on colonies separated from pure culture.

  • Some streptococci have been demonstrated to be affected by the incubation atmosphere; for optimal results, incubate plates in a CO2-enriched or anaerobic environment.

Quality Control

After evaluating the pH, colour, depth, and sterility of the final medium, the following organisms are employed to determine its growth performance.


Gram-negative bacilli may overrun gram-positive cocci on standard blood agar. When gram-positive cocci (Streptococcus species and Staphylococcus species) are present in a mixed culture with gram-negative bacilli, Columbia CNA agar facilitates their isolation. The inclusion of sheep blood aids in Streptococcus species differentiation.

Storage and Shelf Life

The Columbia CNA Agar must be sheltered from light and kept between 4 and 8 degrees Celsius. The medium side should be facing up to prevent excessive moisture accumulation on the agar surface. Under these conditions, the medium has a shelf life of eight weeks from the date of production.

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