Columbia Agar | NCM0013

Columbia Agar | NCM0013

Intended Use
Columbia Agar is used with or without blood for the isolation and cultivation of a wide variety of fastidious
microorganisms. Conforms to Harmonized USP/EP/JP Requirements. Columbia Agar is not intended for
use in the diagnosis of disease or other conditions in humans.

Description
A medium recommended by the Harmonized USP/EP/JP for isolation and identification of Clostridia from
non-sterile products. Conforms to Harmonized USP/EP/JP performance specification. Originally
described as a general purpose nutritious agar base by Ellner et al. at Columbia University that can be
enriched by the addition of sterile blood. The peptone mixture and yeast extract provide a source of
nitrogen, essential vitamins and amino acids. The starch provides a carbon source and sodium chloride
maintains osmotic balance. The Harmonized European Pharmacopoeia states that where necessary,
gentamicin sulfate at a concentration of 20mg/L can be added after sterilization to reduce the growth of
non-target organisms. According to the Harmonized European Pharmacopoeia, Reinforced Medium for
Clostridia is used as a selective enrichment broth, with subculture performed onto Columbia Agar.

Typical Formulation
Pancreatic Digest of Casein 10.0 g/L
Meat Peptic Digest 5.0 g/L
Heart Pancreatic Digest 3.0 g/L
Yeast Extract 5.0 g/L
Maize Starch 1.0 g/L
Sodium Chloride 5.0 g/L
Agar 12.0 g/L
Final pH: 7.3 ± 0.2 at 25 C
Formula may be adjusted and/or supplemented as required to meet performance specifications.

Preparation
1. Dissolve 41 g of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
3. Autoclave at 121°C for 15 minutes.
4. Cool 45-50°C,
Test Procedure
According to the Harmonized US/EP/JP
Quality Control Specifications
Dehydrated Appearance: Powder is homogeneous, free flowing and light beige to beige.
Prepared Appearance: Prepared medium without blood is trace to very slightly hazy and light amber.
With 5% sheep blood the medium is opaque and red.
Expected Cultural Response and USP/EP/JP Growth Promotion Testing: Columbia Agar was
prepared according to label directions and inoculated with the organisms listed below. Cultures wereincubated under appropriate atmosphere at 30 – 35° C and examined for growth at 18 – 48 hours.

Without 5% defibrinated sheep blood:

With 5% defibrinated sheep blood:

This culture was tested at Harmonized USP/EP/JP specified temperatures and incubation times

The organisms listed are the minimum that should be used for quality control testing

Results

Examine Columbia Agar for growth.
For Columbia Agar, supplemented with blood, examine the medium for growth and hemolytic reactions
after 18 – 24, and after 48 hours incubation. There are four types of hemolysis on blood agar media
described as:
1. Alpha hemolysis (α) is the reduction of hemoglobin to methemoglobin in the medium surrounding the
colony. This produces a green discoloration of the medium.
2. Beta hemolysis (β) is the lysis of red blood cells, producing a clear zone surrounding the colony.
3. Gamma hemolysis (γ) indicates no hemolysis. No destruction of red blood cells occurs and there is
no change in the medium.
4. Alpha-prime-hemolysis (α’) is a small zone of complete hemolysis that is surrounded by an area of
partial lysis. 

Expiration

Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not
free flowing, or if appearance has changed from the original color. Expiry applies to medium in its intact
container when stored as directed.

Limitations of the Procedure

1. Due to nutritional variation, some strains may be encountered that grow poorly or fail to grow on this
medium.
2. Hemolytic reactions of some strains of group D streptococci have been shown to be affected by
differences in animal blood. Such strains are beta-hemolytic on horse, human, and rabbit blood agar
and alpha-hemolytic on sheep blood agar.
3. Atmosphere of incubation has been shown to influence hemolytic reactions of beta-hemolytic
streptococci. For optimal performance, incubate blood agar base media under increased CO2 (5 -
10%) in accordance with established laboratory procedures. 

Storage
Store dehydrated culture media at 2-30°C away from direct sunlight. Once opened and recapped, place
container in a low humidity environment at the same storage temperature. Protect from moisture and light
by keeping container tightly closed.