Casman Agar Base

Intended Use

Casman Agar Base is used for the cultivation of fastidious pathogenic organisms, such as Haemophilus influenzae and Neisseria gonorrhoeae, from clinical specimens.

Summary and Explanation

Members of the genus Haemophilus are fastidious microorganisms that require the addition of X and/or V growth factors for in vitro cultivation.1 Neisseria are also fastidious microorganisms with complex growth requirements.2 In 1947, Casman described a blood-enriched medium prepared without an infusion of fresh meat for cultivation of Haemophilus
and gonococci.1 The medium was developed to replace previous formulations that required time-consuming preparations of fresh and heated blood and fresh meat infusion to supply
the nutrients necessary for the growth of these fastidious organisms.2,3

Casman found that nicotinamide interfered with the activity of an enzyme in blood that inactivates V factor (NAD). Using unheated human blood, he found that amount of nicotinamide
required for good growth of H. influenzae was inhibitory to gonococci.2 Therefore, he reduced the nicotinamide to a level that allowed good growth of gonococci. To improve the recovery of H. influenzae on this medium, horse or rabbit blood should be used instead of human blood, since they contain less NADase.4

Principles of the Procedure

Casman Agar Base is a nonselective, peptone-based medium. The peptones and beef extract provide amino acids and other complex nitrogenous nutrients. Yeast extract is a source of the B-complex vitamins. 

Supplementing Casman Agar Base with blood supplies the growth factors required by H. influenzae – hemin, or X factor, and nicotinamide adenine dinucleotide (NAD), or V factor. 
Horse and rabbit bloods are preferred by some laboratories because they are relatively free of NADase, an enzyme that destroys the V factor. The addition of lysed blood stimulates the growth of some strains of N. gonorrhoeae. Nicotinamide is incorporated into the medium to inhibit the nucleotidase of erythrocytes that destroys the V factor.
Cornstarch is incorporated to prevent fatty acids from inhibiting the growth of N. gonorrhoeae and to facilitate β-hemolytic reactions by neutralizing the inhibitory action of dextrose. A small amount of dextrose is added to enhance the growth of pathogenic cocci.

User Quality Control

Identity Specifications
Casman Agar Base
Dehydrated Appearance: Fine, homogeneous, free of extraneousmaterial.
Solution:                       4.3% solution, soluble in purified water upon
                                    boiling. Solution is medium to dark, yellow
                                    to tan, hazy to cloudy, with a moderate to
                                    large amount of cream flocculation.
Prepared Appearance:    Medium to dark, yellow to tan, hazy to
                                   cloudy, with a moderate to large amount
                                   of cream flocculation.
Reaction of 4.3%
Solution at 25°C:           pH 7.3 ± 0.2
Cultural Response
Casman Agar Base
Prepare the medium per label directions. Inoculate and incubate for 42-48 hours at 35 ± 2°C, aerobically for L. monocytogenes and with 3-5% CO2 for all other organisms.

Formula

Casman Agar Base
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 11.5 g
Peptic Digest of Animal Tissue...................................... 5.0 g
Yeast Extract.............................................................. 3.5 g
Beef Extract................................................................ 3.0 g
Nicotinamide............................................................. 0.05 g
p-Aminobenzoic Acid.................................................. 0.05 g
Dextrose.................................................................... 0.5 g
Cornstarch.................................................................. 1.0 g
Sodium Chloride.......................................................... 5.0 g
Agar......................................................................... 13.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 43 g of 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 15 minutes.
4. Cool to 45-50°C and add 5% sterile blood and 0.15% blood solution, made by lysing 1 part of blood with 3 parts of water. Alternatively, add 5% partially lysed blood.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

For a complete discussion on the isolation and identification of Neisseria and Haemophilus, consult appropriate references.5,6

Expected Results

H. influenzae produces colorless to gray, transparent, moist colonies with a characteristic “mousy” odor. N. gonorrhoeae produces small, translucent, raised, moist, colorless to grayishwhite colonies.
Gram staining, biochemical tests and serological procedures should be performed to confirm findings.

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