Columbia Blood Agar Base w/ Hemin

Intended Use:

An enriched base for preparation of chocolate agar, blood agar and for various selective and identification media.

Composition**

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 44.01 grams in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C before adding heat sensitive compounds.

For Blood Agar: Add 5% v/v sterile defibrinated sheep blood to sterile cool base.

For Chocolate Agar: Add 10% v/v sterile defibrinated sheep blood to sterile cool base. Heat to 80°C for 10 minutes with constant agitation.

The medium can be made selective by adding different antimicrobials to sterile base.

For Brucella species: Add rehydrated contents of 1 vial of NPBCVN Selective Supplement (FD005) to 500 ml sterile molten base.

For Campylobacter species: Add rehydrated contents of 1 vial of Blaser-Wang Selective Supplement(FD006) or Butzler Selective Supplement (FD007) or Skirrow Selective Supplement (FD008) or VTCA Selective Supplement (FD090) or Butzler VI Selective Supplement (FD106) to 500 ml sterile molten base along with rehydrated contents of 1 vial of Minerals Growth Supplement (FD009).

For Gardnerella species: Add rehydrated contents of 1 vial of GNA Selective Supplement (FD056) to 500 ml sterile molten base.

For Cocci: Add rehydrated contents of 1 vial of NC Selective Supplement (FD030) or NNP Selective Supplement (FD031) or CO Selective Supplement (FD119) to 500 ml sterile molten base.

Principle And Interpretation

Columbia Agar Base is used as the base for media containing blood and for selective media formulations, which incorporates various combinations of antimicrobial agents as additives. Sheep blood allows detection of hemolytic reactions and supplies the X-factor (hemin) necessary for the growth of many bacterial species but lacks V-factor (Nicotinamide Adenine Dinucleotide), since it contains NADase, which destroys the NAD. Therefore, Haemophilus influenzae, which requires both the X and V-factors, will not grow on this medium. Fildes found that Nutrient Agar supplemented with a digest of sheep blood supplied both of these factors and the medium would support the growth of H. influenzae (1,2,3). The inclusion of bacitracin makesthe enriched Columbia Agar Medium selective for the isolation of Haemophilus species from clinical specimens, especially from upper respiratory tract (4). Columbia Blood Agar Base w/ 1% Agar is used as a base for preparing media containing blood and for selective media formulations in which different combinations of antimicrobial agents are used as additives. Columbia Agar Base supplemented with sheep, rabbit or horse blood derives its superior growth-supporting properties from the combination of peptones prepared from tryptone, peptone and HM peptone B. Cornstarch serves as an energy source and also neutralizes toxic metabolites. Sheep blood permits the detection of haemolysis and also provides heme (X-factor), which is required for the growth of many bacteria. As these media have a relatively high carbohydrate content, beta- haemolytic Streptococci may exhibit a greenish haemolytic reaction, which may be mistaken for alpha haemolysis. Confirmatory tests of all the presumptive colonies are needed.

Columbia Agar Base with added sterile serum provides an efficient medium for Corynebacterium diphtheriae virulence test medium. After following the established technique for C. diphtheriae, lines of toxin-antitoxin precipitation are clearly visible in 48 hours. Many pathogens require carbon dioxide; therefore, plates may be incubated in an atmosphere containing approximately 3-10% CO2.

Precaution: Brucella cultures are highly infective and must be handled carefully; incubate in 5-10% CO2.Campylobacter species are best grown at 42°C in a microaerophillic atmosphere. Plates with Gardnerella supplements plates should be incubated at 35°C for 48 hours containing 7% CO2 (5).

Corn starch serves as an energy source and also neutralizes toxic metabolites. Sheep blood permits the detection of haemolysis and also provides heme (X factor) which is required for the growth of many bacteria. However it is devoid of V factor (Nicotinamide adenine dinucleotide) and hence Haemophilus influenzae which needs both, X and V factors will not grow on this medium. Hemin stimulates growth of various fastidious organisms. As this medium has a relatively high carbohydrate content, beta-haemolytic Streptococci may exhibit a greenish haemolytic reaction which may be mistaken for the alpha haemolysis. Carry out confirmatory tests of all the colonies.

Type of specimen

Clinical samples: Specimens containing aerobic organisms.

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (6,7). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Prepared plates of supplemented media should be used within 18 hours of preparation for utmost selectivity.
2. Further biochemical and serological tests must be performed for confirmation.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance Cream to yellow homogeneous free flowing powder

Gelling Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium Basal medium: Light amber coloured clear to slightly opalescent gel. On standing the molten medium shows haziness. After addition of 5%w/v sterile defibrinated blood : Cherry red coloured opaque gel forms in Petri plates

Reaction Reaction of 4.4% w/v aqueous solution at 25°C. pH: 7.3±0.2

pH 7.10-7.50

Cultural Response

Cultural characteristics observed with added 5% w/v sterile defibrinated blood, after an incubation at 35-37°C for 24-48 hours.

Key: *Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (6,7).

Reference

1. Bailey R. K., Voss J. L. and Smith R. F., 1979, J. Clin. Microbiol., 9; 65
2. Chapin K. C. and Doern G. V., 1983, J. Clin. Microbiol., 17:11
3. Ellner P. P., Stoessel C. J., DrE
4. Fildes P., 1920, Br. J. Exp. Pathol., 1:129.
5. Fildes P., 1921, Br. J. Exp. Pathol., 2:
6. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
7. Jorgensen, J.H., Pfaller, M.A., Carroll, K. C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015)Manual of Clinical Microbiology, 11th Edition. Vol. 1.