Columbia Blood Agar Base EH

Intended Use

Columbia Agar Base, without or with the addition of 5% (or 10%) sheep blood, is a highly nutritious, general-purpose medium for the isolation and cultivation of nonfastidious and fastidious microorganisms from a variety of clinical and nonclinical materials. Columbia Blood Agar Base EH (Enhanced Hemolysis) is used with blood in isolating and cultivating fastidious microorganisms.
Columbia Agar with Fildes Enrichment and Bacitracin is used in qualitative procedures for isolation and cultivation of Haemophilus species from clinical specimens.
Columbia Agar Base meets United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)1-3 performance specifications, where applicable.

Summary and Explanation

Ellner et al.,4 in 1966, reported the development of a blood agar formulation, which has been designated as Columbia Agar. The base achieves the more rapid and luxuriant growth
obtained from casein hydrolysate media with the sharply defined hemolytic reactions, more typical colonial morphology and improved pigment production achieved with media containing 

Columbia Agar Base is utilized as the base for media containing blood and for selective media formulations in which various combinations of antimicrobial agents are used as additives.
Sheep blood allows detection of hemolytic reactions and supplies the X factor (heme) necessary for the growth of many bacterial species but lacks V factor (nicotinamide adenine dinucleotide), since it contains NADase which destroys the NAD. For this reason, Haemophilus influenzae, which requires both the X and V factors, will not grow on this medium. Fildes found that supplementing nutrient agar with a digest of sheep blood supplied both of these factors and the medium would support the growth of H. influenzae.5,6 The inclusion of bacitracin makes the enriched Columbia Agar medium selective for the isolation of Haemophilus species from clinical specimens, especially from the upper respiratory tract.7

Columbia Agar with 5% sheep blood is a general all-purpose enriched primary isolation medium that allows growth of all clinically significant anaerobes and facultative anaerobes.8,9
Columbia Agar supplemented with 5% sheep blood is recommended when processing clinical specimens for unusual organisms, such as Bartonella bacilliformis, the causative agent of
Oroya fever and Peruvian wart.8 Columbia Agar supplemented with 5% sheep blood and 20 μg of ampicillin per mL is used in isolating Aeromonas sp. from stool samples of patients showing clinical symptoms of gastroenteritis.10

Columbia Agar Base is used to prepare Modified Butzler Agar, which is a selective isolation medium for the detection of thermotolerant Campylobacter in food and animal feed.11 Columbia
Agar Base is a component of Oxford Medium and Columbia Blood Agar Base is a component of Modified Oxford Medium, both of which are used to detect Listeria monocytogenes in
food and milk samples.11-14 Columbia Agar is listed as one of the recommended media for the isolation of Clostridia sp. from nonsterile pharmaceutical products.1

User Quality Control

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the
development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Columbia Blood Agar Base EH
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       3.9% solution, soluble in purified water upon
                                    boiling. Solution is light to medium amber, clear
                                   to slightly opalescent.
Prepared Appearance:    Plain – Light to medium amber, clear to slightly
                                   opalescent.
                                   With sheep blood – Medium to bright cherry red,
                                   opaque, no hemolysis.
Reaction of 3.9%
Solution at 25°C:           pH 7.3 ± 0.2
Cultural Response
Columbia Blood Agar Base or Columbia Blood Agar Base EH
Prepare the medium per label directions without (plain) and with 5% sheep blood (SB) for Columbia Blood Agar Base and with 5% sheep blood for Columbia Blood Agar Base EH. Inoculate and incubate at 35 ± 2°C with 5-10% CO2 for 18-48 hours.

Principles of the Procedure

Columbia Agar Base supplemented with sheep, rabbit or horse blood derives its superior growth-supporting properties from the combination of peptones prepared from pancreatic digest
of casein, meat peptic digest and heart pancreatic digest. 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. Sodium chloride maintains osmotic balance in the medium. It should be noted that Columbia Sheep Blood Agar has a relatively high carbohydrate content and, therefore, beta-hemolytic streptococci may produce a greenish hemolytic reaction that may be mistaken for alpha hemolysis. Fildes enrichment is prepared by the action of the enzyme pepsin on defibrinated sheep blood. Bacitracin is a polypeptide antibiotic that is active mainly against gram-positive bacteria.

Formula

Columbia Blood Agar Base EH
Approximate Formula* Per Liter
Pantone..................................................................... 12.0 g
Bitone H Plus................................................................ 6.0 g
Enzymatic Digest of Animal Tissue................................... 3.0 g
Starch.......................................................................... 1.0 g
Sodium Chloride........................................................... 5.0 g
Agar.......................................................................... 12.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water: Columbia Blood Agar Base EH – 39 g. 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. For preparation of blood agar, cool the base to 45-50°C and add 5% sterile, defibrinated blood. Mix well.
5. Test samples of the finished product for performance using stable, typical control cultures.

Sample Collection and Handling

For clinical specimens, refer to laboratory procedures for details on specimen collection and handling.8-10 For food or milk samples, follow appropriate standard methods for details on sample collection and preparation according to sample type and geographic location.11-14 For pharmaceutical samples, refer to USP General Chapter <62> for details on the examination of nonsterile products and tests for isolating Clostridium sp. using Columbia Agar.1

Procedure

Refer to appropriate standard references for details on test methods to obtain isolated colonies from specimens or samples using Columbia Agar.1,8-14 Incubate the plates at 35 ± 2°C for 18-72 hours under appropriate atmospheric conditions, or as instructed in the standard reference.1,8-14 Since many pathogens require carbon dioxide on primary isolation, plates may be incubated in an atmosphere containing approximately 3-10% CO2.

Expected Results

After the recommended incubation period, most plates will show an area of confluent growth. Because the streaking procedure is,
in effect, a “dilution” technique, diminishing numbers of microorganisms are deposited on the streaked areas. Consequently, one or more of these areas should exhibit isolated colonies of the organisms contained in the specimen. Further, growth of each organism may be semi-quantitatively scored on the basis of growth in each of the streaked areas.

*Store at 2-8°C.
†QC testing performed according to USP/EP/JP performance specifications.

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