Horse Blood Agar

Cat. no. A149 Horse Blood Agar 5%, 15x100mm Plate, 17ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Horse Blood Agar is an enriched all-purpose growth medium recommended for the cultivation of nonfastidious and fastidious microorganisms such as Haemophilus spp.

SUMMARY

Tryptic Soy Agar (TSA) is a popular all-purpose growth medium used to support a wide variety of microorganisms. TSA can be used unsupplemented or as a base for media containing mammalian blood. Although the most common form of blood containing media in the clinical laboratory is sheep blood agar, horse blood provides X- (hemin) and V-factors (NAD) required for the growth of fastidious microorganisms such as Haemophilus influenzae . Sheep and human blood are not suitable for this purpose, as they contain enzymes that inactivate NAD. (6)

Horse Blood Agar contains pancreatic digest of casein and soy peptone which provide essential carbon and nitrogen elements to support cell growth. Koenzyme enrichments are a chemically defined supplement that provides NAD (V-factor), amino acids, vitamins, dextrose, ferric ions and coenzymes to promote the growth of fastidious strains. Sodium chloride helps maintain osmotic equilibrium and agar is the solidifying agent. The medium is supplemented with 5% defibrinated horse blood to provide essential growth factors and to facilitate the determination of hemolytic reactions.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 14.5gm
Peptic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Koenzyme Enrichments 1.5ml
Horse Blood 50.0ml
Agar 14.0gm

Final pH 7.4 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for specific information on specimen collection and appropriate methods of culture. (2-6,8)

Method of Use:

1. Prior to inoculation, the medium should be brought to room temperature.

2. Inoculate the medium and streak the specimen to obtain isolated colonies.

3. Incubate at 35ºC. in a moist, CO 2 enriched atmosphere for 18-24 hours. NOTE: H. aegyptius requires a longer incubation period of 2-4 days. H. ducreyi may require up to 9 days incubation, preferably at 33ºC.

INTERPRETATION OF RESULTS

H. influenzae produce small, pale gray, moist non-hemolytic colonies with a characteristic "mousy" odor.

Haemophilus haemolyticus and Haemophilus parahaemolyticus may be similar in appearance to H. influenzae , except that colonies are surrounded by a zone of beta-hemolysis.

H. influenzae and H. parainfluenzae may be differentiated by colony color. (8)

LIMITATIONS

Defibrinated horse blood may give hemolytic reactions different from sheep blood. (5) Some streptococci (e.g. group D) give hemolytic reactions on horse blood, but not on sheep blood and may be mistakenly reported as group A. If a hemolytic reaction is obtained, the organism should be tested with a Bacitracin differentiation disk (Cat. no. Z7021) and grouped serologically or tested by the fluorescent method. Beta-hemolytic streptococci and Haemophilus haemolyticus may be differentiated by performing a Gram stain from pure culture.

The medium is not recommended for use with throat cultures. (6)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, Bacitracin disk (Cat. no. Z7021), other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Haemophilus influenzae
ATCC ® 10211***
B 18-24hr 35°C CO 2 ** Growth
Haemophilus influenzae
ATCC ® 49247
B 18-24hr 35°C CO 2 ** Growth
Streptococcus pyogenes
ATCC ® 19615***
B 18-24hr 35°C CO 2 ** Growth

** Atmosphere of incubation is enriched with 5-10% CO 2 .

*** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

Horse Blood Agar should appear opaque and cherry red in color.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

5. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

6. Krumweide, E. and A.G. Kutter. 1938. A growth inhibitory substance for the influensa group of organisms in the blood of various animal species. The use of the blood of various animals as a selective medium for the detection of hemolytic streptococci in throat cultures. J. Exp. Med. 67:429-441.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Roberts, D.E., E. Higgs and P.J. Cole. 1987. Selective medium that distinguishes Haemophilus influenzae from Haemophilus parainfluenzae in clinical specimens: its value in investigating respiratory sepsis. J. Clin. Pathol. 40:75-76.

ATCC is a registered trademark of the American Type Culture Collection.

030116gr