HAEMOPHILUS ISOLATION AGAR

(w/ Bacitracin and Horse Blood)

INTENDED USE
Remel Haemophilus Isolation Agar (w/ Bacitracin and Horse Blood) is a solid medium recommended for the use in qualitative procedures for
the selective isolation of Haemophilus species from respiratory specimens.

SUMMARY AND EXPLANATION

Haemophilus influenzae, a fastidious gram-negative bacillus, is a common cause of serious diseases of the upper and lower respiratory tract. 1
Microbiological diagnosis is frequently delayed by an inability to recover the organism on primary-plated media due to the overgrowth of
commensal microbial flora. Klein and Blazevic evaluated a selective medium containing horse blood and bacitracin to determine its
usefulness in recovering Haemophilus spp. from respiratory tract specimens.2 Horse blood and rabbit blood do not contain V factor-
inactivating enzymes present in sheep blood and the use of a medium containing intact erythrocytes enables differentiation of hemolytic and
nonhemolytic Haemophilus spp.3 The results of their study demonstrated that Haemophilus spp. were isolated more frequently on the
enriched medium, selective or nonselective, which contained horse blood rather than rabbit blood.

PRINCIPLE
Beef Heart infusion and casein peptone supply the nutritional requirements for all but the very fastidious bacteria. Horse blood supplies both
the X and V factors which are growth requirements for certain organisms, such as Haemophilus influenzae. Horse blood also serves to
distinguish the hemolytic species of Haemophilus from those which are nonhemolytic. Bacitracin inhibits normal flora found in respiratory
cultures, particularly Neisseria spp.4

REAGENTS (CLASSICAL FORMULA)*

Casein Peptone ............................................................... 13.0 g
Sodium Chloride ................................................................ 5.0 g
Yeast Extract ..................................................................... 5.0 g
Beef Heart Infusion ............................................................ 2.0 g
pH 7.4 ± 0.2 @ 25°C
*Adjusted as required to meet performance standards.
NAD .............................................................................. 72.0 mg
Bacitracin ..................................................................... 8250 U
Horse Blood ....................................................................... 5 %
Agar .............................................................................. 17.0 g
Demineralized Water ..................................................1000.0 ml

PROCEDURE
1. Inoculate and streak the specimen as soon as possible after it is received in the laboratory.
2. If a swab specimen is received, roll the swab over a small area of the agar surface and streak for isolation.
3. Incubate in 5-10% CO2 for 24-48 hours at 35-37°C.
4. Examine for typical colonial morphology and hemolysis following established laboratory procedures.

QUALITY CONTROL
All lot numbers of Haemophilus Isolation Agar (w/ Bacitracin and Horse Blood) have been tested using the following quality control organisms
and have been found to be acceptable. Testing of control organisms should be performed in accordance with established laboratory quality
control procedures. If aberrant quality control results are noted, patient results should not be reported.

CONTROL INCUBATION RESULTS

Haemophilus influenzae ATCC® 10211 CO2, 24-48 h @ 35-37°C Growth
Haemophilus parahaemolyticus ATCC® 49700 CO2, 24-48 h @ 35-37°C Growth, beta hemolysis
Staphylococcus aureus ATCC® 25923 Aerobic, 18-24 h @ 35-37°C Inhibition (partial to complete)
Streptococcus sanguinis ATCC® 10556 Aerobic, 18-24 h @ 35-37°C Inhibition (partial to complete)

LIMITATIONS
1. Gram stain and biochemical testing of colonies morphologically representative of Haemophilus spp. is required for presumptive and definitive
identification.3

BIBLIOGRAPHY
1. Chapin, K.C. and G.V Doern. 1983. J. Clin. Microbiol. 17:1163-1165.
2. Klein, M. and D.J. Blazevic. 1970. Am. J. Med. Tech. 36:97-106.
3. Koneman, E.W., S.D. Allen, W.M Janda, P.C. Schreckenberger, and W.C. Winn, Jr. 1997. Color Atlas and Textbook of Diagnostic
Microbiology. 5th ed. Lippincott Williams and Wilkins, Philadelphia, PA.
4. Hovig, B. and E.H. Aandahl. 1969. Acta. Pathol. Microbiol. Scand. 77:676-684

Refer to the front of Remel Technical Manual of Microbiological Media for General Information regarding precautions, product storage and
deterioration, specimen collection, storage and transportation, materials required, quality control, and limitations.

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

IFU 1470, Revised May 5, 2021 Printed in U.S.A.

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