HAEMOPHILUS TEST MEDIUM (HTM) AGAR

Cat. no. G33 HTM Agar, 15x100mm Plate, 25ml 10 plates/bag
Cat. no. H07 HTM Agar, 15x150mm Plate, 72ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Haemophilus Test Medium (HTM) Agar is a solid medium recommended by the Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS) for use in antimicrobial disk susceptibility tests of Haemophilus species. (4)

SUMMARY

Increasing incidence of resistant strains of Haemophilus influenzae has underscored the need for a reliable susceptibility test method for this organism. Modifications of standard procedures have become necessary because of the fastidious nature of H. influenzae . Complex growth additives may cause antagonism of certain antibiotics and have shown poor reproducibility. Jorgensen, et al. described this formula, Haemophilus Test Medium, as an improved growth medium for Haemophilus influenzae . (5) In their study, the HTM compared favorably with the conventional medium, Mueller Hinton with Chocolate Agar. HTM is an uncomplicated growth medium that is transparent to facilitate the reading of susceptibility tests.

FORMULA

Ingredients per liter of deionized water:*

Acid Hydrolysate of Casein 17.5gm
Yeast Extract 5.0gm
Beef Extract 2.0gm
Starch 1.5gm
Hematin 15.0mg
NAD 15.0mg
Agar 17.0gm

Final pH 7.3 +/- 0.1 at 25ºC.

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

STORAGE AND SHELF LIFE

Upon receipt store 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration, (shrinking, cracking or discoloration), or if the expiration date has passed.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. This medium is used in characterizing pure cultures. Direct inoculation of this medium will result in erroneous results. Information on specimen collection may be found in standard reference texts. (1,3)

Method of Use: Refer to CLSI document M2-A. (4)

INTERPRETATION OF RESULTS

Refer to CLSI document M2-A. (4)

LIMITATIONS

Failure to follow CLSI recommended procedures as described in the standard document may result in inaccurate results. (4)

Bacterial suspensions must be prepared according to CLSI recommended procedure and compared with the appropriate turbidity standard to insure reliable results. Higher inoculum concentrations may lead to false resistant results with some beta-lactamase antimicrobics. (4,5)

The plates are to be incubated at 35ºC., in 5-7% CO 2 , for 16-18 hours. A longer incubation time may lead to erroneous results; i.e. zones that are too small.

Disk diffusion methods for HTM have been standardized only for Haemophilus spp. Other organisms should not be tested because results cannot be reliably interpreted.

The use of other strains of H. influenzae for quality control testing other than the strains listed may lead to erroneous results.

A nitrocefin beta-lactamase test should be performed on all Haemophilus isolates. beta-lactamase negative, ampicillin-resistant strains of Haemophilus influenzae are rare in the United States (< 0.5%).

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, incubators, and antimicrobial disks, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Incubation Results
Time Temperature Atmosphere
Haemophilus influenzae
ATCC ® 49247
16-18hr 35°C CO 2 ** Refer to CLSI document M2 for QC and interpretive zone diameters
Haemophilus influenzae
ATCC ® 49766
16-18hr 35°C CO 2 ** Refer to CLSI document M2 for QC and interpretive zone diameters
Haemophilus influenzae
ATCC ® 10211
16-18hr 35°C CO 2 ** Tested for growth only
Escherichia coli
ATCC ® 35218
16-18hr 35°C Aerobic Refer to CLSI document M2 for QC and interpretive zone diameters

User Quality Control

PHYSICAL APPEARANCE

HTM Agar should appear clear, and medium to dark amber in color.

Haemophilus Test Medium

Haemophilus influenzae (ATCC ® 49247) growing on Haemophilus Test Medium (Cat. no. H07) for antimicrobial disk susceptibility test. Incubated in CO 2 for 18 hours at 35ºC.

Haemophilus Test Medium

Uninoculated plate of Haemophilus Test Medium (Cat. no. H07) for antimicrobial disk susceptibility test. Incubated in CO 2 for 18 hours at 35ºC.

PERFORMANCE CHARACTERISTICS

A reproducibility study was performed involving three separate test sites. Ten Haemophilus influenzae isolates, five ATCC ® strains and five clinical isolates, were tested in triplicate at each of the three test sites on three separate days (10x3x3x3).

Clinical disk diffusion testing of HTM at an independent test site resulted in < 4% no growth rate of Haemophilus spp.

Reproducibility results are available from Hardy Diagnostics upon request. Acceptable criteria > 95% reproducibility.

See listed reference for more information on reproducibility of clarithromycin on HTM. (6)

REFERENCES

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

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

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

4. Performance Standards for Antimicrobial Disk Susceptibility Tests, M2-A. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

5. Jorgensen, J.H., et al. 1987. Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J. Clin. Microbiol.; 25:2105-2113.

6. Jones, R.N., et al. 1994. Validation of NCCLS macrolides; azithromycin, clarithromycin and erythromycin interpretive criteria for Haemophilus influenzae tested with Haemophilus Test Medium. Diagn. Microbiol. Infect. Dis.; 18: 243-249.

7. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, Informational Supplement. Approved Standard M100. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.


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