H.T.M. (HAEMOPHILUS TEST MEDIUM) AGAR

Technical Data #1802a / 2013.09.18

HTM is a solid medium used for susceptibility testing of Haemophilus influenzae. Routine methods for the rapidly growing pathogens, as described by Bauer et al. are generally not acceptable. Addition of X and V factors and yeast extract to Mueller-Hinton Agar ensure a satisfactory growth.

 


 

FORMULA
in grams per liter purified filtered water

Beef Extract

2,0

Acid Casein Hydrolysate

17,5

Starch

1,5

Yeast Extract

5,0

Agar

17,0

Enrichments

 

Bovine Hematin

0,015

-NAD

0,015

pH 7,3 +/- 0,1 25 C

This approximate formula may be adjusted and/or enriched to obtain best results.


PRECAUTIONS
This medium is for IN VITRO diagnostic use only.


STORAGE
Store prepared media at 2-8 C protected from direct light and dehydrated powder, in a dry place, in tightly-sealed containers at 2-25 C.


SIGN OF DETERIORATION
Media should not be used if the expiry date has passed. Prepared media should not be used if there are signs of contamination or deterioration (shrinking, cracking, evaporation or discoloration). Do not use dehydrated media if it is caked.


DIRECTIONS
Suspend 38 g of HTM dehydrated media in 1000 ml of purified filtered water. Heat with frequent agitation and boil for one minute. Add a solution of bovine hematin. Sterilize at 121 C for 15 minutes. Cool to 45-50 C. Add a sterile enrichment of -NAD. Mix gently and dispense into sterile Petri dishes.


PROCEDURE
Prior to inoculate, the prepared media should be brought to room temperature.

  1. Into a HTM Broth or Mueller Hinton Broth, with 4-5 similar colonies from a 24 hours chocolate agar growth, prepare a solution and adjust to 0,5 Mc Farland Standard.
  2. Following established procedures, inoculate plates by swabbing the inoculum on the surface three times.
  3. Place appropriate susceptibility disk on the surface and press gently.
  4. Incubate at 35 C with 5% CO2 for 24 hours.


QUALITY CONTROL

Expected results after 24 hrs at 35C W CO2

Organisms

ATCC

NCCLS STANDARDS

Haemophilus influenzae

49247

meets NCCLS Standards

Haemophilus influenzae

49766

meets NCCLS Standards


LIMITATIONS OF METHOD
This medium is only a part of the identification. Other tests may be required.


REFERENCES

  1. Bauer, A.W., W.M.M. Kirby, J.C. Sherris, M. Turck. 1966 Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496
  2. D'Amato, R.F., L. Hochstein 1982 Evaluation of a rapid inoculum preparation method for agar disk diffusion susceptibility tests J. Clin. Microbiol. 15:282-285
  3. Jorgensen, J.H., J.S. Redding, L.A. Maher, A.W. Howell 1987 Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J. Clin. Microbiol. 25:2105-2113
  4. Lennette, E.H., Ballows, A., Hausler, W.J.Jr., and Shadomy, H.J. Manual of Clinical Microbiology. 4th ed. 1985 Washington D.C.: American society for Microbiology.
  5. NCCLS 1990 Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard 4th ed. Document M2-A4 Vol.10, No.7
  6. NCCLS 1994 Performance Standards for Antimicrobial Susceptibility Testing 5th Informational Supplement Document M100-S5 Vol.14, No.16


CATALOGUE NUMBERS

Dehydrated media 500 g QB-39-2115
Hematin solution, 0,015g/L 10 X 5ml 8755
-NAD enrichment, 0,015g/L 10 X 5ml 8750
     
Prepared media    
Plates, 100x15 mm 10/pkg 1702
Plates, 150x15 mm 5/pkg 1802
     
HTM Broth    
Tubes 16x125 mm 10/pkg 2702
     
M-H Broth    
Tubes 16x125 mm 10/pkg 2663

 


 

 

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