MUELLER HINTON AGAR WITH LYSED HORSE BLOOD (LHB)

Cat. no. H98 Mueller Hinton Agar with LHB, 15x150mm Plate, 72ml 10 plates/bag

INTENDED USE

Mueller Hinton Agar with Lysed Horse Blood is intended for veterinary purposes only.

SUMMARY

Mueller and Hinton developed Mueller Hinton Agar in 1941 to be a protein free medium for isolating pathogenic strains of Neisseria . (3) It was found that Mueller Hinton Agar was useful in identifying sulfonimide-resistant and responsive strains of gonococci. (3) Additionally, in recent times this media has been used in standardized antimicrobial disk susceptibility testing, as described by Bauer, Kirby, et al. (1) Barry and Fay investigated the effects of altering the depth of plated Mueller Hinton Agar on disk diffusion testing, and determined a standardized depth of approximately four millimeters to be sufficient. (2) In 1970 Dewees, et al., studied the effect of storage on Mueller Hinton Agar plates used for antimicrobial disk diffusion zone sizes. Their findings indicated commercially manufactured Mueller Hinton Agar plates were suitable for use in routine susceptibility testing. (6)

Mueller Hinton Agar with Lysed Horse Blood is a modification to the traditional Mueller Hinton Agar formulation and recommended by the Clinical Laboratory Standards Institute (CLSI - formerly NCCLS) for veterinary susceptibility testing. It is known that media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides and of trimethoprim, thus yieding smaller and less distinct zones, or even no zone at all, resulting into false-resistant reports. Addition of thymidine phosphorylase or lysed horse blood can improve the clarity of zones and the reliability of sulfonamide and trimethoprim testing of most common pathogens, except enterococci. (7)

FORMULA

Ingredients per liter of deionized water:*

Acid Hydrolysate of Casein 17.5gm
Beef Extract 2.0gm
Starch 1.5gm
Lysed Horse Blood 50.0ml
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

Storage: Upon receipt store plates at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), 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.

Method of Use: For Mueller Hinton Agar with LHB, refer to CLSI document M31-A2. (7)

INTERPRETATION OF RESULTS

For Mueller Hinton Agar with LHB, consult CLSI document M31-A2. (7)

LIMITATIONS

In vitro susceptibility does not necessarily imply in vivo effectiveness.

When using the disk diffusion method, technical human errors may compromise reliability and accuracy. The following errors are common sources encountered in the clinical microbiology laboratory, and must be watched for: improper disk storage, inoculum not properly adjusted (too light or too heavy), incubation temperature deviating from 35-37ºC., use of an increased CO 2 atmosphere, reading plates before or after the full 16-18 hours of incubation, transcribing errors, reader error when measuring zone diameters, deterioration of the McFarland Turbidity Standard, and contamination or mutation in the control strain(s).

The plates are to be incubated at 35ºC. in 5-10% CO 2 for 20-24 hours. A longer incubation time may lead to erroneous results (i.e. zones that are too small).

Mueller Hinton Agar with LHB is not cation adjusted. Variation in the concentration of divalent cations, primarily calcium and magnesium, affects results of aminoglycoside, tetracycline, and colistin tests with certain isolates. A cation content that is too high reduces zone sizes, where as a cation content that is too low has the opposite effect.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, calipers, antimicrobial disks, McFarland 0.5 Turbidity Standard, forceps, 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
Streptococcus pneumoniae
ATCC ® 49619
A 24hr 35°C CO 2 ** Growth

Refer to CLSI publication M31-A2 for additonal information. (7)

User Quality Control

PHYSICAL APPEARANCE

Mueller Hinton Agar with LHB should appear clear, and dark red in color.

S. pneumoniae growing on Mueller Hinton Agar with LHB

Streptococcus pneumoniae (ATCC ® 49619) colonies growing on Mueller Hinton Agar with LHB (Cat. no. H98). Incubated in CO 2 for 24 hours at 35ºC.

Mueller Hinton Agar with LHB

Uninoculated plate of Mueller Hinton Agar with LHB (Cat. no. H98).

REFERENCES

1. Bauer, A.W., W.M.M. Kirby, et al. 1966. Am. J. Clin. Pathol. ; 45:493-496.

2. Dewees, et al. 1970. Effect of storage of Mueller Hinton Agar plates on zone sizes for antimicrobial testing. Appl. Microbiol. ; 30:203.

3. Mueller, J.H. and J. Hinton. 1941. A protein-free medium for primary isolation of the Gonococcus and Meningococcus . Proc. Soc. Exp. Diol. and Med. ; 48:330-333.

4. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22-A2, Vol. 16, No. 16. 1996. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

5. Ryan, K.J., et al. 1970. Disk sensitivity testing. Hosp. Prac. ; 5:91-100.

6. Standard Disk Susceptibility Test. The Federal Register , September 30, 1972; 37(191):20527-20529.

7. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals , M31-A2, Vol. 22, No. 6. 2002. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.


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

030716sw