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Biolife Italiana Srl
Technical Sheet
Ready to use plates of an improved
antimicrobial susceptibility testing medium

Beet Extract
*Adjusted or supplemented to meet performance standard
Final pH 7.3 ± 0.1

Mueller Hinton Agar was originally prepared for the cultivation of gonococci and because of its low
level of p-aminobenzoic acid it was used for sensitivity tests with sulphonamides.
Mueller Hinton Agar is recommended by NCCLS for antimicrobial disc susceptibility test of most
common, rapidly growing bacteria, by the Kirby-Bauer diffusion method. To test strains that fail to
grow satisfactorily on Mueller Hinton Agar, 5% defibrinated sheep blood is added to the melted and
cooled medium. For tests with H. influenzae the Mueller Hinton Agar must be supplemented with
l5mg/L NAD, 15mg/L bovine hematin, 5g/L yeast extract (see Haemophilus Test Agar cat. N°549901).
Biolife Mueller Hinton Agar II is prepared with selected raw materials. It contains low levels of thymine
and thymidine since they affect sulphonamide and trimethoprim MIC values, and controlled levels of
Ca++ and Mg++ to obtain aminoglycosides inhibition zones against P. aereuginosa and tetracycline
inhibition zones against staphylococci within the ranges suggested by NCCLS M100-S12.

Use Mueller Hinton Agar II in plates of 90mm or 140mm. To prepare the inoculum, suspend 4-5
colonies grown in a primary isolation medium in 4-5ml of Tryptic Soy Broth and incubate for 2-6 hours
until the broth culture reaches the same density as the opacimetric standard prepared by adding 0.5ml
of 0.1% barium chloride to 99.5ml of 0.36 N sulphuric acid. Within 15 minutes, dip a sterile swab into
the broth culture, squeeze it against the walls of the tube to remove excess liquid, then streak it over
the surface of the agar plates to obtain a uniform distribution of the inoculum. Leave the plates to dry
then lay the paper discs pressing them onto the surface of the agar.
Incubate at 35ºC for 18 hours then read the inhibition zones by taking in to consideration the zones,
which are completely free of microbial growth and which have distinct borders.
Compare the zone sizes obtained to those reported on the table no. 2 of the NCCLS Document M100-
S12 “Zone Diameter Interpretative Standards" to detect whether the organism is resistant, intermediate
or susceptible to antimicrobial agents.
Store at 2-8° - When stored as directed the plates remain stable until the expiry date shown on the
label. Do not use beyond stated expiry date.

• Bauer, A.W.; Kirby, W.M,M,; Sherris, K.C. & Truck, M. (1966), Amer. Clin. Path, 45, 493-496.
• National Committee for Clinical Laboratory Standard. Performance Standards for Antimicrobial Disk
Susceptibility Tests; Approved Standard M2 A4 - Seventh Edition. American National Standard.
•NCCLS Performance Standard for Antimicrobial Susceptibility Testing; Twelfth Informational
Supplement. NCCLS Document M100 – S12, January 2002.


Mueller Hinton Agar II
20 ready to use plates (90mm)
Mueller Hinton Agar II
5 ready to use plates (140mm)
Biolife Italiana S.r.l. Viale Monza 272, 20128 Milan, Italy. Tel. n° ++39 (0)2 25 209 1, Fax n° ++39 (0)2 25 76 428
E-mail: ; Web:


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