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MUELLER-HINTON 56137 - 63824 - 63901 - 64884 - 64888 MUELLER-HINTON + SHEEP BLOOD 63825 - 63902 MUELLER-HINTON + HORSE BLOOD + β-NAD 63524 ISOLATION MEDIUM FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING

1- INTENDED USE Mueller-Hinton agar is a standardized solid medium recommended for antimicrobial susceptibility testing by the agar diffusion or agar dilution method. 2- PRINCIPLE The composition of Mueller-Hinton medium must be standardized to obtain reliable antimicrobial susceptibility results, with defined concentrations of CaCl2, MgCl2 and ZnCl2, as variations in the concentrations of Mg2+ and Ca2+ cations are known to affect the results obtained (diameters and minimum inhibitory concentrations or MIC) for Pseudomonas aeruginosa with aminoglycosides and for Staphylococci with tetracycline (3, 4, 5, 6). The low concentration of thymidine also decreases regrowth phenomena around trimethoprim-sulphonamide and trimethoprim disks. 3- HOW SUPPLIED • Ready to use medium:

- box of 20 round Petri dishes (90 mm) (MH) code 63824 - box of 10 square Petri dishes (120 mm) (MH) code 63901

• Ready to use medium (to be dispensed): - 6 x 200 ml bottles (MH) code 56137

• Dehydrated medium: - bottle of 500 g code 64884 - drum of 5 kg code 64888

• Ready to use media sheep blood: - box of 20 round Petri dishes (90 mm) (MHB) code 63825 - box of 10 square Petri dishes (120 mm) (MHB) code 63902

• Ready to use media horse blood supplemented with β-NAD: - box of 20 round Petri dishes (90 mm) (MHF) code 63524

4- THEORETICAL COMPOSITION (g/l of distilled water)* Mueller-Hinton, with or without the addition of sheep blood or horse blood, is prepared according to the formula described by W.H.O. (1, 2)

Peptons 3.0 Casein hydrolysate 17.5 Agar 15 Ca2+ 20-25 mg/L Mg2+ 10-12.5 mg/L Final pH 7.4 ± 0.2 Supplements: + 5% sheep blood (for MHB medium) or 5% horse blood (for MHF medium)

β-NAD (for MHF medium only) 20 mg/L β-NAD: β-Nicotinamide Adenine Dinucleotide * formula adapted to ensure the best performances of the medium. Preparation of the medium: Homogenize the powder contained in the bottle. Add 35 grams of dehydrated medium to 1 litre of freshly distilled water. Heat to boiling until dissolution complete. Sterilize in the autoclave at 121°C ± 1°C for 15 minutes. Dispense into sterile tubes or bottles. At the time of use, melt the medium in the boiling water-bath and dispense the entire contents of the bottle or tubes into round Petri dishes. The agar layer must be 4 mm thick. Dry the Petri dishes for 30 minutes at 37°C. The volume of Mueller-Hinton medium required to produce a layer of agar exactly 4 mm thick is 25 ml for a 90 mm Petri dish, 60 ml for a 120 mm square Petri dish, 70 ml for a 150 mm round Petri dish.

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Before dispensing the medium can be supplemented with: • 5% sheep or horse blood for Mueller-Hinton + blood medium, • 5% sodium chloride (NaCl) for Mueller-Hinton + 5% NaCl medium. 5- STORAGE • Ready to use medium: at +2-20°C • Ready to use medium (to be dispensed): at +2-25°C • Ready to use blood medium: at +2-8°C • Dehydrated medium: tightly sealed bottle in a dry place at +15-25°C. The expiry date and batch number are indicated on the packaging. 6- INSTRUCTIONS Material: Material provided: Mueller-Hinton medium with or without the addition of sheep or horse blood. Specific material not provided: • Opacity standard equivalent to Mac Farland 0.5 standard. • Laboratory equipment necessary for antimicrobial susceptibility testing by the agar diffusion method.

Precautions for use: Comply with the instructions of current guidelines (CLSI 7,8, CA-SFM 9, EUCAST 10,11,12). Always observe current techniques and precautions concerning protection against microbiological hazards. Inoculation: From a pure, fresh culture grown on agar medium, prepare a suspension with an opacity equivalent to Mac Farland 0.5 standard. The inoculum standardization protocol is described in the various guidelines published by the CLSI, the CA-SFM or the EUCAST for the agar diffusion method and the agar dilution method. If the medium is stored at 4°C, allow it to adjust to room temperature (18-30°C). If excessive moisture is observed on the surface, dry the dishes for 10 to 30 minutes in the incubator at 35°C before use. DIFFUSION METHOD Inoculation by flooding (method recommended by the CA-SFM): From the standardized inoculum, follow the procedure indicated by the CA-SFM concerning dilution of the initial suspension according to the bacterial species tested. Flood the whole plate with the suspension obtained, then eliminate the excess suspension. Inoculation by streaking (Kirby-Bauer method recommended by the CLSI, CA-SFM and EUCAST): From the standardized inoculum, follow the CLSI, CA-SFM, EUCAST guidelines to inoculate the dish: • Immerse a sterile, non-toxic swab stick into the suspension. • Remove surplus suspension by gently rotating the swab against the walls of the tube. • Inoculate the plate with the swab to obtain a culture of confluent colonies. Apply disks using a dispenser or forceps, pressing lightly. AGAR DILUTION METHOD The inoculation and test procedure are described in the CLSI and CA-SFM guidelines. Incubation: Follow the current CLSI, CA-SFM and EUCAST guidelines. Incubation conditions vary according to the bacterial species tested. 7- INTERPRETATION OF THE RESULTS The interpretation of the antimicrobial susceptibility test using the disk diffusion method or the agar dilution method is described in updates of the CA-SFM, CLSI and EUCAST guidelines. 8- PERFORMANCE/QUALITY CONTROL OF THE TEST • Appearance of ready to use Mueller-Hinton medium: clear to opalescent, amber-coloured agar. • Appearance of dehydrated Mueller-Hinton medium: beige powder. • Appearance of ready to use Mueller-Hinton + blood medium: red agar.

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The growth performances of Mueller Hinton medium with or without sheep or horse blood are verified with the following strains:

STRAINS CULTURE RESULT AFTER 24 hours at 37°C Escherichia coli ATCC 25922 Satisfactory growth Pseudomonas aeruginosa ATCC 27853 Satisfactory growth Staphylococcus aureus ATCC 25923 Satisfactory growth Streptococcus pneumoniae ATCC 49619 Satisfactory growth Haemophilus influenzae NCTC 8468/ CIP 54.94 Satisfactory growth

The composition of the MH medium is verified with the following strains and disks (non-exhaustive list):

STRAINS ANTIMICROBIAL SUSCEPTIBILITY TESTING Escherichia coli ATCC 25922 - Amikacin 30 µg - Ampicillin 10 µg - Tetracycline 30 µg Staphylococcus aureus ATCC 25923 - Amikacin 30 µg - Cephalothin 30 µg - Tetracycline 30 µg Pseudomonas aeruginosa ATCC 27853 - Amikacin 30 µg - Cefotaxime 30 µg - Ciprofloxacin 5 µg

complying with inhibition diameters current specifications

(CLSI and/or CA-SFM) (8, 9).

The composition of the MHB medium is verified with the following strains and disks (non-exhaustive list):

STRAINS ANTIMICROBIAL SUSCEPTIBILITY TESTING Streptococcus pneumoniae ATCC 49619 - Erythromycin 15 µg - Penicillin 10 UI - Tetracycline 30 µg Pseudomonas aeruginosa ATCC 27853 - Amikacin (30 µg) - Cefotaxime (30 µg) - Ciprofloxacin (5 µg)

complying with inhibition diameters current CLSI specifications (8).

The composition of the MHF medium is verified with the following strains and disks (non-exhaustive list):

STRAINS ANTIMICROBIAL SUSCEPTIBILITY TESTING Streptococcus pneumoniae ATCC 49619 - Erythromycin 15 µg - Norfloxacin 10 µg - Rifampin 5 µg - Tetracycline 30 µg Haemophilus influenzae NCTC 8468 / CIP 54.94 - Ampicillin 2 µg - Cefaclor 30 µg - Tetracycline 30 µg

complying with inhibition diameters current EUCAST specifications (12).

9- QUALITY CONTROL OF THE MANUFACTURER All manufactured reagents are prepared according to our Quality System, starting from receipt of raw material to the final commercialization of the product. Each lot is submitted to quality control assessment and is only released to the market, after conforming to pre-defined acceptance criteria. The records relating to production and control of each single lot are kept within Bio-Rad. 10- LIMITS OF USE Pure, fresh cultures must always be used to obtain interpretable results. Some strains may not grow on this medium due to their nutritional requirements. An appropriate medium should be used in this case: HTM for Haemophilus, Mueller-Hinton plus 5% sheep blood for Streptococcus pneumoniae and �haemolytic Streptococci, GC agar for Neisseria gonorrhoeae. A number of factors can affect the results (size of the inoculum, incubation time and atmosphere, etc.). It is therefore essential to comply with the protocol described in the current guidelines (CLSI, CA-SFM.). Mueller-Hinton fresh blood medium is not recommended to test the susceptibility of S. pneumoniae to cotrimoxazole (7).

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11- REFERENCES 1- World Heath Organization Expert Committee on Biological Standardization. 1981. Technical report series 673 (Révision

1981). W.H.O., Geneva – p156-192. 2- World Heath Organization Expert Committee on Biological Standardization. 1992. Technical report series 822. W.H.O.,

Geneva. 3- Casillas, E.,Kenny, M.A., Minshew B.H., Schoenknecht, F. 1981. Effect of ionized calcium and soluble magnesium on the

predictability of the performance of Mueller-Hinton agar susceptibility testing of Pseudomonas aeruginosa with Gentamicin. Antimicrob. Agents Chemother. 19:987-992.

4- Murray, P.R., Zeitinger, J. R. 1983. Evaluation of Mueller-Hinton agar for disk diffusion susceptibility tests. J. Clin. Microbiol. 18: 1269-1271

5. D’Amato, R.F., Thornsberry C., Baker C.N., Kirven, L.A. 1976. Effect of calcium and magnesium ions on the susceptibility of Pseudomonas species to Tetracycline, Gentamicin Polymyxin B, and Carbenicillin. Antimicrob. Agents Chemother. 7: 596-600.

6. D’Amato, R.F., Thornsberry C. 1979. Calcium and Magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results. Current Microbiol.2:135-138

7. Clinical and Laboratory Standards Institute. 2010. Approved standard M2-A10. Performance standards for antimicrobial susceptibility tests, 10th ed. CLSI, Wayne, Pa.

8. Clinical and Laboratory Standards Institute. 2010. CLSI document M100-S19. Performance standards for antimicrobial susceptibility testing, 20th informational supplement, Wayne, Pa.

9. Comité de l’antibiogramme. 2010. French Society of Microbiology. 10. European Committee on Antimicrobial susceptibility testing (EUCAST). 2009. Media preparation for disc diffusion testing

V1.0. 11. European Committee on Antimicrobial susceptibility testing (EUCAST). 2009. EUCAST disk diffusion antimicrobial

susceptibility testing method summary - V1.0. 12. European Committee on Antimicrobial susceptibility testing (EUCAST). 2010. EUCAST QC Tables V1.1.

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