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BD BBL™ Mueller Hinton Agar with 5% Sheep Blood 111-251801-N-00, December 2014 QUALITY CONTROL PROCEDURES BBLTM Muel er Hinton Agar with 5% Sheep Blood is recommended for disc diffusion susceptibility testing of Streptococcus pneumoniae with selected agents; i.e., chloramphenicol, erythromycin, ofloxacin, tetracycline and vancomycin, in addition to oxacil in screening for susceptibility to penicil in, as standardized by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS). II. PERFORMANCE TEST PROCEDURE Inoculate representative samples of the cultures listed below. Preparation of inoculum. The S. pneumoniae culture for Muel er Hinton Agar with 5% Sheep Blood is prepared by suspending sufficient growth from an 18- to 20-h culture on TrypticaseTM Soy Agar with 5% Sheep Blood (TSA II) into Muel er Hinton II Broth to achieve the desired turbidity (comparable to a 0.5 McFarland turbidity standard). Within 15 min after adjusting the turbidity of the inoculum, dip a sterile swab into the broth suspension. Rotate the swab several times on the inside wal of the tube above the fluid level to remove excess inoculum from the swab. Inoculate the surface of the plate by streaking the swab over the surface of the plate. Repeat this procedure two more times, rotating the plate 60 degrees each time. Replace the lid of the plate and al ow inoculum to be absorbed for at least 3 min, but no longer than 15 min, before applying the Sensi-DiscTM antimicrobial susceptibility test discs. Place the appropriate discs onto the respective cultures. Incubate plates at 35 ± 2°C in 5-7% CO2 within 15 min after the discs are applied. 2. Examine Muel er Hinton Agar with 5% Sheep Blood plates after 20-24 h. Measure the zone diameters of the complete zones of inhibition to the nearest mm. The endpoint should be taken as the area showing no obvious visible growth, excluding faint growth of tiny colonies which can be detected with difficulty at the edge of the zone of inhibition. Expected Results a. Test Organism * Streptococcus pneumoniae . . . . . ATCCTM 49619 Zone sizes should fal within the ranges of acceptable zone diameter quality control limits specified by the Clinical and Laboratory Standards Institute (CLSI), formerly NCCLS. These limits are published in CLSI Document M100-S23 (M2)1 Supplemental tables containing revised tables of antimicrobial discs and interpretive standards are published periodical y. The latest tables should be consulted for current recommendations. See the "NOTE" under "RESULTS" in the PRODUCT INFORMATION section for additional information. *Recommended organism strain for User Quality Control. III. ADDITIONAL QUALITY CONTROL 1. Examine plates as described under "Product Deterioration." 2. Visual y examine representative plates to assure that any existing physical defects wil not interfere Determine the pH potentiometrical y at room temperature for adherence to the specification of 7.3 ± 0.2 for the blood-containing medium. 4. Note the firmness of plates during the inoculation procedure. Incubate uninoculated representative plates at 30 ± 1°C in an aerobic atmosphere for 84h and examine for microbial contamination. PRODUCT INFORMATION IV. INTENDED USE BBL Muel er Hinton Agar with 5% Sheep Blood is recommended for antimicrobial disc diffusion susceptibility testing of S. pneumoniae with selected agents; i.e., chloramphenicol, erythromycin, ofloxacin, tetracycline, and vancomycin, in addition to oxacil in screening for susceptibility to penicil in, as standardized by the Clinical and Laboratory Standards Institute (CLSI), formerly NCCLS.1 V. SUMMARY AND EXPLANATION Because clinical microbiology laboratories in the early 1960s were using a wide variety of procedures for determining the susceptibility of bacteria to antibiotics and chemotherapeutic agents, Bauer, Kirby and others developed a standardized procedure in which Muel er Hinton agar was selected as the test medium.2,3 A subsequent international col aborative study confirmed the value of Muel er Hinton agar for this purpose because of the relatively good reproducibility of the medium, the simplicity of its formula, and the wealth of experimental data that had been accumulated using this medium.4 Unsupplemented Muel er Hinton agar, although adequate for susceptibility testing of rapidly growing aerobic pathogens, was not adequate for more fastidious organisms such as S. pneumoniae. The CLSI Document M2-A11 recommends Muel er Hinton agar supplemented with 5% defibrinated sheep blood.1 Details of quality control procedures and interpretive criteria for use with S. pneumoniae and other Streptococcus spp. Are contained in supplemental tables.5 VI. PRINCIPLES OF THE PROCEDURE The Bauer-Kirby procedure is based on the diffusion through an agar gel of antimicrobial substances which are impregnated on paper discs.6 In the test procedure, a standardized suspension of the organism is swabbed over the entire surface of the medium. Paper discs impregnated with specified amounts of antibiotic or other antimicrobial agents are then placed on the surface of the medium, the plate is incubated, and zones of inhibition around each disc are measured. For S. pneumoniae, the determination as to whether the organism is susceptible, intermediate or resistant to an agent is made by comparing zone sizes to those in the CLSI Document M100-S23 (M2),5 which is included with CLSI Document M2-A11.1 Mueller Hinton Agar with 5% Sheep Blood Approximate Formula* Per Liter Purified Water Beef Extract . Acid Hydrolysate of Casein . Sheep Blood, defibrinated . *Adjusted and/or supplemented as required to meet performance criteria. Muel er Hinton Agar with 5% Sheep Blood Prepared Plates are medium to dark red in appearance. Warnings and Precautions: For in vitro Diagnostic Use in Taiwan. If excessive moisture is observed, invert the bottom over an off-set lid and al ow to air dry in order to prevent formation of a seal between the top and bottom of the plate during incubation. Observe aseptic techniques and established precautions against microbiological hazards throughout al procedures. After use, prepared plates, specimen containers and other contaminated materials must be sterilized by autoclaving before discarding. Storage Instructions: On receipt, store plates in the dark at 2-8°C. Avoid freezing and overheating. Do not open until ready to use. Minimize exposure to light. Prepared plates stored in their original sleeve wrapping at 2-8°C until just prior to use may be inoculated up to the expiration date and incubated for recommended incubation times. Al ow the medium to warm to room temperature before inoculation. Product Deterioration: Do not use plates if they show evidence of microbial contamination, discoloration, drying, cracking or other signs of deterioration. VIII. SPECIMEN COLLECTION AND HANDLING This product is not intended for use directly with specimens or mixed cultures. The organism to be tested must first be in pure culture. A Gram stain and a presumptive identification of S. pneumoniae are recommended.1 Material Provided: Muel er Hinton Agar with 5% Sheep Blood Materials Required But Not Provided: Inoculum broth in 5 mL amounts, such as Muel er Hinton II Broth for preparation of standard inoculum. A 0.5 McFarland barium sulfate standard for adjustment of inoculum, or prepared by adding 0.5 mL of 0.048M BaCl2 [1.175% w/v BaCl2・2H2O] to 99.5 mL of 0.18M [0.36N] H2SO4 [1% v/v]). A photometric device for adjusting the turbidity of the inoculum suspension to be equivalent to the 0.5 McFarland standard. As an alternative to the above materials (1-3), the BBLTM PromptTM Inoculation System (volumetric inoculum preparation device) can be used.7 Control culture – S. pneumoniae ATCC 49619. Paper discs impregnated with specified amounts of antimicrobial agents, such as BBL Sensi-Disc susceptibility test discs. Dispensing device, such as the Sensi-Disc 6 – or 12 - Place Dispenser. Device for measuring or interpreting zone diameters to the nearest whole mil imeter, such as a sliding caliper or a ruler.1 An incubator that produces an atmosphere containing 5-7% CO2, or another device that produces a similar CO2-enriched atmosphere. 10. Ancil ary culture media, reagents and laboratory equipment as required. Test Procedure: The direct colony suspension method should be used when testing S. pneumoniae.1 Observe aseptic techniques. Suspend growth from an overnight (16-18 h) sheep blood agar plate in saline or broth, such as Muel er Hinton II Broth. Adjust the turbidity to be equivalent to the 0.5 McFarland barium sulfate standard. For the diluent, use sterile broth or sterile saline. The turbidity of the standard and the test inoculum should be compared by holding both tubes in front of a white background with finely drawn black lines or a photometric device can be used. Alternative methods of inoculum preparation involving devices that permit direct standardization of inocula without adjustment of turbidity, such as the BBL Prompt Inoculation System, have been found to be acceptable for routine testing purposes.7 Within 15 min of adjusting the turbidity of the inoculum, dip a sterile cotton swab into the properly diluted inoculum and rotate it firmly several times against the upper inside wal of the tube to express excess fluid. Inoculate onto Muel er Hinton Agar with 5% Sheep Blood by streaking the entire agar surface of the plate three times, rotating the plate 60° between streakings to obtain even inoculation. As a final step, swab the rim of the agar bed. Replace the lid of the plate and hold the plate at room temperature for at least 3 min, but no longer than 15 min, to al ow surface moisture to be absorbed before applying the drug-impregnated discs. Use no more than nine discs per 150 mm plate, or four discs per 100 mm plate. Incubate for 20-24 h at 35°C in an atmosphere of 5-7% CO2. User Quality Control: The control culture, S. pneumoniae ATCC 49619, should be included each time a susceptibility test is performed or weekly if satisfactory performance can be documented according to the CLSI standard.1 The correct zone diameters wil be found in CLSI Document M100-S23 (M2),5 which is included with CLSI Document M2-A11.1 Quality Control requirements must be performed in accordance with applicable local, state and/or federal regulations or accreditation requirements and your laboratory's standard Quality Control procedures. It is recommended that the user refer to pertinent CLSI (formerly NCCLS) guidance and CLIA regulations for appropriate Quality Control practices. With Muel er Hinton Agar with 5% Sheep Blood, the zone of growth inhibition should be measured, not the zone of inhibition of hemolysis. The zones are measured from the upper surface of the agar il uminated with reflected light, with the cover removed. Zone diameters should be compared with those in CLSI Document M100-S23 (M2), which provides interpretive criteria.5 Results obtained may then be reported as resistant, intermediate or susceptible. Isolates of S. pneumoniae with oxacil in zone diameters of ≥ 20 mm are susceptible (MIC ≤ 0.06 µg/mL) to penicil in. CLSI Document M2-A11 should be consulted for other antimicrobial agents to which penicil in-susceptible isolates may also be considered susceptible.1 See "Limitations of the Procedure" NOTE: Supplemental tables to CLSI Document M2-A11, containing revised tables of antimicrobial discs and interpretive standards are published periodical y. The latest tables should be consulted for current recommendations. The complete standard and supplemental tables can be ordered from the Clinical and Laboratory Standards Institute, 940 West Val ey Road, Suite 1400, Wayne, PA 19087-1898. Telephone: (610) 688-0100. XI. LIMITATIONS OF THE PROCEDURE Isolates of S. pneumoniae with oxacil in zone sizes of ≥ 20 mm are susceptible to penicil in (MIC ≤ 0.06 µg/mL), as wel as to other β-lactam agents. The disc test does not, however, distinguish penicil in intermediate strains (i.e., MICs = 0.12 to 1.0 µg/mL) from strains that are penicil in resistant (i.e., MICs ≥ 2.0 µg/mL). Furthermore, a smal percentage of susceptible strains wil also have zone sizes of ≤ 19 mm in the oxacil in screening test; i.e., they wil be falsely categorized as resistant or intermediate.8,9 For these reasons, a penicil in MIC should be determined on al isolates of S. pneumoniae with oxacil in zones of ≤ 19 mm.1 With some organism-antimicrobial agent combinations, the inhibition zone may not have a sharply demarcated edge, which could lead to incorrect interpretation. Various factors have been identified as influencing disc diffusion susceptibility tests. These include the medium, agar depth, disc potency, inoculum concentration, age of inoculum, and pH.4,6 Incorrect inoculum concentration may produce incorrect results. Zones of inhibition may be too smal if the inoculum is too heavy and they may be too large and difficult to measure if the inoculum is too light. Improper storage of antimicrobial discs may cause a loss of potency and a falsely resistant result. In vitro susceptibility of an organism to a specific antimicrobial agent does not necessarily mean that the agent wil be effective in vivo. Consult appropriate references for guidance in the interpretation of results.6,10 As indicated in "PERFORMANCE CHARACTERISTICS," the use of cefaclor, cefprozil and trimethoprim/sulfamethoxazole on Muel er Hinton Agar with 5% Sheep Blood when testing S. pneumoniae is not recommended. Alternatively, susceptibility of S. pneumoniae to trimethoprim/ sulfamethoxazole can be determined by the broth dilution (MIC) method using Muel er Hinton II Broth (Cation-Adjusted) with Lysed Horse Blood.11 XII. PERFORMANCE CHARACTERISTICS Antimicrobial disc diffusion susceptibility testing using the quality control strain recommended by NCCLS Document M2-A5,12 S. pneumoniae ATCC 49619, was performed in-house with cefaclor, cefprozil, chloramphenicol, erythromycin, ofloxacin, tetracycline, trimethoprim/sulfamethoxazole and vancomycin. Fol owing the test procedures described in M2-A5, twenty tests with the quality control strain and eight antimicrobic discs were performed over a period of 10 test days. For al eight antimicrobics, 100% (160/160) of the zone sizes fel within the expected zone size ranges published in Table 3C of NCCLS Document M100-S6.13 The standard deviation for tetracycline was less than 1 mm, and for al other antimicrobics, less than 2 mm.14 Reproducibility studies (3x/day for 3 days) were done at two field sites with the antimicrobics listed above against S. pneumoniae ATCC 49619 and nine additional wel -characterized S. pneumoniae strains. Zone diameter interpretive standards from Table 2C of NCCLS Document M2-A5 and Supplement M100-S6 were fol owed for each antimicrobic. Testing with chloramphenicol, erythromycin, ofloxacin, tetracycline, and vancomycin resulted in over 95% Category Agreement with the NCCLS reference method. Testing with trimethoprim/sulfamethoxazole resulted in 90% Category Agreement with the NCCLS reference method.14 Reproducibility could not be determined for cefaclor and cefprozil due to the absence of interpretive standards for these two antimicrobics in Table 2C of NCCLS Document M100-S6.13 Based on the studies outlined above, the use of cefaclor, ceprozil or trimethoprim/sulfamethoxazole is not recommended on this medium when testing S. pneumoniae. Additional y, references in the literature15 report excessive interpretive errors in disc diffusion testing with trimethoprim/sulfamethoxazole on Muel er Hinton sheep blood agar plates. XIII. AVAILABILITY BD BBLTM Muel er Hinton Agar with 5% Sheep Blood, Ctn. of 24 plates XIV. REFERENCES Clinical and Laboratory Standards Institute (formerly NCCLS). 2012. Approved Standard: M2-A11. Performance standards for antimicrobial disk susceptibility tests, 9th ed. Clinical and Laboratory Standards Institute, Wayne, Pa. Bauer, A.W., W.M.M. Kirby, J.C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496. Ryan, K.J., F.D. Schoenknecht, and W.M.M. Kirby. 1970. Disc sensitivity testing. Hospital Practice 5:91-100. Ericsson, H.M., and J.C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international col aborative study. Acta Pathol. Microbiol. Scand. Sec. B, Suppl. 217. Clinical and Laboratory Standards Institute. 2013. Performance standards for antimicrobial susceptibility testing; fifteenth informational supplement, M100-S23 (M2). Clinical and Laboratory Standards Institute, Wayne, Pa. Jorgensen, J.H. and J.D. Turnidge. 2003. Susceptibility test methods: dilution and disk diffusion methods, p. 1108-1127. In P.R. Murray, E.J. Baron, J.H. Jorgensen, M.A. Pfal er, and R.H. Yolken (ed.), Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C. Baker, C.N., C. Thornsberry, and R.W. Hawkinson. 1983. Inoculum standardization in antimicrobial susceptibility testing: evaluation of overnight agar cultures and the rapid inoculum standardization system. J. Clin. Microbiol. 17:450-457. Swenson, J.M., B.C. Hil , and C. Thornsberry. 1986. Screening pneumococci for penicil in resistance. J. Clin. Microbiol. 24:749-752. Hindler, J.A., and J.A. Swenson. 2000. Susceptibility tests of fastidious bacteria, p. 1544-1554. In P.R. Murray, E.J. Baron, M.A. Pfal er, F.C. Tenover, and R.H. Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 10. Neumann, M.A., D.F. Sahm, C. Thornsberry, J.E. McGowan, Jr. 1991. Cumitech 6A, New developments in antimicrobial agent susceptibility testing: a practical guide. Coordinating ed., J.E. McGowan, Jr. American Society of Microbiology, Washington, D.C. 11. Clinical and Laboratory Standards Institute (formerly, NCCLS). 2012. Approved Standard M7-A9: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobical y, 6th ed. Clinical and Laboratory Standards Institute, Wayne, Pa. 12. National Committee for Clinical Laboratory Standards. 1993. Approved Standard: M2-A5. Performance standards for antimicrobial disk susceptibility tests, 5th ed. National Committee for Clinical Laboratory Standards, Vil anova, Pa. 13. National Committee for Clinical Laboratory Standards. 1995. Sixth informational supplement: M100-S6. Performance standards for antimicrobial susceptibility testing. National Committee for Clinical Laboratory Standards, Wayne, Pa. 14. Data on file at Becton Dickinson. 15. Jorgensen, J.J. 1994. Detection of antimicrobial resistance in Streptococcus pneumoniae by use of standardized susceptibility testing methods and recently developed interpretive criteria. Clin. Microbiol. Newsl. 16(13)97-104. Nippon Becton Dickinson Company, Ltd. 1 Aza Gotanda, Tsuchifune, Fukushima City, Fukushima, Japan e-mail : [email protected] WEB : http://www.bd.com/jp/ ATCC is a trademark of the American Type Culture Col ection. BO, BO Logo, and al other trademarks are property of Becton, Dickinson and Company. 2014 BD.

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