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Chapter 5.15 : Preparation of Broth Microdilution MIC Trays

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Abstract:

Broth microdilution MIC trays may be prepared in-house for performance of quantitative antimicrobial susceptibility studies. If large numbers of trays are required, dilutions of the antimicrobial agents are made in large volumes and generally dispensed in 0.1-ml amounts into microdilution trays by using a commercial dispensing apparatus. For individual test trays, dilutions of the required antimicrobial agents may be accomplished with an eight-channel micropipette. The procedure described here is based on 96-well trays containing 0.1 ml per well. Not every aspect of broth microdilution tray preparation has been standardized, and for some steps, there are multiple ways of accomplishing identical results. The method presented here conforms closely to that described by the NCCLS ( ) and is the procedure that has been successfully used with a Quick Spense (Sandy Springs In strument Co.) dispenser at the clinical microbiology laboratories of the University of California—Los Angeles for nearly two decades. It assumes that the layout of the tray in terms of positioning respective concentrations of each antimicrobial agent has been previously determined. The method is detailed for preparation of approximately 1,900 trays, but recommendations for preparing fewer trays are included.

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Image of Broth Microdilution MIC Tray Preparation QC Log
Broth Microdilution MIC Tray Preparation QC Log

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Image of Broth Microdilution MIC Tray Preparation QC Log
Broth Microdilution MIC Tray Preparation QC Log

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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References

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1. Hwang, J. M. D.,, T. E. Piccinini,, C. J. Lammel,, W. K. Hadley,, and G. F. Brooks. 1986. Effect of storage temperature on the stability of antimicrobial agents in MIC trays. J. Clin. Microbiol. 23:959961.
2.NCCLS. 2003. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 6th ed. Approved standard M7-A6. NCCLS, Wayne, Pa.
3. Nickolai, D. J.,, C. J. Lammel,, B. A. Byford,, J. H. Morris,, E. B. Kaplan,, W. K. Hadley,, and G. F. Brooks. 1985. Effects of storage temperature and pH on stability of 11 betalactam antibiotics in MIC trays. J. Clin. Microbiol. 21:366370.

Tables

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Generally, serial twofold dilutions are tested.

For extended-spectrum beta-lactamase screening test.

Additional dilutions may be tested to define endpoint more exactly.

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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See Appendix 5.15-5 for definitions of abbreviations. For QC values, see NCCLS. 2003. , 6th ed. Approved standard M7-A6. NCCLS, Wayne, Pa.

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Table 5.15-A1

Preparation of antimicrobial dilutions: primary scheme

Sufficient volumes for preparation of trays: 20 ml for approximately 150 trays (0.1 ml per well), 50 ml for 400 trays, 100 ml for 900 trays, 150 ml for 1,400 trays, and 200 ml for 1,900 trays.

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Table 5.15-A2

Preparation of antimicrobial dilutions: alternate scheme

Data are based on information in NCCLS. 2003. , 6th ed. Approved standard M7-A6. NCCLS, Wayne, Pa.

5.12 ml of 10,000-µg/ml antimicrobial solution + 4.88 ml of CAMHB =5,120 µg/ml.

Use multiples of these figures to obtain desired volumes of intermediate antimicrobial solutions.

Use CAMHB rather than deionized water (as in Table 5.15-A1 ) as diluent to avoid the diluting effect of large volumes of water in CAMHB.

Add intermediate solution and CAMHB to achieve final concentrations in reservoir tube as follows.

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Stock Solution QC

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Example Stock Solution QC

Except for data on (UCLA U), values are from NCCLS. 2003. 6th ed. Approved standard M7-A6. NCCLS, Wayne, Pa.

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Worksheet for Microdilution MIC Trays

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15
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Example of microdilution MIC tray layout for gram-negative organisms ,,

This represents a sample layout only and is not a recommendation from the author.

See Appendix 5.15-5 for antimicrobial agents represented by abbreviations.

Numbers are final concentrations (micrograms per milliliter).

Citation: Garcia L. 2010. Preparation of Broth Microdilution MIC Trays, p 202-219. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.15

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