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Chapter 5.21 : 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 CLSI ( ) and is the procedure that has been successfully used with a Quick Spense (Sandy Springs Instrument Co.) and AutoSpense IIe (Halpro, Inc.) dispensers at the clinical microbiology laboratories of the University of California, Los Angeles for nearly three 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 1,800 trays, but recommendations for preparing fewer trays are included.

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
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Image of Broth Microdilution MIC Tray Preparation QC Log
Broth Microdilution MIC Tray Preparation QC Log

Broth Microdilution MIC Tray Preparation QC Log

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
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Run Day Operations Checks

Run Day Operations Checks

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
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Image of Stock Solution QC
Stock Solution QC

Stock Solution QC

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
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Image of Example Stock Solution QC
Example Stock Solution QC

Example Stock Solution QC

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
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References

/content/book/10.1128/9781555818814.chap5.21
1. CLSI. 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 10th ed. CLSI document M07-A10. CLSI, Wayne, PA.
2. CLSI. 2015. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. CLSI document M100-S25. CLSI, Wayne, PA.
3. Hwang JMD, Piccinini TE, Lammel CJ, Hadley WK, Brooks GF. 1986. Effect of storage temperature on the stability of antimicrobial agents in MIC trays. J Clin Microbiol 23:959961.
4. Nickolai DJ, Lammel CJ, Byford BA, Morris JH, Kaplan EB, Hadley WK, Brooks GF. 1985. Effects of storage temperature and pH on stability of 11 beta-lactam antibiotics in MIC trays. J Clin Microbiol 21:366370.
5. Hindler JF, Tamashiro L. 2010. Preparation of broth microdilution MIC trays, p5.15.1–5.15.18. In Garcia LS (ed), Clinical Microbiology Procedures Handbook, 3 ed. ASM Press, Washington, DC.

Tables

Generic image for table
Sample listing of antimicrobial agents, abbreviations, and concentrations that might be included in microdilution MIC trays

Sample listing of antimicrobial agents, abbreviations, and concentrations that might be included in microdilution MIC trays

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
Generic image for table
ATCC QC organisms that have on-scale endpoints for the agents and concentrations depicted in example in Appendix 5.21–4

ATCC QC organisms that have on-scale endpoints for the agents and concentrations depicted in example in Appendix 5.21–4

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
Generic image for table
Table 5.21–A1

Preparation of antimicrobial dilutions: primary scheme

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
Generic image for table
Table 5.21–A2

Preparation of antimicrobial dilutions: alternative scheme

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
Generic image for table
Untitled

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
Generic image for table
Worksheet for Microdilution MIC Trays

Worksheet for Microdilution MIC Trays

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21
Generic image for table
Example of microdilution MIC tray layout for Gram-negative organismsa,b,c

Example of microdilution MIC tray layout for Gram-negative organisms

Citation: Leber A. 2016. Preparation of Broth Microdilution MIC Trays, p 5.21.1-5.21.19. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch5.21

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