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Chapter 5.12 : Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods

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Abstract:

Microbiologists commonly use a two-dimensional, two-agent broth microdilution checkerboard to evaluate combinations of antimicrobial agents against microorganisms. A broth macrodilution limited series, agar dilution method, or disk diffusion method can also be used. Test methods are based on NCCLS broth dilution susceptibility methods ( ) for evaluating the inhibitory or bactericidal activity ( ) of specific concentrations in combination at a fixed time. In vitro interactions are calculated algebraically and interpreted as synergistic, indifferent, or antagonistic depending on whether the antibacterial activity of the combination is greater than, equivalent to, or less than, respectively, the activities of the individual agents. In vitro interactions can also be represented geometrically with isobolograms ( ).

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Shaded squares indicate growth present in tube.

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Shaded squares indicate growth present in tube.

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
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References

/content/book/10.1128/9781555817435.chap5.12
1. Berenbaum, M. C. 1978. A method for testing synergy with any number of agents. J. Infect. Dis. 137:122130.
2. Eliopoulos, G. M.,, and C. T. Eliopoulos. 1988. Antibiotic combinations: should they be tested? Clin. Microbiol. Rev. 1:139156.
3. Eliopoulos, G. M.,, and R. C. Moellering, Jr., 1996. Antimicrobial combinations, p. 330396. In V. Lorian (ed.), Antibiotics in Laboratory Medicine, 4th ed. The Williams & Wilkins Co., Baltimore, Md.
4.NCCLS. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved guideline M26-A. NCCLS, Wayne, Pa.
5.NCCLS. 2003. Methods for Dilution Susceptibility Tests for Bacteria That Grow Aerobically, 6th ed. Approved standard M7-A6. NCCLS, Wayne, Pa.
6. Rahal, J. J., Jr. 1978. Antibiotic combinations: the clinical relevance of synergy and antagonism. Medicine 57:179195.
7. Weinstein, A. J. 1977. Principles of concomitant antibiotic therapy. Med. J. Aust. Spec. Suppl. 2:1922.
1. Eliopoulos, G. M.,, and R. C. Moellering, Jr., 1991. Antimicrobial combinations, p. 432492, Fig. 13.2. In V. Lorian (ed.), Antibiotics in Laboratory Medicine, 3rd ed. The Williams & Wilkins Co., Baltimore, Md.
1. Eliopoulos, G. M.,, and R. C. Moellering, Jr., 1996. Antimicrobial combinations, p. 330396. In V. Lorian (ed.), Antibiotics in Laboratory Medicine, 4th ed. The Williams & Wilkins Co., Baltimore, Md.

Tables

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Examples of reported combination interactions

In vitro reports outnumber in vivo documentation. Documentation is found in the following references: G. M. Eliopoulos and C. T. Eliopoulos. 1988. Antibiotic combinations: should they be tested? 1:139–156; G. M. Eliopoulos and R. C. Moellering, Jr. 1996. Antimicrobial combinations, p. 330–396. V. Lorian (ed.), , 4th ed. The Williams & Wilkins Co., Baltimore, Md.; and J. J. Rahal, Jr. 1978. Antibiotic combinations: the clinical relevance of synergy and antagonism. 57:179–195.

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
Generic image for table

Example of format of broth microdilution checkerboard panel

Well A1, drug-free growth control; well H12, sterility control. Abbreviations: PP 0.5, 0.5 µg of piperacillin per ml; AK 1, 1 µg of amikacin per ml; etc.

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
Generic image for table

Dilution schematics for two-agent broth microdilution checkerboard

Diluent is usually CAMHB. It is combined with 1 ml of source. These values can be multiplied to obtain sufficient final working volumes.

Intermediate working concentration is prepared at twice the concentration stated on panel configuration.

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12
Generic image for table

Dilution schematics for two-agent broth macrodilution limited checkerboard

These values can be multiplied to obtain sufficient final working volumes.

Diluent is usually CAMHB.

Intermediate working concentration is prepared at four times the concentration stated on schematic.

Citation: Garcia L. 2010. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, p 140-162. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch5.12

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