Korean J Lab Med.  2009 Aug;29(4):324-330. 10.3343/kjlm.2009.29.4.324.

The Optimal Wavelength of Spectrophotometric Broth Microdilution Antifungal Susceptibility Test for Flucytosine and Three Azoles

• Lee JY
• Lee EJ
• Kim SH
• Jeong HS
• Oh SH
• Kim HR
• Lee JN
• Chang CL
• Kho WG
• Shin JH

• KMID: 1096926
• DOI: http://doi.org/10.3343/kjlm.2009.29.4.324

Abstract

BACKGROUND: There is no guideline for the appropriate wavelength at which to measure the optical density (OD) value in broth microdilution antifungal susceptibility testing, although a spectrophotometric reading method is commonly used. The present study aimed to analyze the difference in the OD values over the range of visible light and to ascertain the optimal wavelength for the spectrophotometric method of microdilution testing.
METHODS
We measured the OD of background blank controls of broth medium, antifungal agents, and inocula of five type strains using a Synergy HT multi-detection microplate reader at 5-nm intervals from 380 nm to 760 nm. We also estimated the OD differences between the 50% of growth control and blank control.
RESULTS
The OD of the blank control showed a parabola shape with two peaks and steadily decreased at longer wavelengths. The curves of the antifungal agent were similar to those of blank controls, and the influence of each antifungal agent on the OD was minimal. For the difference in OD between 50% of growth control and the blank control, the curve was the opposite of the blank control, and the OD increased steadily at the wavelengths above 600 nm.
CONCLUSIONS
The range between 600 nm and 700 nm was the optimal wavelength for broth microdilution antifungal susceptibility testing, although any wavelength within the visible light spectrum can be used.

Keyword

Antifungal susceptibility test; Broth microdilution method; Spectrophotometer; Wavelength

MeSH Terms

Antifungal Agents/*chemistry
Azoles/*chemistry
Culture Media/*chemistry
Flucytosine/*chemistry
Microbial Sensitivity Tests
Spectrophotometry/*methods

Figure

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  Fig. 1. Absorbance spectrum curve of blank control well. Abbreviation: OD, optical density.

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  Fig. 2. Absorbance spectrum curves of antifungal agent-containing wells. Abbreviation: See Fig. 1.

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  Fig. 3. Absorbance spectrum curves according to wavelength of five type strains after incubation at 35°C for 48 hr. Abbreviation: See Fig. 1.

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  Fig. 4. Difference between half OD of growth control and the OD of the blank control. Abbreviation: See Fig. 1.

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