In recent years, the emerging of fungal resistance has highlighted the need for in vitro susceptibility testing. Establishing clinical correlation with the antifungal profile is important for antifungal drug discovery. Given this, Creative Biolabs' professional antifungal drug discovery team provides you various testing procedures for in vitro susceptibility testing by both broth- and agar-based methods.

Clinical and Laboratory Standards Institute (CLSI) and the Subcommittee of European Committee on Antimicrobial Susceptibility Testing (EUCAST) have established two standards for Antifungal Susceptibility Testing (AFST). The standard methods described in both two documents treat minimum inhibitory concentration (MIC) as an index to determine the susceptibility. The MIC is defined as the lowest concentration of an antimicrobial agent that causes a reduction in visible growth.

Creative Biolabs provides both broth-based and agar-based assays for evaluating MIC. For broth-based assays, broth macrodilution and broth microdilution are provided. For agar-based assays, disk diffusion, Etest, and colorimetric assays are provided. We also suggest an automatic high throughput assay by using luciferase-tagged adenylate kinase (AK) as an indicator.

Broth Dilution Assay

In Vitro Susceptibility Testing Figure 1. A broth microdilution susceptibility panel containing 98 reagent wells and a disposable tray inoculator. (Jorgensen et al. 2009)

We provide broth microdilution method for antifungal susceptibility testing. Two-fold dilutions of antifungals (e.g. 1, 2, 4, 8, and 16 µg/mL) are dispensed in a liquid growth medium. The final fungal inoculum concentration is 0.4×104 to 5×105 CFU/mL depending on different fungal species. After inoculation for a certain period, we examine fungal growth as evidenced by turbidity (for yeast) or no-growth on agar (for filamentous fungi) according to different fungal species and antifungal drugs. MIC is the lowest concentration at which no growth is observed or the lowest concentration in which an over 50% decrease in turbidity is noted.

We provide standard small, disposable, plastic “microdilution” trays containing 96 wells which largely miniaturizing and mechanizing the test. Our automatic testing system makes broth dilution testing practical and popular.

Antimicrobial Gradient Assay

In Vitro Susceptibility Testing Figure 2. A Staphylococcus aureus isolate tested by the Etest gradient diffusion method. (Jorgensen et al. 2009)

In this assay, we use Etest gradient diffusion method to determine susceptibility. Antifungal agents are deposited on plastic strips within a defined range of concentrations. The MIC is read by the intersection of the lower part of the ellipse inhibition zone with the test strip. We can compare the results obtained by Etest to those determined by broth microdilution to assess in vitro resistance.

Disk Diffusion Test

In Vitro Susceptibility Testing Figure 3. A disk diffusion test with an isolate of Escherichia coli from a urine culture. (Jorgensen et al. 2009)

We provide well-standardized disk diffusion susceptibility test. Fungal inoculum is applied to the surface of a large (150 mm diameter) Mueller-Hinton agar plate. Commercially-prepared, fixed concentration, paper antibiotic disks are placed on the inoculated agar surface (Figure 3). The growth inhibition zones of each antibiotic disk are measured. The result of the disk diffusion test is a qualitative (susceptible, intermediate or resistant) description rather than a quantitive MIC.

Automatic Systems

Our high throughput automatic system can standardize the testing process and get the results in a short period. We suggest a high throughput assay by using luciferase-tagged adenylate kinase (AK), and the results are measured by luminescence. Up to 99 panels can be measured simultaneously, and we monitor each panel every 20 min by both turbidometric and colorimetric (oxidation-reduction indicator) growth detection.

Creative Biolabs will be with you at every step of your way. For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference

  1. Jorgensen JH and Ferraro MJ (2009) “Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices” Infect Dis 49:1749-1755
  2. Perkhofer S, Mrazek C, Hartl L, et al. (2010). “In Vitro Susceptibility Testing in Fungi: What is its Role in Clinical Practice?” Curr Infect Dis Rep 12(6):401-408. doi: 10.1007/s11908-010-0134-z.

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