Standardization of Operator-Dependent Variables Affecting Precision and Accuracy of the Disk Diffusion Method for...

Area of Science:

• Microbiology
• Clinical Diagnostics
• Bacterial Susceptibility Testing

Background:

• Disk diffusion antibiotic susceptibility testing (AST) is crucial for guiding antimicrobial therapy.
• Inoculum density and plate streaking are critical, yet variable, parameters in disk diffusion AST.
• Automated imaging improves reading precision, but inoculum and streaking standardization require further investigation.

Purpose of the Study:

• To evaluate the impact of photometrically controlled inoculum preparation on standardization.
• To assess the effectiveness of automated inoculation in improving disk diffusion standardization.
• To compare manual and automated methods for inoculum preparation and plate streaking in AST.

Main Methods:

• Bacterial suspensions (E. coli, S. aureus) were prepared using visual turbidity standards and a photometer (Densichek).
• Colony-forming unit (CFU) counts were determined for prepared suspensions.
• Operators inoculated agar plates using regular cotton swabs, dry flocked swabs, or an automated streaking device.

Main Results:

• Photometric preparation (Densichek) yielded significantly different CFU counts compared to visual preparation (P < 0.001).
• Visual preparation resulted in CFU counts closer to the CLSI/EUCAST target (10^8 CFU/ml).
• Automated streaking significantly reduced interoperator variability and total methodological variation compared to manual methods.

Conclusions:

• Photometric inoculum preparation did not consistently improve standardization over visual methods.
• Dry flocked swabs reduced interoperator variability in streaking compared to cotton swabs.
• Automated inoculation significantly enhances the standardization of disk diffusion AST by minimizing variations.