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J Lab Physicians. 2019 Jul-Sep;11(3):220-228. doi: 10.4103/JLP.JLP_11_19.

Development, optimization, standardization, and validation of a simple in-house agar gradient method to determine minimum inhibitory concentration of vancomycin for Staphylococcus aureus.

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Department of Clinical Microbiology, Super Specialty Pediatric Hospital and Post Graduate Teaching Institute, Noida, Uttar Pradesh, India.
Department of Clinical Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India.
Department of Clinical Microbiology, Maulana Azad Medical College and Lok Nayak Hospital, New Delhi, India.



The Clinical and Laboratory Standards Institute recommends reporting minimum inhibitory concentration (MIC) values of vancomycin for Staphylococcus aureus. Commercial MIC strips are expensive, and the traditional broth microdilution method is cumbersome. With this background, we attempted to develop and standardize an in-house agar gradient method to determine MIC values of vancomycin for S. aureus.


To develop and validate an in-house vancomycin MIC strip, based on simple agar gradient method for S. aureus as per bioassay development guidelines.


Filter paper gradient strips were made in house and impregnated with varying concentrations of vancomycin to create an antibiotic gradient. During standardization, MICs of ninety clinical strains of S. aureus and ATCC 29213 were tested by the broth microdilution and commercial strip followed by the in-house strip. During the validation stage, MICs of ninety different clinical strains of S. aureus and ATCC 29213 were determined by the in-house strip followed by MIC detection by broth microdilution and commercial strips. A reading of more than ± 1log2 dilution compared with broth microdilution was considered as an outlier.


During the initial stage, there were 7/90 outliers in the clinical strains, and no outliers were seen with the ATCC 29213 control strain. Corrective action included increasing precaution during the antibiotic impregnation on the strip. During validation stage, only 4/90 outliers were observed in the clinical strains. The commercial strips had 29/90 among clinical and 15/30 outliers in the control strain during the prevalidation phase. Despite maintaining cold chain during the validation phase, the outliers for commercial strip were 18/90 and 4/30 for clinical and control strains, respectively.


Reporting vancomycin MIC for S. aureus may be attempted using the in-house method after validating it with a gold standard broth microdilution method and quality control as per protocol.


AMR; Epsilometer test; antimicrobial testing; in-house agar gradient method; minimum inhibitory concentration; vancomycin

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