Table 1.
Variables for the preliminary considerations for optimization and standardization of the in-house agar gradient strip
| Variations | Preventive actions taken to rule out variations |
|---|---|
| The sample: Bacterial strains including the control strain ATCC 29213 and test isolates | |
| Storage of reference strain | ATCC 29213 was maintained and revived as per the CLSI protocol |
| Purity and age of isolate | Pure isolates from fresh overnight growth from nonselective media were used |
| Identification | Test strains of Staphylococcus aureus were identified by standard identification tests[6] |
| Bacterial inoculum | Bacterial inoculum with turbidity matched to that of 0.5 McFarland commercially available turbidity standard was used for broth microdilution, commercial, and in-house agar gradient strips as per the CLSI and manufacturer’s instructions |
| The assay system: The in-house MIC strip on Mueller Hinton agar | |
| Strip dimensions | The length, width, and holes were made at equal dimensions. Thinner, thicker strips or those with holes made at unequal distances were discarded |
| Antibiotic impregnation | |
| Solvent quality | Double-distilled sterile (autoclaved) water was used |
| Antibiotic powder | Vancomycin powder was acquired from the WHO GMP, CE, and ISO 13485-certified company |
| Quantity | Calibrated electronic scale and pipettes used |
| Pipetting errors while impregnating the strip | If the volume of the antibiotic solution in all the eight tips was not equal at the time of aspiration of the antibiotic solution, the tips were readjusted to fit properly to avoid air entry during aspiration. If the impregnated drops were of unequal size after diffusion on the strip, the strip was discarded |
| Storage | Up to ten strips were stored in universal container with rubber washer and a silica desiccator at 2°C–8°C for up to 14 days |
| Shelf life | Being in-house strips, with all the components readily available, shelf life beyond 14 days was not evaluated |
| Result interpretation: MIC values | |
| Observer variations | Specific objective parameters were defined for reading the breakpoint MICs for the in-house strips |
| All MIC readings on the in-house strip were taken by one observer only, and parameters were followed to rule out intraobserver variation | |
| Incubation conditions | 35°C±2°C in ambient air for 24 h |
CLSI=Clinical laboratory standards institute, GMP=Good manufacturing practices, CE=Conformité européene, ISO=International organization for standardization, MIC=Minimum inhibitory concentration