Stokes Disc Diffusion Method: Principle, Procedure, Results

Stokes disc diffusion technique varies from Kirby Bauer disc diffusion in the use of both control and test strain on the same plate. Stokes disc diffusion technique is not as highly standardized as the Kirby-Bauer technique. Also, it is applicable in laboratories particularly when the exact amount of antimicrobial in a disc cannot be guaranteed due to difficulties in obtaining discs and storing them correctly or when the other conditions required for the Kirby-Bauer technique cannot be met.

Comparative disc diffusion techniques based on the Stokes method are still in wide use in the majority of laboratories in the UK,  to determine antibiotic susceptibility. The stokes method allows each individual isolate to be compared with sensitive control of the same or similar species which is subjected to the same technical conditions of medium, incubation time, atmosphere, temperature, and disc content.  As control and test organisms are adjacent on the same plate the difference between respective zone sizes can be measured directly.

Procedure 

1. Select at least 3-5 well-isolated colonies of the same morphological type of both test and control strains from the culture. Then, touch the top of each colony with a loop. After that, transfer the growth into a tube containing 4-5 ml of a suitable broth medium, like tryptic soy broth.
2. Incubate the broth culture at 37°C until it achieves or exceeds the turbidity of the 0.5 McFarland standard (usually 2-6 hours)
3. Then, adjust the turbidity of the actively growing broth culture with sterile saline or broth to obtain turbidity optically comparable to that of the 0.5 McFarland standard. This results in a suspension containing approximately 1 to 2 x 10⁸ CFU/ml for E.coli ATCC 25922.  To perform this step properly, either use a photometric device or, use adequate light visually compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines.
4. Within 15 minutes after adjusting the turbidity of both tubes, dip sterile cotton swabs into each of the adjusted suspensions.  Rotate the swabs rotated several times. Then press firmly on the inside wall of the tube above the fluid level.  This will remove excess inoculum from the swab.
5. A dried Müeller-Hinton agar plate is divided into 3 halves.
6. Inoculate the dried surface of a Müeller-Hinton agar plate with the control strains evenly across the upper and lower thirds of the plate. Then, inoculate the test strains between the control, with a distance of not more than 5mm on each side of the control strain.
7. Allow the inocula to dry for few minutes with the lid.
8. Place antimicrobial discs in the gap between the test and control strain using a sterile forceps and press gently.
9. Within 30 mins of applying the discs, incubate the plates aerobically at 35-37 °C for 18-24 hrs.

Inoculation of Test Plates

NOTE: Extremes in inoculum density must be avoided.  Never use undiluted overnight broth cultures or other unstandardized inocula for streaking plates.

Interpretation of results

Measure the radius of the inhibition zone from the edge of the disc to the edge of the zone.

1. Sensitive (S): Zone radius is wider than or equal to, or not more than 3mm smaller than the control.
2. Intermediate (I): Zone Radius is > 2 mm but smaller than the control by > 3mm.
3. Resistant (R): No zone of inhibition or zone radius measures 2mm or less

Advantages of Stokes Method

1. The control strain and test strain can be checked on the same plate.
2. More reliable for the quality testing of discs.
3. The effect of variation of the environmental condition like temperature, time affects both simultaneously thus minimizing error
4. Errors due to using too heavy or light inoculums will be detected.

References

1. Gosden, P. E., Andrews, J. M., Bowker, K. E., Holt, H. A., MacGowan, A. P., Reeves, D. S., Sunderland, J., & Wise, R. (1998). Comparison of the modified Stokes’ method of susceptibility testing with results obtained using MIC methods and British Society of Antimicrobial Chemotherapy breakpoints. The Journal of antimicrobial chemotherapy, 42(2), 161–169. https://doi.org/10.1093/jac/42.2.161 
2. Donnelly J. P. (1986). Comparison of methods for the interpretation of disk diffusion susceptibility tests for augmentin. Diagnostic microbiology and infectious disease, 5(3), 255–263. https://doi.org/10.1016/0732-8893(86)90009-x