Stokes Disc Diffusion Method: Introduction, Principle, Procedure and Result Interpretation

Stokes disc diffusion method

Introduction of Stokes Disc Diffusion Method

Stokes disc diffusion method as shown above picture. Antibiotic Sensitivity Testing (AST) is a determination of the least amount of an antimicrobial chemotherapeutic agent that will inhibit the growth of microorganisms in vitro and it is achieved by following methods 1. Disk diffusion method: A. Kirby Bauer method, B. Stokes method 2. MIC: A. Broth dilution method B. Agar dilution method and 3. E-test. Stokes disc diffusion method varies from Kirby Bauer disc diffusion in the use of both control and test strain on the same plate. A set of standard strains are used as control strains depending on the organism to be tested. The control strains are E. coli NCTC 10414 for testing coliform bacilli from the urinary tract, Pseudomonas aeruginosa NCTC 10662 against aminoglycosides. Comparative disc diffusion techniques based on the Stokes method is still in wide use in a developed country like the UK, to determine antibiotic susceptibility and therefore it is also useful in developing countries.

Stokes disc diffusion method is of two types-

  1. Stokes disc diffusion method  and
  2. Modified Stokes disc diffusion method

Stokes disc diffusion method (conventional): The test organism is inoculated on the central one-third and controls on the upper and lower thirds of the plate.

Modified Stokes disc diffusion method: In this method, the test bacterium is inoculated over the upper and lower thirds of the plate and control on the central one-third as shown above picture.

Principle of Stokes Disc Diffusion Method

In this Stokes disc diffusion method discs are applied between the standard and test inocula, so that zone inhibition (ZOI) formed around each disc are composed of standard and test bacteria. The diffusion of antibiotic takes place and thus the susceptibility of those organisms to the antibiotics are known by measuring zone size.

Requirements for the Test

  • MHA plate
  • Antimicrobial discs
  • Sterile cotton swabs
  • Sterile forceps
  • Control strains depending on the bacterium tested-S. aureus NCTC 6571 or E. coli NCTC 10414 or P. aeruginosa NCTC 10662
  • Bunsen burner
  • Inoculating loop
  • O.5 McFarland Std. or Mc Farland Densitometer
  • Incubator
  • Wickerham Card

Procedure of Stokes Disc Diffusion Method

  1. Take 3-5 well-isolated colonies of the same morphological type of both test and control strains. Transfer the colonies into a tube containing 4 to 5 ml of a  tryptic soy broth.
  2. Incubate the broth culture at 37°C until it achieves the turbidity of the 0.5 McFarland standard and it usually takes 2 to 6 hours.
  3. Adjust the turbidity of the actively growing broth culture with sterile saline or tryptic soy broth when high but in low turbidity further, incubate to obtain turbidity optically comparable to that of the 0.5 McFarland standard. This standard is equivalent to a suspension containing approximately 1 to 2 x 108 CFU/ml for E.coli ATCC 25922.
  4. To perform this step properly, either read inoculum in McFarland densitometer or, if done visually, adequate light is needed to visually compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines called Wickerham card.
  5. Dip sterile cotton swabs into each of the adjusted suspensions (within 15 minutes after adjusting the turbidity).
  6. Rotate the swabs several times and pressed firmly on the inside wall of the tube above the fluid level and this will remove excess inoculum from the swab.
  7. Dry the inoculation plates with the lid open so that there should be free droplets of moisture on the surface.
  8. Apply the control culture in two bands on either of the plates leaving a central band uninoculated with the help of sterile swabs.
  9. Apply the test organism in the central portion without touching either side and this is applicable for conventional Stokes disc diffusion method whereas modified Stokes disc diffusion method reverse of steps 9 and 10.
  10. Apply the antibiotics discs with forceps on the line between test and control organisms and press gently to ensure even contact with the medium. Note: there should be a minimum distance of 2 cm between two discs.
  11.  Incubate the plates overnight aerobically at 35-37 °C.

Result Interpretation of Stokes Disc Diffusion Method

Measure the radius of the inhibition zone from the edge of the disc to the edge of the zone as shown above image-

Sensitive (S)– The zone size of the test strain is larger than or equal to the control strain. If the zone size of the test bacterium is smaller than that of the control, the difference between the two should not be more than 3 mm.

Intermediate(I)– zone size of the test strain is > 2 mm but smaller than the control by > 3 mm.

Resistant(R)– The zone size of the test strain is smaller than 2 mm.

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. Both control and test organism is the same environment i.e. the effect of variation of an environmental condition like temperature, time affect both simultaneously thus minimizing error
  4. Errors due to using too heavy or light inocula will be detected.
  5. The strips with multiple Antibiotics can be tested on a single plate.

Disadvantages of Stokes Disc Diffusion Method

  1. Stokes disc diffusion technique is not as highly standardized as the Kirby Bauer technique and is used 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.
  2. A low number of antibiotics are tested in a  plate.

Keynotes on Stokes Disc Diffusion Method

  1. Four discs can be accommodated on an  85 mm circular plate.
  2. Extremes in inoculum density must be avoided. Never use undiluted overnight broth cultures or other un-standardized inocula for streaking plates.
  3. For inoculation, a rotatory plating method can also be used wherein the control strain is applied on the outer periphery and the test strain is applied in the central portion. In such a method, six discs can be put on an 85 mm circular plate.

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Bibliography

  1. https://www.sciencedirect.com/science/article/abs/pii/073288938690009X
  2. https://jcp.bmj.com/content/jclinpath/37/9/1059.full.pdf
  3. http://www.pharmascholars.com/articles_pdfs/issues/2124638525_060302-1657.pdf
  4. Watermark.silverchair.com/420161.pdf
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