Stokes Disc Diffusion Method Principle, Procedure, Result.

Stokes Disc Diffusion Method

• Stokes disc diffusion technique differs in comparison to Kirby Bauer disc diffusion in the application of control and test strain on the same plate.
• Stokes disc diffusion method isn’t as well-standardized as Kirby-Bauer’s method and is utilized in labs especially where the exact amount of antimicrobial present in discs isn’t known because of the difficulty in getting discs and correctly storing them or when other requirements needed for the Kirby-Bauer method cannot be fulfilled.
• Comparative disc diffusion techniques based upon techniques based on the Stokes method are being used extensively in the majority laboratories in the UK for determining susceptibility to antibiotics.
• The Stokes’ method allows each isolate to be compared against sensitive control of the same species that are subjected to the same conditions for medium, incubation duration and temperature, atmosphere and disc contents.
• Because test and control organisms share the same plate, the differences between their sizes in the respective zones can be determined directly.

Principle of Stokes Disc Diffusion Method

In this method, the Stokes disc diffusion discs are placed between normal and the test inocula in order to create zones of inhibition (ZOI) that form around each disc comprise of test and standard bacteria. The antibiotic’s diffusion occurs and the susceptibility of the organisms to antibiotics is measured by measuring the size of the zone.

Purposes of Stokes Disc Diffusion Method

The Stokes Disc Diffusion Method is a laboratory technique used for several purposes, including:

1. Determining antimicrobial susceptibility: The primary purpose of the Stokes Disc Diffusion Method is to determine the antimicrobial susceptibility of a microorganism to various antibiotics or other antimicrobial agents. The test helps to identify which antibiotics or agents are effective in treating an infection caused by the microbe.
2. Quality control of antibiotics: The Stokes Disc Diffusion Method is also used to ensure the quality and potency of antibiotics. The test is performed on antibiotic products to determine their efficacy against specific microorganisms.
3. Surveillance of antibiotic resistance: The test is used to monitor the emergence of antibiotic resistance in microbial populations. The results of the test can help to identify trends in antibiotic resistance and guide appropriate treatment strategies.
4. Research: The Stokes Disc Diffusion Method is used in research studies to investigate the mechanisms of antibiotic resistance and to evaluate the effectiveness of new antimicrobial agents.

Overall, the Stokes Disc Diffusion Method is a valuable tool for determining the susceptibility or resistance of microorganisms to various antimicrobial agents. This information can be used to guide appropriate treatment strategies and to monitor the emergence of antibiotic resistance.

Types of Stokes Disc Diffusion Method

Stokes Disc Diffusion Method is divided into two classes base on inoculation method of organism such as-

1. Stokes disc diffusion method (conventional): The test organism is infected in the middle third of the plate, with controls in the upper and bottom thirds.
2. Modified Stokes disc diffusion method: As depicted in the image, the test bacteria is inoculated on the upper and lower thirds of the plate, with a control on the middle third.

Other types of Stokes Disc Diffusion Method

1. Kirby-Bauer Method: This is the most commonly used method of Stokes Disc Diffusion. In this method, paper discs impregnated with specific antibiotics or other antimicrobial agents are placed on a culture plate containing the test organism. The diameter of the zone of inhibition (clear zone around the disc where no growth of the organism occurs) is measured and compared to a standardized chart to determine the susceptibility or resistance of the microbe to the tested agent.
2. E-test Method: This method uses a plastic strip impregnated with a gradient of antibiotic concentrations. The strip is placed on the agar surface inoculated with the test organism, and the lowest concentration of the antibiotic at which there is no growth of the organism is recorded as the minimum inhibitory concentration (MIC).
3. M.I.C.Evaluator Strips: Similar to the E-test method, M.I.C.Evaluator strips contain a gradient of antibiotic concentrations on a plastic strip, but in this case, the strip is placed in a liquid culture rather than on an agar surface. The lowest concentration of the antibiotic at which there is no visible growth of the organism is recorded as the MIC.
4. Broth Dilution Method: In this method, the antimicrobial agent is diluted in liquid culture medium, and the test organism is inoculated into the medium at a specific concentration. The tubes are incubated, and the lowest concentration of the antimicrobial agent that inhibits growth of the organism is recorded as the MIC.

These different methods of the Stokes Disc Diffusion Method can be used to determine the susceptibility or resistance of a microorganism to various antimicrobial agents, which can guide appropriate treatment strategies.

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 – How do you perform disc diffusion method?

1. Colonies should be well-isolated and with the same morphological form of both control and test strains. Transfer the colonies to tubes that contain up to 5ml of tryptic soy broth.
2. The broth should be incubated at 37°C until it attains the turbidity of 0.5 McFarland standard and it generally takes between 2 and 6 hours.
3. Change the turbidity level of the broth that is growing culture by using sterile saline, or tryptic soybean broth if the turbidity is high, but it is low. further, let it incubate until you achieve an optically similar turbidity to that from that of the 0.5 McFarland standards. This standard corresponds to a suspension that contains between 1 and 2 x 1 108 CFU/ml of E.coli ATCC 25922.
4. For this procedure to be done correctly you must either read the inoculum using a McFarland densitometer , or visual method, if you choose to do it sufficient light is required to see the inoculum tube as well as it’s 0.5 McFarland standard against a card that has white background and contrasting black lines, also known as Wickerham card.
5. Inject sterile cotton swabs of each of the suspensions that have been adjusted (within 15 minutes of adjusting the volume of turbidity).
6. The swabs should be rotated multiple times before pressing them on the surface of the tube, above the fluid level . This will eliminate any excess inoculum from the swabs.
7. Dry the inoculation plates by letting the lid opened so that there are no droplets of water over the surfaces.
8. Place the control culture on two bands on each of the plates, leaving one central band that is not inoculated the aid of sterilized wipes.
9. Place the test organism in the middle of the area without touching any side, and this can be done with the conventional Stokes disc diffusion technique, whereas the modified Stokes disc diffusion technique is the reverses of steps 9 and 10.
10. Place the discs of antimicrobials using forceps along the line between the control and test organisms. Press gently to ensure that they are in touch with medium. Be aware that there must be a an absolute minimum distance of 2 centimeters between the two disks.
11. The plates should be incubated overnight aerobically at 35 to 37 degC.

Result Interpretation of Stokes Disc Diffusion Method

Determine your inhibition zones radius starting from to the edges of the disc until its edge, as illustrated in the above image.

• Sensitive (S)–  The size of the zone of the strain being tested is greater in comparison to that of the control strain. If the measurement of the bacterium is less than the control strain, the difference must not exceed 3 millimeters.
• Intermediate(I)– The Zone size of the strain test is greater than 2 millimeters, but less that the test strain by more than 3 millimeters.
• Resistant(R)–  The area dimension of the test strain is not greater than 2 millimeters.

Advantages of Stokes Disc Diffusion Method

• The test strain and the control strain can be compared with the help of the identical plate.
• More reliable for quality tests of discs.
• Control and test organisms are the same environmental i.e. the effects of changes in an environmental factor like temperatureand time affect both simultaneously , thereby minimizing errors.
• Incorrect use of heavy or light inoculas will be identified.
• The strips that contain several Antibiotics are tested on one plate.

Disadvantages of Stokes Disc Diffusion Method

• Stokes disc diffusion isn’t as well-standardized like it is the Kirby Bauer technique, but it is utilized in laboratories, especially where the exact amount of antimicrobial contained in the disc cannot be determined because of difficulties in obtaining discs and properly storing them or when other requirements necessary for the Kirby-Bauer procedure cannot be fulfilled.
• An insignificant amount of antibiotics are tested on the form of a plate.

Keynotes on Stokes Disc Diffusion Method

• Four discs can be seated on an 85mm circular plate.
• Extremes in the density of inoculum are to be prevented. Do not use overnight broth that is undiluted cultures or any other inocula that is not standardized that streaks plates.
• To inoculate, a rotating plating technique may be employed where the strain for control is applied around the perimeter, while the test strain is placed in the center. In this way there are six discs that can be placed on an 85 mm circular plate.

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