Disk Diffusion Method for Antibiotic Susceptibility Testing
Disk diffusion is the most widely used antibiotic susceptibility testing (AST) method in microbiology laboratories because of its low cost and ease of performance and applicability to numerous bacterial species and antibiotics. Creative Biolabs specializes in live biotherapeutic product (LBP) development. Our expert scientists and talented technical support team have got you covered all the way, including antibiotic susceptibility testing.
Overview of Disk Diffusion Test
The disk diffusion method of susceptibility testing allows the categorization of most bacterial isolates as susceptible, intermediate, or resistant to a variety of antimicrobial agents. The drugs in the disk diffuse spread through agar. As the distance from the disk increases, the antimicrobial concentration decreases logarithmically, creating a drug concentration gradient in the agar medium around each disk. Concomitant with the diffusion of the drug, the bacteria that are inoculated onto the surface and are not inhibited by the concentration of the antimicrobial agent in the agar continue to multiply until a lawn of growth is visible. In the region where the drug concentration is inhibited, no growth occurs, forming a zone of inhibition around each disk.
Disk Diffusion Test Protocol
1. Preparation of Mueller-Hinton Plate
a. Allow an MH agar plate to come to room temperature.
b. If the liquid is visible on the surface of the agar, turn the plate upside down, cover it half-open, and allow the excess liquid to flow out of the agar and evaporate.
c. Appropriately label each MH agar plate for each organism to be tested.
2. Preparation of Inoculum
a. Contact 4 or 5 isolated colonies to be tested using sterile inoculum rings or needles.
b. Suspend the organism in 2 mL of sterile saline.
c. Vortex the saline tube to create a smooth suspension.
d. Adjust the turbidity of this suspension to a 0.5 McFarland standard.
e. Use this suspension within 15 minutes of preparation.
3. Inoculation of the MH Plate
a. Dip a sterile swab into the inoculum tube.
b. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid.
c. The dried surface of an MH agar plate is inoculated with a swab and the entire agar surface is streaked three times. Rotate the plate about 60 degrees each time to ensure that the inoculum is evenly distributed.
d. Leave the lid slightly open and let the plate sit at room temperature for 3-5 min, but no more than 15 minutes, to dry the surface.
4. Placement of the Antibiotic Disks
a. Place the appropriate antimicrobial-impregnated disks on the surface of the agar.
b. Once all disks are in place, replace the cap, flip the plate, and place it in an air incubator at 35°C for 16 to 18 hours.
5. Incubation of the Plates
A temperature range of 35°C ±2°C is required.
6. Measuring Zone Sizes
After incubation, use a ruler or caliper to measure the zone sizes to the nearest millimeter, disk diameter is included in the measurement.
7. Interpretation and Reporting of the Results
a. Using the published CLSI guidelines, determine the susceptibility or resistance of the organism to each drug tested.
b. For each drug, indicate on the recording sheet whether the zone size is susceptible (S), intermediate (I), or resistant (R) based on the interpretation chart.
Fig.1 Scheme of the agar disk diffusion method.1
Advantages and Disadvantages
The disk diffusion test has several advantages:
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It is technically simple to perform and very reproducible.
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The reagents are relatively inexpensive.
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It does not require any special equipment.
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It provides susceptibility category results that are easily understood by clinicians.
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It is flexible regarding the selection of antimicrobial agents for testing.
A potential disadvantage of disk diffusion susceptibility testing is that it provides only qualitative and not quantitative results.
The agar disk diffusion is commonly used for rapid analysis of the growth of common microorganisms such as bacteria, fungi, and yeasts. The antimicrobial sensitivity profile of the strain(s) present in the LBP is of the highest importance. Adequate options must be available so that patients can be treated with effective antibiotics in the event of an accidental infection or an allergy to the LBP. Following the LBP development process, Creative Biolabs provides AST services to meet requirements in different stages with rapid turn-around time and 100% on-time delivery rate. Please contact us for more detail to start your project.
Reference
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Correa, Matías Guerrero, et al. "Antimicrobial metal-based nanoparticles: A review on their synthesis, types and antimicrobial action." Beilstein journal of nanotechnology 11.1 (2020): 1450-1469. Distributed Under Open Access license CC BY 4.0, without modification.
For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.
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