What is the Difference Between Agar Well and Disk Diffusion Method

The main difference between agar well and disk diffusion method is that agar well diffusion uses an antimicrobial agent in a hole punched into the agar plate, whereas the disk diffusion method uses filter paper disks containing the antimicrobial compound placed on the agar surface. 

Agar well and disk diffusion method are two diffusion methods in use to evaluate in vitro antimicrobial activity of an extracted or pure compound.      

Key Areas Covered

1. What is Agar Well Diffusion Method
– Definition, Features, Process
2. What is Disk Diffusion Method
– Definition, Features, Process
3. Similarities Between Agar Well and Disk Diffusion Method
– Outline of Common Features
4. Difference Between Agar Well and Disk Diffusion Method
– Comparison of Key Differences

Key Terms 

Agar Well Diffusion Method, Disc Diffusion Method

What is Agar Well Diffusion Method

The agar well diffusion method is a common method to evaluate the antimicrobial activity of plants and other compounds. In this method, first, the microbial inoculum is spread over an agar plate. Then, antimicrobial agents are placed in holes punched into the agar. The diameter of the holes is 6 to 8 mm. Also, the volume of the hole can be 20–100 µL. Finally, agar plates are incubated for the growth of microorganisms. In this process, the diffusion of the antimicrobial agent in the agar inhibits the growth of the microorganisms or kills them.

Figure 1: Agar Diffusion Method

However, the agar well diffusion method and other diffusion methods are not suitable for determining the minimum inhibitory concentration (MIC). It means that the diffusion methods cannot quantify the antimicrobial agents diffused into the agar. Nevertheless, we can calculate the approximate antimicrobial concentration by comparing the inhibition zones with algorithms.  

What is Disk Diffusion Method

The disk diffusion method is the official method for antimicrobial susceptibility testing for bacteria and yeasts. In the disk diffusion method, filter paper disks containing a desired concentration of antimicrobial agent are placed on the surface of the agar plate. The diameter of the filter papers is about 6 mm. Also, the agar plate contains the inoculated microorganism. The inhibition of the growth of microorganisms can be observed after incubation. Similar to the agar well diffusion method, the antimicrobial agent diffuses through the agar and inhibits the growth of the microorganism.

Figure 2: Disk Diffusion Method

Similarities Between Agar Well and Disk Diffusion Method

• Agar well and disc diffusion method are important for evaluating in vitro antimicrobial activity of pure or extracted compounds.
• In both methods, they use the diffusion of the antimicrobial compound through the gel, which prevents the growth or kills microorganisms.
• They use agar plates to grow microorganisms for testing.
• Besides, both tests are inappropriate for determining the minimum inhibitory concentration (MIC).
• Also, they are simple and low-cost methods that can use numerous microorganisms and antimicrobial agents for testing. 

Difference Between Agar Well and Disk Diffusion Method

Definition

The agar well diffusion method is a widely used method to evaluate the antimicrobial activity of plants or microbial extracts. Meanwhile, the disk diffusion method refers to a method to determine microbial susceptibility to antibiotics using filter paper disks.

Method

Agar well diffusion method uses punched wells with antimicrobial agents, while the disk diffusion method uses a filter paper disk. 

Process

Agar well diffusion method adds antimicrobial agents in punched wells in an inoculum to test microbial growth, while the disk diffusion method uses filter paper disks containing antimicrobial agents in an inoculum to test microbial growth. 

Conclusion

In brief, agar well and disk diffusion method are two diffusion methods to test the activity of antimicrobial agents that are pure or extracted. Agar well diffusion method uses punched wells to fill antimicrobial agents in an inoculum. In comparison, the disk diffusion method uses filter paper with the antimicrobial agent in the inoculum to test the growth of microorganisms. Therefore, the method is the main difference between the agar well and the disk diffusion method.  

References:

1. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitroevaluating antimicrobial activity: A review. J Pharm Anal. 2016 Apr;6(2):71-79. doi: 10.1016/j.jpha.2015.11.005. Epub 2015 Dec 2. PMID: 29403965; PMCID: PMC5762448

Image Courtesy:

1. “Agar Diffusion Method 1” By Sommer36 – Own work (CC-BY SA 4.0) via Commons Wikimedia
2. “KB test” By NOAA – Own Work (Public Domain) via Commons Wikimedia