Antimicrobial drug development is a complicated process which requires a thorough knowledge of the microbiological testing procedures. Among these microbiological tests, Minimum Bactericidal Concentration (MBC), Zone of Inhibition (ZOI), and Minimum Inhibitory Concentration (MIC) assays are the basic antimicrobial tests which are performed frequently by drug developers. These tests provide important data regarding the effectiveness of any proposed new drug. This article briefly explains these test methods, their importance and application in the development of new drugs.
Zone of Inhibition (ZOI)
What is ZOI?
Zone of Inhibition is an assay that is conducted to test the efficacy of an antimicrobial agent in inhibiting the growth of specific bacteria. It refers to the clear area where there is no visible microbial growth which appears around the antimicrobial agents being tested on an agar plate. This zone indicates the effectiveness of the antimicrobial agent in restricting the growth of the microorganism.
How is ZOI Measured?
ZOI is measured using a circular filter paper disc impregnated with the test substance. This filter paper is placed on an agar plate that is inoculated with a specific bacterial strain. This setup is then incubated for a period of 24-48 hours. After incubation, the agar plate is examined for clear zones around the antimicrobial disc. The presence of a zone of clearance indicates that the test substance is effective against the target bacteria. Absence of this zone indicates otherwise. The diameter of the inhibition zone around the disc is measured in millimeters. Larger zones indicate greater antimicrobial activity.
Importance in Drug Development
The ZOI test is particularly suitable as a rapid screening tool for antimicrobial agents for drug developers. It can help in finding those compounds which have significant antimicrobial activity. It is simple and inexpensive, therefore, most appropriate for conducting the tests with new drugs. One important thing, however, that needs to be kept in mind is the fact that while ZOI does give a visual and quantitative measure of the efficacy, it does not provide data on whether the bacteria are killed or merely inhibited from growing.
Minimum Inhibitory Concentration (MIC): Quantifying Inhibition
What is MIC?
Minimum Inhibitory Concentration (MIC) is defined as the least concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. MIC is of critical value for susceptibility studies of bacteria, fungi, and other pathogens towards antimicrobial compounds.
MIC Testing Methods
Broth Dilution
The broth dilution is considered a common technique to determine MIC. The test may be done in tubes or in a microdilution format using a 96-well plate. Generally, this method involves exposure of the bacteria to different concentrations of the antimicrobial agent in a liquid medium which is followed by spectrophotometric analysis of bacterial growth.
Agar Dilution
Agar dilution is similar to broth dilution but is carried out on a solid medium. Different concentrations of antimicrobial agents to be tested are incorporated into an agar medium and the plates are inoculated with a standardized amount of bacteria. The bacterial colonies are counted to determine the inhibitory effect of different concentrations of antimicrobial agents.
E-test
E test is a process widely used to determine the minimum inhibitory concentration (MIC) of antibiotics against microorganisms; it is also known as the epsilometer test. This is a quantitative technique where the plastic strip impregnated with defined concentrations of antibiotics is placed on the bacterial inoculum swabbed over the surface of an agar plate. As growth continues, an elliptical zone of inhibition develops around the strip, showing the antibiotic effectiveness. The MIC is read directly from the strip, which is where the edge of the inhibition zone intersects with the antibiotic gradient. The E test is unique for simplicity, accuracy, and the ability to provide precise MIC values, making it a very critical tool not only in clinical use but also in microbiological research for the evaluation of resistance of microbes against antibacterial drugs.
MIC’s Role in Drug Development
Determining MIC is important for developing effective drug formulation. It helps determine the therapeutic concentration of the drug, ensuring that the administered concentration is safe and effective. It is also very essential in the clinical phase of drug trials since the MIC data can help in determining dosage to be given for optimizing the therapeutic outcome.
Minimum Bactericidal Concentration (MBC): Ensuring Bacterial Eradication
What is MBC?
Minimum Bactericidal Concentration (MBC) is considered as the lowest concentration of an antimicrobial that would kill 99.9% of the original bacterial inoculum. It is an indispensable test for determining if a drug is bactericidal or bacteriostatic. Unlike the Minimum Inhibitory Concentration (MIC), which aims at the inhibition of bacterial growth, MBC denotes the given agent’s ability to kill bacteria completely.
How is MBC Determined?
MBC testing is usually performed after MIC tests. Bacterial suspensions corresponding to the MIC and slightly above of the candidate compound are sub-cultured on media free from antibiotics to check whether the bacteria were inhibited or killed. The concentration at which there is no regrowth of the bacteria is noted as MBC.
Importance of MBC in Drug Development
MBC is particularly important in scenarios where the complete elimination of bacteria is crucial, such as in invasive infections. It helps in differentiating antibiotics that are suitable for treating critical bacterial infections from those which can just minimize the growth of bacteria.
Role of MIS
At Microbe Investigations Switzerland, we specialize in providing critical microbial testing services, including Zone of Inhibition (ZOI), Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) assays. These tests are essential for new drug developers aiming to assess the efficacy of antimicrobial agents. Our team of experienced microbiologists uses cutting-edge techniques to deliver accurate and reliable results, ensuring your drug development processes are informed and effective.
To enhance your understanding of ZOI, MIC, and MBC testing, or to discuss how our services can support your drug development efforts, please contact our experts today.
