Dilution Method for Antibiotic Susceptibility Testing
Dilution is one of the earliest tools in microbiological practice, allowing the growth and identification of bacterial populations in suspension. Antibiotic susceptibility testing (AST) using agar and broth dilution is one of the oldest methods used to determine the minimum inhibitory concentration (MIC). In addition to MICs measurements, broth dilution has the advantage of obtaining a minimum bactericidal concentration (MBC), which is the minimum concentration of an antimicrobial substance that kills 99.9% of bacteria. Creative Biolabs has 10+ years of experience providing CRO services with a skilled team of scientists who will work with you in developing your live biotherapeutic product (LBP), including proforming AST for LBP by dilution method.
Dilution Methods for AST at Creative Biolabs
The two basic types of dilution are microdilution and macrodilution, wherein broth and agar are the most commonly used mediums. Broth and agar dilution susceptibility testing methods are used to determine the minimal concentration, usually in micrograms per milliliter, of an antimicrobial agent required to inhibit or kill a microorganism. The lowest concentration that inhibits the visible growth of an organism is designated the MIC.
In broth dilution, consecutive two-fold dilutions (1, 2, 4, 8, and 12 μL) of antibiotics are made and dispensed into micro-centrifuge tubes containing bacterial growth medium, followed by making up the final volume by adding the medium and incubating overnight at 35℃. Finally, growth tests are performed by setting breakpoints through medium turbidity. Microbial contamination or population heterogeneity is more readily detected by the agar method than by broth methods.
Fig.1 Flow diagram for undertaking a microdilution MIC study. (Gemma, 2019)
In agar dilution, antibiotics are diluted into the agar medium, followed by plate formation and application of bacterial cells to the surface of the agar plate. Dilution testing by the agar method is a well-standardized, reliable susceptibility testing technique that may be used as a reference for evaluating other testing methods. Microbial contamination or population heterogeneity is more readily detected by the agar method than by broth methods.
Fig.2 Scheme of the agar dilution method. (Correa, 2020)
Advantages and Disadvantages
Dilution methods offer flexibility in the sense that the standard medium used to test frequently encountered organisms may be supplemented or even replaced with another medium to allow accurate testing of certain fastidious bacterial species that may not be reliably tested by disk diffusion. The advantages of these dilution processes are repeatability and cost-effectiveness. However, it is labor-intensive and expensive by nature. Apart from that, other potential limitations include experimental space, tedious dilution steps (macrodilution), the possibility of false-positive results due to long incubation times, chances of cross-contamination, bacterial incompatibility for growth, and the inability of discriminating viable and nonviable bacteria.
Creative Biolabs is a customer-driven organization that is oriented toward quick knowledgeable responses to your LBP development needs. You can rely on our know-how, understanding, experience, and passion for providing the best quality solutions, in a reliable manner with quick turnaround times and the utmost respect for customer confidentiality. If you are interested in our dilution method for AST services, please contact us for more information.
References
-
Gemma, C.C.; et al. 3.04 - Antibacterial Nanoparticles. Comprehensive Nanoscience and Nanotechnology (Second Edition). 2019, Pages 65-82
-
Correa, M.G.; et al. Antimicrobial metal-based nanoparticles: a review on their synthesis, types and antimicrobial action. Beilstein journal of nanotechnology. 2020, 11(1): 1450-1469.
For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.
Related Services
