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“QUEST FOR THE BEST”
METHODS TO EVALUATE THE EFFICACY OF ANTIMICROBIAL AGENTS USED IN
PERIODONTAL THERAPY.
DIFFUSION METHOD
AGAR DISK DIFFUSION METHOD
Fastidious bacterial pathogens like streptococci, Haemophilus influenzae, Haemophilus parainfluenzae, Neisseria gonorrhoeae, and Neisseria meningitidis can be accurately tested using
specific culture media, various incubation conditions, and interpretive criteria for inhibition zones
The results provide qualitative information about the susceptibility of the microorganism to the tested antimicrobial agent, categorizing them as susceptible, intermediate or resistant..
This method cannot distinguish bactericidal and bacteriostatic effects, and it is not appropriate for determining the minimum inhibitory concentration (MIC) for all microorganisms and
antibiotics.
AGAR PLUG DIFFUSION METHOD
This method is used to demonstrate antagonism between microorganisms.
AGAR WELL DIFFUSION METHOD
Commonly used technique for evaluating antimicrobial activity of plant or microbial extracts.
The procedure involves spreading a microbial inoculum over the agar surface, then punching a 6-8 mm hole in the agar.
A volume of the antimicrobial agent or extract solution is added to the well and the agar plates are incubated under suitable conditions.
The antimicrobial agent diffuses into the agar medium, inhibiting the growth of the microbial strain being tested.
DILUTION METHOD
BROTH DILUTION METHOD-
Broth micro- or macro-dilution is a basic antimicrobial susceptibility testing method that involves diluting an antimicrobial agent in liquid growth medium in tubes or 96-well microtitration
plates, inoculating the tubes or wells with a microbial inoculum, incubating them, and determining the minimum inhibitory concentration (MIC).
The microdilution method is advantageous due to its reproducibility and economy of reagents and space. However, the final result is significantly influenced by the approach, which must be
carefully controlled for reproducible results.
Broth dilution has been standardized by CLSI for testing bacteria that grow aerobically, yeast, and filamentous fungi.
AGAR DILUTION METHOD -
Preferred when multiple isolates are being tested against a single compound or if the compound being tested masks the detection of microbial growth in liquid medium
Often recommended for fastidious organisms such as anaerobes and Helicobacter species and for antifungal agent-drugs combinations against Candida sp., Aspergillus, Fusarium and
dermatophytes.
When compared disk-diffusion and broth microdilution methods give excellent results.
TIME KILL TEST
Best method to determine bactericidal or fungicidal effects of antimicrobial agents.
It provides information on the dynamic interaction between the agent and the microbe
This test can reveal time-dependent or concentration-dependent effects
The test is well-standardized for bacteria and is performed in broth culture medium
The test involves three tubes with varying concentrations of the agent and a growth control
The percentage of dead cells is calculated by determining the number of living cells of each tube using the agar plate count method
The test can determine synergism or antagonism between drugs in combinations
ATP BIOLUMINESCENCE ASSAY
Quantifies ATP produced by bacteria or fungi to estimate microbial population in a sample.
Dluciferin in the presence of the ATP undergoes conversion by luciferase to oxyluciferin that generates light. The quantity of the emitted light is measured by a luminometer and expressed as
relative light unit (RLU) which can be converted into RLU/mole of ATP.
There is a linear relationship between cell viability and luminescence measured.
This test has applications in cytotoxicity tests, biofilm evaluation, drug screening on Leishmania, antibacterial and antifungal testing also used for antimicrobial testing in vivo or in situ.
Rapidity is the major advantage .
ANTIMICROBIAL GRADIENT METHOD
This method is used to determine the minimum inhibitory concentration (MIC) value of antibiotics, antifungals, and antimycobacterials.
combines the principles of dilution and diffusion methods by creating a concentration gradient of the antimicrobial agent tested in the agar medium.
Etests are a commercial version of this technique.
MIC value is determined at the intersection of the strip and the growth inhibition ellipse.
The Etest strip can also be used to investigate the antimicrobial interaction between two drugs.
This approach becomes costly if numerous drugs are tested.
CONCLUSION- Microbial infections are currently a significant clinical threat that can cause severe morbidity and mortality. This is primarily due to the emergence of
microbial resistance to existing antimicrobial agents. As a result, the development of new antimicrobial agents and methods for antimicrobial susceptibility testing are
crucial and continue to be actively pursued.
