Evaluation Methods for Bacteriostatic and Bactericidal Actions
Bacteriostatic and Bactericidal Actions refer to how antimicrobials work: bacteriostatic inhibits bacterial growth, while bactericidal kills bacteria directly.
Bacteriostatic vs. Bactericidal Actions
Bacteriostatic Action:
Inhibits the growth and reproduction of bacteria without killing them.
If the bacteriostatic agent is removed, bacteria can resume growth.
Bactericidal Action:
Kills bacteria directly, reducing the bacterial count.
Evaluation Methods for Bacteriostatic and Bactericidal Actions
1. Tube Dilution Method
Purpose:
To determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of an antimicrobial agent.
Procedure:
Preparation of Dilutions: Prepare serial dilutions of the antimicrobial agent in a liquid growth medium.
Inoculation: Inoculate each tube with a standardized number of the test organism.
Incubation: Incubate the tubes under suitable conditions for the organism.
MIC Determination:
After incubation, observe the tubes for visible growth.
The MIC is the lowest concentration of the antimicrobial agent that inhibits visible growth.
MBC Determination:
From the tubes showing no visible growth, subculture aliquots onto fresh agar plates without the antimicrobial agent.
Incubate the plates.
The MBC is the lowest concentration that shows no growth on the agar plates.
Interpretation:
MIC indicates bacteriostatic activity (inhibition of growth).
MBC indicates bactericidal activity (killing of bacteria).
2. Agar Plate Method (Disk-Diffusion and E-test)
Disk-Diffusion Method:
Inoculation: Spread a standardized inoculum of the test organism on the surface of an agar plate.
Application of Disks: Place paper disks impregnated with the antimicrobial agent on the agar surface.
Incubation: Incubate the plate under suitable conditions.
Observation: Measure the diameter of the zone of inhibition around each disk.
E-test:
Inoculation: Spread a standardized inoculum of the test organism on the surface of an agar plate.
Application of Strips: Place an E-test strip with a gradient of the antimicrobial agent on the agar surface.
Incubation: Incubate the plate under suitable conditions.
Observation:
Read the MIC value at the point where the zone of inhibition intersects the strip.
Interpretation:
The size of the inhibition zone (disk-diffusion) or the MIC value (E-test) indicates the antimicrobial activity.
3. Cup-Plate Method
Purpose:
To evaluate the antimicrobial activity by measuring the inhibition zone.
Procedure:
Preparation: Pour a layer of agar medium into a Petri dish and allow it to solidify. Punch wells (cups) into the agar.
Inoculation: Add a standardized inoculum of the test organism to the agar surface.
Addition of Antimicrobial Agent: Fill the wells with the antimicrobial agent.
Incubation: Incubate the plate under suitable conditions.
Observation:
Measure the diameter of the zone of inhibition around each well.
Interpretation:
The size of the inhibition zone correlates with the antimicrobial activity.
4. Phenol Coefficient Method
Purpose:
To compare the efficacy of a test disinfectant with that of phenol.
Procedure:
Preparation: Prepare serial dilutions of the test disinfectant and phenol.
Inoculation: Inoculate each dilution with a standardized number of the test organism.
Contact Time: Allow a fixed contact time (e.g., 5 and 10 minutes).
Neutralization and Subculture: Neutralize the disinfectant action by subculturing aliquots into a fresh growth medium.
Observation:
Observe and record the highest dilution that kills the organism in 10 minutes but not in 5 minutes.
Calculation:
Calculate the phenol coefficient as the ratio of the highest effective dilution of the test disinfectant to that of phenol.
Interpretation:
A phenol coefficient greater than 1 indicates that the test disinfectant is more effective than phenol.
5. Kelsey-Sykes Test
Purpose:
To evaluate the efficacy of disinfectants under simulated practical conditions.
Procedure:
Preparation: Mix the test disinfectant with a suspension of the test organism.
Contact Time: Allow the mixture to stand for a specific contact time (e.g., 8 minutes).
Neutralization: At intervals, withdraw samples and add to a neutralizing medium.
Subculture: Subculture the neutralized samples into a fresh growth medium.
Incubation: Incubate the cultures.
Observation:
Observe for growth to determine the bactericidal action.
Interpretation:
The absence of growth indicates bactericidal activity, while the presence of growth indicates bacteriostatic activity or resistance.
Factors Affecting Evaluation
Inoculum Size: Larger bacterial populations may require higher antimicrobial concentrations.
pH and Temperature: Can influence antimicrobial efficacy.
Presence of Biofilms: Bacteria in biofilms are more resistant, affecting test outcomes.
Medium Composition: Nutrient availability and ions can impact bacterial susceptibility