Introduction
Microbiological assays are analytical methods used to measure the concentration or potency of a substance by its effect on microorganisms.
These assays are pivotal in the pharmaceutical industry for the standardization of antibiotics, vitamins, amino acids, and for the assessment of new antibiotics.
They rely on the response of microorganisms to specific substances, allowing quantification based on microbial growth inhibition or stimulation.
Principles of Microbiological Assays
1) Biological Activity Measurement
Basis: Microbiological assays measure the biological activity of a substance rather than its chemical concentration.
Response Relationship: They rely on a linear relationship between the logarithm of the concentration of the substance and the biological response (e.g., inhibition zone size, turbidity).
2) Use of Test Organisms
Selection: Specific microorganisms are chosen based on their sensitivity to the substance being assayed.
Standardization: Test organisms must be of a standardized strain and in optimal physiological state.
3) Quantitative Analysis
Comparison with Standards: The activity of the test sample is compared against a reference standard of known potency.
Dose-Response Curve: Constructed to establish the relationship between concentration and response.
Methods of Different Microbiological Assays
Microbiological assays can be broadly classified into:
Diffusion Methods
Turbidimetric (Tube) Methods
Bioautographic Methods
1) Diffusion Methods
These methods involve the diffusion of an antibiotic from a reservoir through a solid or semi-solid medium to inhibit the growth of microorganisms.
A. Cylinder-Plate Method (Cup-Plate Method)
a) Principle:
Antibiotic diffuses from a cylindrical cup into the agar medium inoculated with a microorganism.
Procedure:
Prepare agar plates inoculated with the test organism.
Place stainless steel cylinders or cups on the agar surface.
Fill cups with known concentrations of standard and test solutions.
Incubate plates under suitable conditions.
Measure the zones of inhibition around each cup.
c) Application:
Used for antibiotics like penicillin.
B. Paper Disk Diffusion Method
Principle:
Similar to the cylinder-plate method but uses paper disks impregnated with the antibiotic.
Procedure:
Place paper disks soaked with standard and test solutions on the inoculated agar surface.
Incubate and measure inhibition zones.
Application:
Commonly used for antibiotic susceptibility testing.
Advantages of Diffusion Methods
Simple and inexpensive.
Suitable for substances that diffuse well in agar.
Limitations
Diffusion rate may vary with molecular size and agar properties.
Less precise than turbidimetric methods.
2) Turbidimetric (Tube) Methods
Principle
Based on the inhibition of microbial growth in a liquid medium containing the test substance.
The extent of inhibition is measured by the reduction in turbidity compared to controls.
Procedure
Prepare a series of tubes with varying concentrations of the test and standard solutions.
Inoculate each tube with a standardized microbial suspension.
Incubate under optimal conditions.
Measure turbidity using a spectrophotometer or nephelometer.
Advantages
Rapid and more precise than diffusion methods.
Suitable for substances that do not diffuse well in agar.
Limitations
Turbidity measurements can be affected by the presence of precipitates or colored substances.
Requires careful standardization of inoculum and incubation conditions.
3) Bioautographic Methods
Principle
Combines chromatography and microbiological assay.
Used to identify and quantify antimicrobial substances in complex mixtures.
Procedure
Separate the components of a mixture using chromatography (e.g., TLC).
Transfer the chromatogram onto an agar plate inoculated with a test organism.
Incubate and observe zones of inhibition corresponding to antimicrobial substances.
Application
Screening natural products for antibiotic activity.
Identifying active components in complex mixture.