Principles and methods of different microbiological assay

Introduction to microbiological assay

• 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

• 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).

• 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.

• 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 of Microbiological assay

• 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)

• 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.

• 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 of Microbiological assay

• • 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 of Microbiological assay

• • 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 mixtures.

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