Introduction
Penicillin and streptomycin are important antibiotics used to treat bacterial infections. To ensure their potency and quality, microbiological assays are performed. These assays compare the inhibitory effect of the antibiotic sample with that of a standard antibiotic preparation having a known activity. The result helps determine whether the antibiotic is effective, stable and suitable for therapeutic use.
Principle of Microbiological Assay
The assay measures the ability of an antibiotic to inhibit the growth of a suitable microorganism. The zone of inhibition (in solid media) or reduction in turbidity (in liquid media) reflects the antibiotic concentration. Results from the sample are compared with standard concentrations to calculate potency.
Methods Used in Microbiological Assay
Two official methods are commonly used:
- Cylinder Plate Method (Cup Plate Method)
- Turbidimetric Method (Tube Assay Method)
Cylinder Plate (Cup Plate) Method
This method is based on diffusion of the antibiotic from a vertical cylinder into solid agar. The antibiotic diffuses through the agar and inhibits the test organism, forming a clear zone around the cylinder.
Procedure
- Prepare sterile Petri plates containing nutrient agar.
- Spread standardised inoculum of test organism uniformly over the agar surface.
- Using a sterile borer, make cylindrical wells in the agar.
- Add different concentrations of standard and test antibiotic solutions into each well.
- Incubate plates at 37°C for 24 hours.
- Measure the diameters of the inhibition zones.
The size of the zone is directly related to the antibiotic concentration. A standard curve is prepared to determine potency of the unknown sample.
Applications
- Highly suitable for penicillin and streptomycin.
- Provides clear, measurable zones.
- Useful for parallel or multiple sample testing.
Turbidimetric (Tube Assay) Method
This method depends on inhibition of microbial growth in a fluid medium. If the antibiotic is effective, the culture remains clear. If ineffective, the medium becomes turbid due to bacterial proliferation.
Principle
The turbidity of the solution is measured using a spectrophotometer. Lower turbidity indicates stronger antibiotic activity. By comparing absorbance values of test and standard solutions, potency is calculated.
Procedure
- Prepare five or more different concentrations of the standard antibiotic.
- Prepare similar dilutions of the test sample.
- Add the bacterial inoculum to tubes containing antibiotic solutions.
- Incubate at 37°C for 3–4 hours for growth to occur.
- Add dilute formaldehyde to stop growth.
- Measure absorbance at around 530 nm.
- Compare with standard curve to calculate potency.
Advantages
- Rapid results within a few hours.
- Economical and simple to perform.
- Suitable for liquid samples.
Preparation of Stock Solutions
Accurate preparation of stock solutions is essential for reliable assay results.
- Weigh required quantity of standard antibiotic.
- Dissolve in specified solvent.
- Prepare a concentrated stock solution.
- Store in a refrigerator and use within allowed time.
- On assay day, prepare serial dilutions (e.g., 1:1.25 stepwise for cup plate method).
- Ensure the middle dilution matches the recommended median concentration.
Test Microorganisms
Each antibiotic has a specific recommended test organism, usually from ATCC (American Type Culture Collection). The microorganisms are maintained on agar slants and sub-cultured weekly.
Examples
- Penicillin: Staphylococcus aureus (ATCC 29737)
- Streptomycin: Klebsiella pneumoniae (ATCC 10031)
Media for inoculum preparation must support optimum bacterial growth. pH adjustments are made using 1M HCl or 1M NaOH.
Important Notes on the Turbidimetric Method
- Common test tubes and an unmodified spectrophotometer can be used.
- Growth curves in replicate tubes remain uniform.
- Suitable for inexpensive and routine analysis.
- Works for liquid cultures and agar plug diffusion tests.
Factors Affecting Accuracy
- Correct preparation and storage of stock solution.
- Proper inoculum standardization.
- Use of freshly prepared media.
- Maintaining recommended pH and temperature.
- Precise incubation time.
Interpretation of Results
- Cylinder Plate Method: Larger zones = higher antibiotic potency.
- Turbidimetric Method: Lower absorbance = higher antibiotic activity.
- Comparison with the standard curve gives exact potency of the unknown sample.
Key Points
- Microbiological assay ensures antibiotic strength and stability.
- Penicillin uses S. aureus, while streptomycin uses K. pneumoniae.
- Cylinder plate and turbidimetric methods are official pharmacopeial procedures.
- Accurate stock preparation and standardized inoculum are essential for valid results.
Detailed Notes:
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