Microbiological assays of Antibiotics and Vitamins

Microbiological assays of Antibiotics and Vitamins

Contents

•       Assay of antibiotics

–      Streptomycin

–      Penicillin

•       Assay of vitamins

–      Vitamin B12 (Cyanocobalamine)

–      Vitamin B2 (Riboflavin)

Learning objectives

At the end of this lecture, student will be able to:

•       Explain the principle of microbiological assay of antibiotics

•       Explain the principle of microbiological assay of vitamins

Assay of antibiotics

•       Method?

•       Both Method A (cup plate) and Method B (Turbidimetric) can be used

•       Method A – Depends on measurement of zone of inhibition

•       Method B – Depends on measurement of  turbidity

Assay of Streptomycin

Method	Method A (Cup plate method) Method B (Turbidimetric method)
Standard Stock solution Conc	Streptomycin sulphate - 1mg/ml
Solvent used for stock solution	Water
Median dose	1µg/ml (for cup plate method) 30 µg/ml  (for turbidimetric method
Test Organism	Bacillus subtilis
Test Media	Peptone Yeast extract Beef extract Agar pH after sterilization 7.8 to 8.0
Incubation temperature	32 - 35⁰C

 

Assay of Penicillin

Method	Method A (Cup plate method) Method B (Turbidimetric method)
Standard Stock solution Conc	Crystalline salt of Benzyl penicillin -1000 Units/ml
Solvent used for stock solution	Potassium phosphate buffer
Median dose	1 Unit/ml
Test Organism	Staphylococcus aureus
Test Media	Peptone Yeast extract Beef extract Agar pH after sterilization 6.6 ± 0.1
Incubation temperature	32 - 35⁰C

 

Microbiological assay of Vitamins

Principle

•       Vitamins and amino acids are essential for the growth of microorganisms.

•       The basis of this assay is to measure the ability of test organism to utilize the substance being assayed under a proper nutritional condition.

•       The organisms require these growth factors (vitamins & amino acids) in micro or nanograms.

•       The response (growth of test organism) is proportional to the dose (amount of factor) added to medium.

•       Nutrient medium will contain an abundance of all nutrients essential for growth of the organism except the test substance

•       On adding small amounts of the test substance to tubes, growth takes place

•       Growth depends on the quantity of test substance added

Microbiological assay of Cyanocobalamine

About Vit B₁₂:

•       Also known as cyanocobalamin

•       It is a water soluble vitamin

•       Structure is similar to that of heme where the iron is replaced with cobalt as a centre of molecule

•       Its main  sources are liver, eggs, milk, meat & fish

•       Vit B₁₂ deficiency causes Macrolyticanemia and Pernicious anemia.

•       National Research Council,USA recommends a daily in take of about 5mg of vitB₁₂

Principle of assay:

•       The test organism selected must be capable of utilizing free cyanocobalamin

•       Lactobacillus Liechmanni is found to satisfy the requirements

•       Gram negative bacilli, non-pathogenic, easy to culture & easily available

•       Isolated from milk, cheese, &  other dairy products

•       Assay is performed by using either titrimetric or turbidimetric method.

Method	Method B (Turbidimetric method) Or Titrimetric method
Standard Stock solution Conc	Standard cobalamine stock solution 0.01ng to 0.04ng per ml
Solvent used for stock solution	Water
Median dose	1 Unit/ml
Test Organism	Lactobacillus leichamannii
Test Media	Contains complex nutrients except vit B2
Incubation temperature	37⁰C

 

Microbiological assay of Riboflavin

Method	Method B (Turbidimetric method) Or Titrimetric method
Standard Stock solution Conc	Standard cobalamine stock solution 0.01ng to 0.04ng per ml
Solvent used for stock solution	Water
Median dose	1 Unit/ml
Test Organism	Lactobacillus casei
Test Media	Contains complex nutrients except vit B12
Incubation temperature	37⁰C

 

Titrimetric Method

Principle

•       Estimation of amounts of acid produced by Lactobacillus by titrimetric method

•       It is a much slower method compared to turbidimetric

•       It takes almost 3 days for the acids to build up

•       Titrimetric procedures have been largely replaced by turbidimetric procedure

Summary

•       Antibiotic assay – determines growth inhibitory property

•       Antibiotic assay can be carried out by cup plate or turbidometric method

•       Vitamin assay determines growth promoting ability of vitamins

•       Vitamin assay  is carried out by turbidometric method

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