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

 For PDF Notes Click on Download Button

PPT File


Post a Comment

0 Comments