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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