1. MICROBIOLOGICAL ASSAY OF
VITAMIN B2 AND B12
G.Pavani

2. •
Microbiological assay of vitamins is a type of
biological assay performed with the aid of
microorganisms.
Many therapeutic agents, which either inhibit the
growth of microorganisms or are essential for their
growth are standardized by microbial assay.
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3. WHAT IS THE UNDERLYING PRINCIPLE?
• 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 response (growth of test organism) is
proportional to the dose (amount of factor)
added to medium.

4. HOW DO WE DO IT ?
The Indian pharmacopoeia gives two methods :
• The cylinder-plate (cup-plate) method
• The turbidimetric (tube assay) method

5. ASSAY OF VITAMIN B12

6. A
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QUICK REVIEW OF VIT. B12
Also known as cyanocobalamin. It’s a water soluble
vitamin.
• Its main sources are liver, eggs, milk, meat & fish.
VitB12 deficiency causes Macrolyticanemia,
pernicious anemia.
National Research Council, USA recommends a
daily intake of about 5mg of vit B12
is
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7. Principle:
Microbial assay of Vit-B12 is based on comparison
of growth of micro organism test .conc of vit-B12 is
examined with known conc of standard. Assay of
vit-B12 is based on turbidometric method.

8. PREREQUISITES FOR THE ASSAY:
•Test organism: LACTOBACILLUS LEICHMANNII
It is easily available, non pathogenic and easily
culturable
Isolated from milk, cheese and other dairy
products
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9. PRECAUTIONS
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Great care must be taken to avoid contamination
All the glasswares must be free from detergents
and other chemicals.
Glasswares must be heated to 2500C for at least 1
hr before use.
The whole experiment must be carried out under
proper aseptic condition
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10. REAGENTS/ Solutions
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Standard Vit-B12 stock solution
A solution of cyanocobalamin of concentration 1.0 microgram per ml is
made using 25% ethanol. dilute stock solution to prepare a solution of
conc. 0.01-0.04 microgram/ml . Prepare freshly.
Test Solution
Accurate amount of material to be assayed is taken & dissolved in water,
Dil HCl or NaOH is added to adjust ph at 6.0.
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11. Sterile SUSPENSION MEDIUM
Prepare a 100ml solution by mixing equal volumes of BASAL
MEDIUM STOCK SOLUTION and distilled water.
After preparation, b0th the medium is kept for sterilisation
for 15min @ 121 deg centigrade by autoclave.

13. PREPARATION OF INOCULUM
Preparation of suspension
1. Transfer a loop full of Lactobacillus liechmannii from a recent/freshly
subculture into two tubes (T1 & T2) each containing 10ml of sterile
culture medium.
2. Incubate the two tubes (T1 & T2) for 18-24hrs @ 37Centi grade. After
incubation centrifuge the tube T1.
3. To this test tube T1 add 10ml of sterile suspension medium to make a
suspension of the cells that settle down. Centrifuge it.
4.once again to this test tubes add 10ml of sterile suspension medium to
make a suspension of the cells that settle down. Centrifuge it.
Preparation of inoculum
5. Now aseptically transfer 1ml of the above prepared suspension to 10ml
sterile suspension medium. This suspension is used as inoculum.

14. WHAT IS THE PROCEDURE?
Assay of vitamin B12 can be carried out
methods
by two
1.
2.
Titrimetric method
Turbidimetric method

15. Preparation of Calibration and test solutions:

16. Titrimetric method:

17. INTERPRETATION OF RESULTS FOR TITRIMETRY
(con-mcg.ml Vs Volume of titrant).

18. Turbidimetric method:
After incubation Measure the turbidity of all these
solutions at 540nm using spectrophotometer.
Calculate the conc of test solution using calibration
curve graph (con-mcg.ml Vs Absorbance).

19. ASSAY OF VITAMIN B2

20. •
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VITAMINB2- riboflavin
Also known as riboflavin
Dietary sources include yeast, liver, beef, eggs,
whole-grain products, peas, beet, peanuts, etc
Deficiency may lead to blurred vision, dermatitis
and anemia.

21. PREREQUISITES
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LACTOBACILLUS CASEI
Basal medium
Stock solutions
Inoculum
is used for the assay

22. BASAL MEDIUM (RIBOFLAVIN FREE)

23. RIBOFLAVIN SOLUTIONS

24. INOCULUM
• Into a 10 ml centrifuge tube add 5 ml basal
medium and 5 ml riboflavin solution (0.2
microgram per ml) so that it would
microgram of riboflavin
Sterilize at 121 deg centigrade for 15min.
contain 1
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Add LACTOBACILLUS CASAEI
Incubate at 37oC for 24 hrs
to it
Centrifuge it and decant off the supernatant
liquid

25. •
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Suspend the bacterial cells in 10 ml sterile saline
Centrifuge again and suspend the bacterial cells
in 10 ml sterile saline
Inoculum is ready•

26. PROCEDURE
• Fill 7 test tubes with 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0
ml of standard riboflavin solution
Make up the volume in each test tube up to 5 ml
using distilled water
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• Add 5 ml of riboflavin
each of he test tubes
Sterilize
free basal medium in
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27. • Take another 5 test tubes and fill them with 0.5,
1.0, 1.5, 2.0, 2.5 ml of the test sample
Make up the volume in each test tube up to 5 ml
using distilled water
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• Add 5 ml of riboflavin
each of he test tubes
Sterilize
free basal medium in
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28. • Cool all the test tubes to room temperature and
then inoculate each test tube with a drop (0.6
ml) of inoculum
Incubate at 37oC for 72 hrs
Titrate the contents of each test tube separately
against 0.1N NaOH to pH 6.8
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29. Turbidity metric method
The B vitamin riboflavin can be assayed by UV/VIS
spectrophotometer at 440 nm.

30. ALTERNATIVELY....
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Even LACTOBACILLUS RHAMNOSUS is used
Only things that change are composition of basal
medium and concentration of riboflavin solution
used
The most effective assay range is between
0.025-0.15 microgram per 10 ml
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31. THANKYOU