Microbiological assay of vitamins is a biological assay method performed with the help of microorganisms. Vitamins and amino acids are the essential components for the growth of microorganisms. The basis of this assay is to measure the ability of the test organism to utilize the substance being assayed under a proper nutritional condition. Some examples of microorganisms used for bioassay of vitamins are listed in Table.
Table: Microorganisms for vitamin bioassay
|
Microorganism |
Vitamin |
|
Lactobacillus
casei |
Biotin, Folic
acid, riboflavin |
|
L.
leichmannii |
Cyanocobalamin |
|
L. arabinosus |
Nicotinic
acid |
|
L. viridans |
Thiamine |
Materials required: A stock solution, inoculum media,
and assay medium are required. Finally, a standard curve is obtained.
Assay of Vitamin B12: Vitamin B12 is also known as
cyanocobalamin. It is a water-soluble vitamin. Its main sources are liver,
eggs, milk, meat, and fish. Vitamin B12 deficiency causes Macrolytic anemia and
pernicious anemia.
Principle: The test organism selected is
Lactobacillus liechmannii which is capable of utilizing free cyanocobalamin. The
assay is performed by using either the titrimetric or turbidimetric method.
Preparation of Standard Stock Solution: An accurately
weighed amount of Cyanocobalamin reference standard is added to sufficient 25%
ethanol (resulting in a solution containing 1.0 µ g of cyanocobalamin per ml)
and stored in the refrigerator. Further dilutions of this stock solution (1 µg/
ml) are made by adding 1 ml stock solution to 99 ml purified water (1 ml = 10
ng) and further adding 1 ml of the above solution to 199 ml purified water (1
ml = 0.05 ng).
Test Solution: Accurate amount of vitamin to be
assayed is taken and dissolved in water, dilute HCl or NaOH is added to adjust
pH at 6.0 and make up the volume with water up to the mark.
Inoculum Preparation: Transfer a loop full of
Lactobacillus liechmannii from a recent sub-culture into two tubes each
containing 10 ml of sterile culture medium.
Composition of culture media:
Yeast extract - 0.75 gm
Peptone - 0.75 gm
Dextrose - 1 gm
Potassium dihydrogen phosphate - 0.2 gm
Tomato juice filtrate - 10 ml
Sorbitan monooleate solution - 1 ml
Water up to - 100 ml
Media is incubated for 18 to 24 hours at 37oC.
The culture is centrifuged and the supernatant fluid is under aseptic
condition. These cultured cells are suspended into 10 ml of sterile suspension
of Basal medium stock solution and again centrifuged and decanted off
supernatant fluid. The cells are uniformly suspended in 10 ml of sterile medium
and then aseptically transferred 1 ml of the cell suspension to 10 ml of
sterile medium and mixed. Finally, this resulting cell suspension is taken as
inoculum.
Assay Procedure: In clean ten test tubes, add 0, 0.5,
1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5 ml of standard cyanocobalamin
solution. To each tube, 5 ml of Basal medium solution and the volume of each is
adjusted to 10 ml by water. In another four test tubes add 1, 2, 3, and 4 ml of
test solution which is to be assayed. To each of this also added 5 ml of Basal
medium stock solution and adjusted volume to 10 ml with water. All test tubes were
sterilized in an autoclave at 121oC for 15 minutes after that cooled
the test tubes were at room temperature. Inoculate a drop of inoculum prepared
of Lactobacillus liechmannii and incubate the test tubes for 64 to 72 hours at a
temperature range of 30 to 37oC. After the incubation period
titrated contents of each test tube with 0.05 N NaOH using bromothymol blue as an
indicator until a green color endpoint. Calculate the readings.
The average titration values of each level of both standard
and test solutions are determined and plotted on a graph considering average
titration values (in ml) of 0.05 N NaOH against the concentration of standard
cyanocobalamin solution. A linear graph is obtained by interpolating the
standard curve to determine the concentration as activity per ml of vitamin
B12. From the graph, the concentration of the test solution of cyanocobalamin
is calculated (Fig.).
Fig.: Standard curve for Vitamin B12
Assay of Calcium Pantothenate: It refers to the B
complex vitamins (or vitamin B complex).
Standardized Stock Solution: Each ml of the stock
solution consists of 50 mcg of calcium pantothenate. It is prepared by
dissolving 50 mg of calcium pantothenate in 500 ml of double-distilled water as
per British Pharmacopeia Chemical Reference Standard; 10 ml of 0.2 M acetic
acid, 100 ml of 1.6% (w/v) sodium acetate; and volume made upto 1 liter with
distilled water.
Standard Solution: The standard solution should
contain approximately 0.04 mcg of calcium pantothenate in 1 ml, and is duly
prepared by diluting the Standard Stock Solution.
Test Solution: The test solution contains nearly the
same equivalent amount of calcium pantothenate as present in the Standard
Solution i.e., 0.4 mcg/ml prepared in double-distilled water.
Culture Medium: The culture medium is composed of the
following solutions and ingredients:
(i) Casein hydrolysate solution: 25 ml
(ii) Cysteine-tryptophane solution: 25 ml
(iii) Polysorbate-80 solution: 0.25 ml
(iv) Dextrose (anhydrous): 10 g
(v) Sodium acetate (anhydrous): 5 g
(vi) Adenine-guanine-uracil solution: 5 ml
(vii) Riboflavin-Thiamine hydrochloride-Biotin Solution: 5
ml
(viii) PABA-Niacin-Pyridoxine hydrochloride solution: 5 ml
(ix) Calcium pantothenate solution A: 5 ml
(x) Calcium pantothenate solution B: 5 ml
The culture medium is usually prepared by dissolving both
anhydrous dextrose and sodium acetate in previously mixed solutions and the pH
is carefully adjusted to 6.8 with 1 M NaOH solution. The final volume is duly
made up to 250 ml with distilled water and mixed thoroughly.
Stock Culture of Organism: The stock culture of the organism
may be prepared by dissolving 2 g water-soluble yeast extract in 100 ml
distilled water, 500 mg anhydrous dextrose, 500 mg anhydrous sodium acetate,
and 1.5 g agar. The resulting mixture is heated gently so as to dissolve the
agar. Now, 10 ml of hot solution is transferred to test tubes and sterilized at
121°C. The stab culture is now prepared duly in three tubes employing
Lactobacillus plantarum, incubated at 30 to 37°C for 16 to 24 hours, and stored
in a refrigerator.
Preparation of Inoculum: The cells consequently
obtained from the stock culture, organism (Lactobacillus Plantarum) are
transferred to a sterile tube containing 10 ml of the culture medium and incubated
at 30 to 37°C for a duration of 16–24 hours. Methodology. The various steps
involved are namely:
(i) Standard Solution is added to five test tubes in varying
amounts viz., 1, 2, 3, 4, and 5 ml in duplicate.
(ii) To each of the five above test tubes plus another four
similar tubes without any standard solution, 5 ml of culture medium, and the
final volume made up to 10 ml with distilled water.
(iii) Now, volumes of test solution corresponding to either
three or more of the levels as taken above, are incorporated carefully to
similar test tubes, in duplicate.
(iv) To each test tube 5 ml of the medium solution and
volume is made up to 10 ml with distilled water.
(v) Tubes of both the series are duly heated in an autoclave
at 121°C for 5 minutes, cooled to ambient temperature, and added 1 drop of
inoculum to each tube except two of the four tubes that specifically have no
‘standard solution’ (i.e., the uninoculated tubes), and mixed thoroughly. The
tubes are adequately incubated at 121°C at 30–37°C for 16– 24 hours.
(vi) Absorbance of the various tubes is measured with a
spectrophotometer at a wavelength ranging between 540–660 nm. A standard
concentration-response curve is plotted between the transmittance Vs log ml
(volume) of the standard solution in each tube. Finally, the exact
concentration of the calcium pantothenate in the ‘test sample’ is determined
with the help of the standard concentration-response curve.