This document describes microbiological assays for Vitamin B6 and Vitamin B12. For the Vitamin B12 assay, it uses E. coli M200 and measures zone diameters to determine Vitamin B12 concentrations. Standard solutions of cyanocobalamin are prepared and tested alongside sample solutions. The Vitamin B6 assay uses Kloeckera brevis yeast and measures zone diameters from standard pyridoxine solutions and processed sample solutions to generate a standard curve and determine Vitamin B6 content. Procedures for culture maintenance, inoculum preparation, sample digestion, and running the cup plate assay are provided.

1. Microbiological
assay of Vitamin
B₆
and Vitamin B₁₂
GAGANDEEP
M. Pharm
Pharmaceutical Analysis

2. Vitamins
– A vitamin is an organic molecule (or related set of molecules) which is
an essential micronutrient—that is, a substance which an organism needs in
small quantities for the proper functioning of its metabolism but cannot
synthesize, either at all or in sufficient quantities, and therefore must obtain
through its diet.
– The 13 vitamins required by humans are: Vitamin A, B₁, B₂,B₃, B₅, B₆, B₇, B₉, B₁₂,
C, D, E and K.

3. B Vitamins
– B vitamins are a class of water-soluble vitamins that play important roles
in cell metabolism. Though these vitamins share similar names, they
are chemically distinct compounds which often coexist in the same foods. In
general, dietary supplements containing all eight are referred to as a vitamin B
complex.
– Each B vitamin is either a cofactor (generally a coenzyme) for key metabolic
processes or is a precursor needed to make one.

4. List of B Vitamins
Vitamin B Name
Vitamin B₁ Thiamine
Vitamin B₂ Riboflavin
Vitamin B₃ Niacin
Vitamin B₅ Pantothenic Acid
Vitamin B₆ Pyridoxal, Pyridoxine and Pyridoxamine
Vitamin B₇ Biotin
Vitamin B₉ Folate
Vitamin B₁₂ Cyanocobalamin or Methylcobalamin

5. Microbiological assay of Vitamin
B₁₂
Organism used: E. coli M200
Method used: Agar plate method
Culture maintenance medium : Nutrient agar
Sr. no. Component Amount (per 100mL)
1 Dipotassium hydrogen phosphate 0.7g
2 Potassium dihydrogen phosphate 0.3g
3 Sodium citrate 0.05g
4 Magnesium sulfate 0.01g
5 Ammonium sulfate 0.1g
6 Sodium chloride 0.005g
7 Distilled water 100mL

6. Dissolve the ingredients separately to avoid precipitation & shake and mix the solution. If
necessary, adjust the pH of the solution to 7.0 ± 0.1. Add 1.5 gm
of agar powder and sterilize at 15 lbs pressure for 20 minutes.
Dextrose Solution : Prepare 40% w/v solution of Dextrose using distilled water. Sterilize at 15 lbs
pressure for 15 minutes. Cool and store in the refrigerator.
Inoculum medium :
Nutrient broth – 1.3 gm
Distilled water - 100 ml
Adjust the pH of the solution to 7.0 ± 0.1. Dispense in screw capped test tubes and autoclave at
15 lbs pressure for 15 minutes. Cool & store at 4°C.
Preparation of Seeding Inoculum :
Using a bacteriological loop inoculate a test tube of inoculum medium or NB with growth from a
fresh agar slope & incubate at 37°C. for 16-18 hours. Store
at 4°C. Centrifuge required amount of inoculum medium for 20 min at 2200rpm & Suspend the
cells in 5 ml of sterile saline centrifuge for 10 min at 2200
rpm and decant the supernatant. Repeat the process. Finally, suspend the cells in 1 ml of sterile
saline and mix well and use it as inoculum.

7. Preparation of Assay Plates :
To 100 ml of sterile and cooled at 45°C Vitamin B12 assay medium, add 1.0 ml of 40% sterile
dextrose solution and shake well. Add 1 ml of inoculum of
E.coli M200 of required thickness. Mix gently but thoroughly. Distribute 25 to 30 ml of the
inoculated medium in sterile petri plates. Allow to set the medium
& store in refrigerator until use. Plates should be used on the same day or within one day.
Preparation of Agar Cups :
A standard 8.0 mm diameter borer is taken. Dip the borer in Isopropyl alcohol and burn the
remaining Isopropyl alcohol from borer on flame, cool the borer
properly and bore cups in preseeded agar plates. Bore four cups per plates.
Preparation of Standard Stock Solution :
Weigh accurately 10 mg of crystalline Cyanocobalamin powder (dark red colored). Transfer it to 10
ml volumetric flask and make up the volume to 10 ml.
Take 1 ml and dilute it to 100 ml with water. Take the reading of this solution at 361 nm and calculate
the conc. taking E1 % as 0.207. Calculate exactly the
conc. of vitamin B12/ml.
Calculation :
O.D. at 361
-------------- X 100 = X mcg/ml.
0.207
Accordingly calculate X ml [about 10 ml of this solution] dilute to 100 ml to get [1 mcg/ml]. This is
one mcg /ml stock, use within one month.

8. Preparation of Standard Dilutions :
Take 5 ml of Std. stock solution & make up the volume to 50 ml, treat it as Standard High
(SH).Take 5 ml. of Std. High dilution and make up the volume to
50 ml. Treat it as Standard Low (SL).
Preparation of Test Dilution :
Weigh and transfer a sample quantity eq. to 5 mcg. to 50 ml volumetric flask. Add distilled water,
shake vigorously and dilute to 50 ml with distilled
Water(TH). Further dilute 5 ml of the TH to 50 ml with distilled water[TL]. Application of standard
and Test Dilutions : With the help sterile micropipette tips
apply 100 μl of different dilution to different cups. Mark every cup with proper dilution and keep
the plates at low temperature (around 10°C) for 10-20
minutes for diffusion.
Incubation of Plates : Incubate the plates at 30-37°C for overnight (18-24 hours).
Measurement of Diameter :
Measure the diameter of every zone of growth from three different sides. Note down every
reading.

9. Microbiological Assay of the
Vitamin B₆ Complex
Organism used: Kloeckera brevis
Method used: Cup Plate Agar method
Composition of Stock medium used:
pH Conditions : 4.8 to 5.0 by Bromocresol Green
Storage temperature: 4°C
Component Amount
Malt Extract (10%) 100ml
Difco Yeast Extract 0.2g
Glucose 2g
Pyridoxine HCl 1mg

10. Composition of Basal Medium used:
Component Amount
Charcoal treated 10%
malt extract
20.0mL
L-asparaginase 1g
Ammonium Sulfate 1g
Pot. Dihydrogen
phosphate
0.6g
Tripotassium citrate 5.0g
Citric acid 1g
Pot. Chloride 0.5g
Calcium chloride 0.2g
L-arginine HCl 20mg
L-histidine HCL 10mg
DL-isoleucine 20mg
Component Amount
L-lysine HCl 20mg
DL-methionine 20mg
DL-tryptophan 20mg
DL-valine 20mg
Niacin 5mg
Calcium pantothenate 5mg
Thiamine HCl 5mg
Biotin 40μg
Inositol 30mg
Inorganic salt solution 6.3mL
Water Upto 100mL

11. Assay Procedure:
Standard curve is plotted b y taking concentrations of Pyridoxine as:
 0.000 μg/mL
 0.001 μg/mL
 0.002 μg/mL
 0.004 μg/mL
 0.006 μg/mL
 0.008 μg/mL

12. Inoculum preparation:
Take 1mL liquid stock culture
Add 10mL stock culture media
Incubate at 25-30°C for 24 hours
Maintain culture at 4°C then.

13. Sample preparation:
Take 2-5 g of sample
Add upto 50mL of 0.05N HCl
Add Bromocresol green as indicator
Add 0.3g of Takadiastase enzyme
Maintain final conc between 0.001-0.002μg/mL of Pyridoxine

14. References
– Microbiological Assay of Cyanocobalamin or Vitamin B12 : Pharmaceutical
Guidelines. (www.pharmaguidelines.com – assessed on 3/9/2018)
– E. C. Barton Wright, The Microbiological Assay of the Vitamin 336 Complex
(Pyridoxine, Pyridoxal and Pyridoxamine) with Kloeckera Brevis , ANALYST, April,
1971, Vol. 96, Page no. 314-318.