microbiological assay of cyanocobalamin or vitamin B12

1.0 OBJECTIVE
To describe the procedure for determine the assay of Cyanocobalamin or vitamin b-12 by biological method using E. Coli mutant MTCC -452.
2.0 PURPOSE
It is the policy of (Analytical Division) that a written procedure shall be followed for determine the assay of Cyanocobalamin or vitamin b-12 by biological method using E. Coli mutant MTCC -452.

3.0 SCOPE
This SOP shall be applicable for in Microbiology Laboratory

4.0 RESPONSIBILITY
Persons along with their responsibilities are given below

Sr. No	Designation	Responsibility
01	Microbiologist	Preparation & revision of SOP.
02	Officer-QA	Training, Distribution and retrieval of SOP.
03	Quality Manager	Approval & Implementation of SOP.

5.0 PROCEDURE
5.1 Method : Cup plate method

5.2 Culture maintenance medium : B-12 culture agar (E.coli maintenance medium) M-185
5.3 B12 Assay Agar Himedia M 110
5.4 Assay Medium :

Sr. No	Ingredients	Quantity / 100 ml.
1	Dipotassium Phosphate	1.29 g
3	Anhydrous Citric acid A.R.	1.1 g
4	Sodium metabisulphate A.R	1.0 g
7	Distilled Water	100 ml

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. Sterilize at 15 lbs pressure for 20 minutes.
5.5 Inoculum medium : Buffer Peptone Water
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 4OC.
5.6 Preparation of Culture Suspension:
Streak the culture on B-12 maintenance agar slant (Himedia M 185) and on the day use inoculate loop full culture in 20 ml of peptone water and incubate 30-35 ºC for 3-4 hours.
5.7 preparation of assay plates:
Vitamin B12 assay medium (M110) is sterilized at 121ºC for 25 minutes ,and cooled to 40-45ºC, Add 3-4 ml of inoculum of E.coli mutant MTCC 452 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.
5.8 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.

5.9 Preparation of Standard Stock Solution :
Weigh accurately 25 mg of crystalline Cyanocobalmin powder (dark red coloured). Transfer it to 1000 ml volumetric flask and make up the volume to 1000 ml (A). Take the reading of this solution at 361 nm and calculate the conc. taking E 1 % as 0.207. Calculate exactly the conc. of vitamin B12/ ml .Take 2 ml and dilute it to 200 ml (A) with water. Take 2 ml (A) and dilute it to 20 ml (B). Take 10 ml (B) and dilute it to 20 ml(C) with water.

5.10 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.
6.0 Preparation of Standard Dilutions :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.
6.1 Protocol Of Standard Dilution:
25 mg—————————–1000 ml (Stock solution)
A) 2 ml(Stock solution)—————200 ml
B) 2 ml (A)———————————-20 ml (Standard Higher)
C) 10 ml (B)——————————–20 ml (Standard Lower)

6.2 Incubation of Plates :
Incubate the plates at 30-37°C for overnight (18-24 hours).
6.3 Measurement of Diameter :
Measure the diameter of every zone of growth from three different sides. Note down every reading.

6.4 Calculations :
Sum up three readings of individual dilution.
Formula –
Percent potency (%P) = Antilog (2 + a log I) Where,

(TH + TL) – (SH + SL)
a = ———————————–
(TH – TL) + (SH – SL)

6.5 Ratio of Dilution = I = 1 : 2
log 2 = 0.3010
Calculations :

6.6 Protocol of Dilutions :
A 25 mg ——> 1000 ml
B 2 ml(A) ——-> 200ml
SH 2ml (B) ——> 20 ml 0.25 mcg/ml
SL 10 ml (Stock) ———-> 20 ml 0.125 mcg/ml
7.0 ACCEPTANCE LIMIT: Not less than 90 percent.

8.0 ABBREVIATIONS & DEFINITION
QA Quality Assurance
SOP Standard Operating Procedure
QM Quality Microbiology
FR Format
S. No. Serial Number
ml Milliliter
mcg microgram
°C Degree centigrade