microbiological assay of cyanocobalamin or vitamin B12

microbiological assay of cyanocobalamin or vitamin B12


To describe the procedure for determine the assay of Cyanocobalamin or vitamin b-12 by biological method using E. Coli mutant MTCC -452.
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.

This SOP shall be applicable for in Microbiology Laboratory

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.

5.1 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.
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.

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

sop for calibration and validation of micro autoclave

sop for Sterility failure investigation

cleaning and operation of discard autoclave

sop for operation of fogger machine

sop for Biological assay of lactic acid bacillus

sop for preparation of culture inoculum

STP for sterility testing of sterile gloves

sop for Operation and calibration of active air sampler

sop for transfer of material for testing and sampling in sterile area

entry & exit procedure in microbiology laboratory

Growth Promotion Test In Microbiology Laboratory

Operation of B.O.D in Microbiology Laboratory

Operation of Horizontal Laminar Air Flow in the microbiology laboratory

Operation and cleaning of Pass Box.

Operation and cleaning of air sampler

Cleaning and Sterilization of Glassware

Analysis of water for microbial load in microbiology lab

Operation and temperature monitoring of Refrigerator

Fumigation of Microbiology Laboratory.

Entry & Exit procedure In Sterility Area

SOP for Microbial analysis of Raw Material Finished Products

SOP for Operation & Calibration of pH meter in Micro Department

SOP for Operation & Calibration of pH meter in Micro Department

SOP Operation and calibration of Hot Air Oven In Microbiology

SOP for operation cleaning & calibration of Digital colony counter

SOP for Operation And Cleaning of Microscope

sop for Media Preparation and Consumption

sop for Receipt Storage and Usage of Culture Media

sop for Cleaning Sanitization And Disinfection In Microbiology

sop for Environmental monitoring of all the Classified area

sop for Handling and Sub culturing of Microbial cultures

sop for Media Growth Promotion Test and various Microbiological test

sop for BOD incubator operation and cleaning

sop sampling of water for microbiological analysis

sop for Disinfectant Efficacy Test

sop for for cleaning and operation of vortex mixture

sop for Temperature & Relative Humidity Monitoring

sop for Operation and Calibration of Heating Block

sop for Sterility Testing of Microbiology

sop for Disposal of Culture Media

sop for Drain point of Microbiology

sop for entry & exit procedure In Microbial limit test and Biosafety

sop for Gram Staining of Bacteria in Microbiology Laboratory

sop for Monitoring of Compressed Air/gases for microbiological analysis

sop for BET (Bacterial Endotoxin) test in Microbiology

sop for receipt storage and Determining the population of Biological indicators

sop for qualification of analyst microbiologist

sop for Bioburden test of Packing materials in Microbiology Laboratory

sop for microbiological assay of erythromycin antibiotic

sop for liquid particle counter

sop for operation and calibration of digital zone reader

sop for monitoring of ultraviolet efficiency LAF and pass box

microbiological assay of cyanocobalamin or vitamin B12

gowning procedure for microbiological testing area

swab testing of various surfaces for bioburden determination

sop for endotoxin challenge test

Hold time study protocol for sterilized media

sop for personnel Qualification protocol for aseptic area

sop for sampling and testing of drain water

Sop for Operation of Airborne Particle Counter

sop for Inoculum Preparation

sop for Validation protocol of steam sterilizer autoclave

sop for pathogen detection from drain point

Sop for Analysis of Raw water Purified water water for injection and pure steam water

sop for preservatives efficacy test

sop for collection and preservation of in house isolated microorganisms

sop for Operation Calibration and Maintenance of Micropipette

Sop for UV Efficacy Test

sop for gram staining

Sop for swab testing

sop for microbiological testing of water


sop for depyrogenation of apparatus

sop for media preparation

sop for fertility test growth promotion test of media

sop for Operation and cleaning of moist heat sterilizer

sop for monitoring by active air sampler

sop for swab sampling and testing for clean rooms in production area

sop for monitoring in microbiology laboratory

sop for Fumigation of aseptic area and microbiology lab

sop for monitoring of personnel in aseptic area

sop for maintenance of cultures

sop for Operation and cleaning of laminar bench

preparation of settle plates

sop for monitoring of pure steam

sop for entry and exit procedure to m.l.t and b.e.t room

sop for storage of and use of media

sop for disposal of microbiological media and cleaning of microbiological glassware

Leave a Comment

error: Content is protected !!