Microbiological Assay of Cyanocobalamin or Vitamin B12

Method : Agar cup method

Culture maintenance medium : Nutrient agar

Assay Medium :

Sr.No	Ingredients	Quantity / 100 ml.
1	Dipotassium Hydrogen Phosphate G.R	0.7 g
2	Potassium Dihydrogen Phosphate G.R	0.3 g
3	Sodium Citrate A.R.	0.05 g
4	Magnesium Sulphate A.R	0.01 g
5	Ammonium Sulphate A.R	0.1 g
6	Sodium Chloride A.R	0.005 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.  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 (A.R.) 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.

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. (*Addition from the inoculum suspension will depend upon culture activity that should be validated by the Microbiologist.)

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 Cyanocobalmin powder (dark red coloured). 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.

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.

Calculations :

Sum up three readings of individual dilution.

Formula -

Assay %  =  Antilog (2 ± a log I)

Where,

                 (TH + TL) - (SH + SL)

a     =  -----------------------------------

                 (TH - TL) + (SH - SL)

Ratio of Dilution = I = 1:10

    log I = 1

Calculations :

Actual % = Mean % x Ft x Fs

                 Sample to be taken           

Ft    =  --------------------------

                 Sample taken

               Std. to be taken

Fs    =  ---------------------

                 Std. taken

                         Actual %  x  Actual Content (Considering overages i.e. 2.5)

mcg / 5ml    =  ----------------------------------------------------------------

                                                                100

                             mcg / 5ml

 % of L.A.   =  ---------------------   X  100

                             L.A.[1 mcg]

Protocol of Dilutions :

A           10 mg   ------> 10 ml                           1 mg/ml.

B           1ml(A) -------> 100ml                         10 mcg/ml

Stock : 10ml (B) ------> 100 ml                         1mcg/ml

SH         5 ml (Stock) ----------> 50 ml             0.1mcg/ml

SL         5 ml (SH)      ----------> 50 ml             0.01mcg/ml

Test Dilutions :

TH      Sample eq. to 5 mcg ------> 50 ml               0.1mcg/ml

TL        5 ml (TH)   ----------------> 50 ml             0.01mcg/ml