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
No comments:
Post a Comment
Please don't spam. Comments having links would not be published.