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Ms Media PDF

The document describes how to prepare stock solutions that are used to make tissue culture media such as Murashige and Skoog (MS) medium. Concentrated stock solutions of minerals, vitamins, and growth regulators are prepared by dissolving precise amounts of compounds in solution at high concentrations. These stock solutions can then be diluted in precise volumes to prepare the desired amount and formulation of tissue culture medium. The document provides examples of calculating the amounts needed for stock solutions of various compounds as well as calculating the volumes of stock solutions required to prepare a given volume and formulation of tissue culture medium.

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309 views7 pages

Ms Media PDF

The document describes how to prepare stock solutions that are used to make tissue culture media such as Murashige and Skoog (MS) medium. Concentrated stock solutions of minerals, vitamins, and growth regulators are prepared by dissolving precise amounts of compounds in solution at high concentrations. These stock solutions can then be diluted in precise volumes to prepare the desired amount and formulation of tissue culture medium. The document provides examples of calculating the amounts needed for stock solutions of various compounds as well as calculating the volumes of stock solutions required to prepare a given volume and formulation of tissue culture medium.

Uploaded by

tin
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Media preparation

Tissue culture medium consists of many items that are classified into micro- and
macroelements, carbon source, vitamins, amino acids, growth regulators and undefined
supplements. Many formulations are now available for minerals and vitamins of which
Murashige and Skoog (1962) medium (MS medium) is the most commonly used medium in
tissue culture labs. To avoid weighing and dissolving each of these items whenever we are
going to prepare tissue culture medium, most of these items are prepared as separate
concentrated stock solutions that are mixed and diluted with water before use.

How to prepare and use stock solution


To Prepare A ml stock soln (B X)
Dissolve (Concn in medium (mg/ml) X A X B) mg In total volume A ml.

Ex1: 50 ml Magnesium Sulfate Stock Solution (100X)

Each liter of MS medium contains 370 mg MgSO4 .7 H2O


i.e. each ml medium contains 370 / 1000 = 0.37 mg
Dissolve (0.37 X 50 X 100) = 1850 mg in total volume 50 ml.

The volume (V ml) required from this stock to prepare V’ ml Ms medium is calculated
according to the following equation:
NV=N’V’
N is the concentration (expressed in any unit i.e X, mg/L or mg/ml) of MgSO4.7 H2O in
stock.
N’ is the required concentration (expressed in the same unit used with N) of MgSO4.7 H2O
in medium.

1
V is the volume (expressed in L or ml) of MgSO4 .7 H2O stock required.
V’ is the volume (expressed in the same unit used with V) of medium to be prepared.

If we want to prepare 1000 ml medium we need


V ml of stock = (N’V’)/N = (0.37 X 1000) / 37 = 10.0 ml

Ex2: 100 ml Vitamins (Thiamine, Pyridoxine and Nicotinic Acid) Stock Solution (1000X)
Thiamine
Each liter MS medium contains 0.1 mg
i.e. each ml medium contains 0.0001 mg.
Wt required for stock = 0.0001 X 100 X 1000 = 10 mg
Pyridoxine
Each liter MS medium contains 0.5 mg
i.e. each ml medium contains 0.0005 mg.
Wt required for stock = 0.0005 X 100 X 1000 = 50 mg
Nicotinic Acid
Each liter MS medium contains 0.5 mg
i.e. each ml medium contains 0.0005 mg.
Wt required for stock = 0.0005 X 100 X 1000 = 50 mg

Dissolve 10 mg Thiamine, 50 mg Pyridoxine and 50 mg Nicotinic Acid in 100 ml total


volume.

If we want to prepare 1000 ml medium we need


Based on thiamine
V ml of stock = (N’V’)/N = (0.0001 X 1000)/0.1 = 1.0 ml

2
Minerals, either alone or along with some vitamins, are available as powder or liquid from
which certain amount are dissolved or diluted with water to prepare any amount of
medium.

Stock Solutions for Growth Regulators


1 mg/ml = 1000 mg/L
X mg in X ml total solution

1 µM/ml (1µM = Mol Wt / 1 000 000)


X µM in X ml total solution

How to Use Stock Solutions for Growth Regulators

N V = N’ V’

Ex: Preparation of 1 liter medium containing 4.5 mg/L BA. Using stock solution (1 mg/ml):
Each liter of medium contains 4.5 mg
i.e. each ml medium contains 4.5 / 1000 = 0.0045 mg
Concn. of medium = 4.5 mg/L OR 0.0045 mg/ml

Each liter stock contains 1000 mg


i.e. each ml stock contains 1000 / 1000 = 1 mg
Concn. of stock = 1000 mg/L OR 1 mg/ml

V ml of stock = (N’V’)/N = (0.0045 X 1000)/1 = 4.5 ml

3
Preparation of 250 ml MS medium
The medium consists of inorganic salts and vitamins of MS medium, 30 g/L sucrose, 100
mg/L myo-inositol and 8 g/L agar Using:
1. powdered MS medium (salts and vitamins) 4.4 g/L
2. Sucrose
3. myo-inositol stock solution (10 mg/ml).
4. Agar
The pH is 5.8
In 250 ml clean beaker, put 100 ml distilled water and dissolve:
1.1 g powdered MS medium
7.5 g sucrose
2.5 ml myo-inositol stock solution (10 mg/ml)
Up to 225 ml with distilled water
Adjust pH to 5.8
Up to 250 ml with distilled water
Add 2 g Agar and boil with continuous stirring till disappearance of Agar
Divide into 5 jars
Autoclave for 20 minutes at 121 oC.

Preparation of 250 ml MS medium supplemented with 1 mg/L 2,4-D


The medium consists of inorganic salts and vitamins of MS medium, 30 g/L sucrose, 100
mg/L myo-inositol, 1 mg/L 2,4-D and 8 g/L agar Using:
1. powdered MS medium (salts and vitamins) 4.4 g/L
2. Sucrose
3. myo-inositol stock solution (10 mg/ml).
4. 2,4-D (1.0 mg/ml).
5. Agar
The pH is 5.8
4
Preparation of 250 ml MS medium supplemented with 4.5 mg/L BA
The medium consists of inorganic salts and vitamins of MS medium, 30 g/L sucrose, 100
mg/L myo-inositol, 4.5 mg/L BA and 8 g/L agar Using:
6. powdered MS medium (salts and vitamins) 4.4 g/L
7. Sucrose
8. myo-inositol stock solution (10 mg/ml).
9. BA (1.0 mg/ml).
10. Agar
The pH is 5.8

5
6
Q1: MS medium contains 332.2 mg/L CaCl2 . 2H2O and 1900 mg/L KNO3
Describe how to make 250 ml stock solution (50 X) containing both substances.

Calculate the volume required from this stock to prepare 750 ml MS medium.

Calculate the volume required from this stock to prepare 750 ml B5 medium.

Q2: You are provided with:


1. powdered MS medium (salts and vitamins)
2. Sucrose
3. myo-inositol stock solution (10 mg/ml).
4. BA (1 mg/ml)
5. IAA (1 mg/ml)
6. Agar
Describe preparation of 250 ml MS medium (salts and vitamins) supplemented 30 g/L
sucrose, 100 mg/L myo-inositol, BA (2, 4 or 6 mg/L either alone or in combination with
0.1 mg/L IAA) and 8 g/L agar. The pH is 5.8.

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