A P P E N D I X -V I I I

COMPOSITION OF USED MEDIA AND CHEMICAL REAGENTS

1. NITROGEN FREE BROMOTHYMOL BLUE (NFB) MEDIUM
Dobereiner et al (1976)
Same media was also used to check the effect of temperature and pH.
Semi solid medium
Ingredients G/LITRE
Malic acid 5.0 g
KOH - 4.0 g
K2HPO4 0.5 g
FeS0 4 7H20 0.05 g
M nS0 4 7H20 0.01 g
MgS047H20 - 0.01 g
NaCl 0.02 g
CaCl2 0.01 g
Na2M o0 4 0.002 g
D/W 1000 ml
Bromothymol blue 2.0 ml
0.5% alcoholic solution
Agar Agar 1.75 g
pH adjusted to 6.6 to 7.0
For solid NFB medium
Semi solid NFB medium
Agar 2%
Yeast extract 0.005 %

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2. BMS AGAR (POTATO INFUSION AGAR MEDIUM)
Components gms/litre
Potatoes 200 g
Mallic acid 2.5
KOH 2.0
Cane sugar 2.5
Biotine 2.1
Cook washed potatoes and filter through cotton. Prepare potassium malate by
dissolving 2.5g malate in 50ml water adding 2 drops of Bromothymol blue (0.5%
ethanol) and 2 g of KOH. Adjust pH to green colour. Add malate, sugar, biotine to
potato filtrate and dilute to 1000ml. Solid media is obtained by adding 2% agar-
agar.
3. R. C. MEDIUM *
•

Azospirillum cultures were isolated (Red, Scarlet colony) on the Roderquez

Caceras (1982) Medium with the following composition.
Components Gm/1.
k 2h po 4 0.5
MgS04 0.2
NaCl 0.1
Yeast Extract 0.5
FeCl36H20 0.015
D. malic acid 5.0
KOH 4.8
Agar 20.0
pH (adjusted by 0.1NKOH) 7.0
Distilled water 1000ml
Medium was autoclaved at 121 °C for 15 minutes per litre, 15ml of 1:400
sterile aqueous solution of congo red was added aseptieally just before plating or
use.
4. M EDIA USED FOR ACIDIFICATION OF PEPTONE BASED SUGAR

M EDIUM
Ingredients G/LITRE

Peptone 2.0
M gS047H20 1.0
(NH4)2S04 1.0
FeCl3 6H20 0.002
M nS04 0.002
Bromothymolblue dissolved in KOH 0.025

The medium made upto the Volume o f 950ml adjusted to pH - 7.0 and
sterlized by autoclaving at 15 lbs. After it has cooled , 50ml o f 20% (w/v) solution
o f glucose is added aseptically. The development o f yellow colour during

incubation for 96 hours at 37°C indicates acidification. Slight growth in the form o f
a sediment can be detected by agitating the tubes.
5. OKO N’S M EDIUM : (Used for testing organic acid utilization)
Ingredients G/LITRE
K 2H P 04 - 6.0 g
KH2P 0 4 - 4.0 g
M gS047H20 - 0.2 g
NaCl - 0.1 g
CaCl2 - 0.02 g
FeCl3 - 0.01 g
Na2M o04 2 H20 - 0.002 g
Distilled water - 1000 ml
Adjust pH to 6.8 with NaOH and sterilized by autoclaving.
Organic acids are added separately (0.5%).
6. KJELDHAL NESSELERIZATION METHOD :
REAGENTS AND PROCEDURE
The following reagents are used
i) Acid digestion mixture : dissolve selenium oxide (Se02, 1g) in distilled
water (50ml) in a 100ml flask and add MAR grade concentrated sulphuric acid
slowly to 100ml.
ii) Nessler’s Solution : Iodine (11.3g) is dissolved in water (10ml)
containing 15g potassium iodide. To mercury (15g) in a glass bottle is added most
of the iodine solution, the bottle is stoppered and the mixture shaken with cooling in
a water bath until the supernatant liquid loses its yellow colour. The supernatant is
then decanted into a flask (100ml) and a drop is tested for the presence of free
iodine with 1% (w/v) starch solution. If no colour is obtained more of the iodine
solution is added until a drop of the mixture gives a faint blue colour with starch.
The solution is then diluted to 100ml and poured into 10% (w/v) sodium hydroxide
solution (485ml). The turbid solution is filtered or the precipitate allowed to settle
before use. Note that the alkaline solution should be stored in a hard glass bottle
closed with a rubber stopper.
iii) Standard nitrogen solution : dry Analar ammonium chloride (153mg) is
dissolved in 100ml of distilled water in a Volumetric flask. 25ml of this solution
and 10ml N sulphuric acid are diluted to 1000ml to provide a solution of which 1ml
is equivalent to 10 fig nitrogen.

Procedure
Samples (0.5 ml) of a solution and blanks of distilled water are measured into
Pyrex test tubes ( 6 in. X 5/8 in.) and acid catlyst solution (0.2 ml) added to each.
The tubes are heated gently until water evaporates off and then more strongly so
that the acid boils. After 4 h the tubes are removed from the heater and allowed to
cool, to each is added distilled water (7 ml) and Neeler’s reagent (3 ml) and after
standing for exactly 5 min at room temperature the extinction values of the
solutions are measured at 465 nm in a spectrophotometer set to give 100 %

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transmission with the diluted blank digests. It is advisable to measure the extinction
values of the diluted blank digests against distilled water to check that the values are
reproducible. Duplicate Volumes of the standard ammonium chloride solution
equivalent to 5, 10 and 20 jug of nitrogen are diluted to 7 ml with water, treated with
Nesler’s reagent (3 ml) and the extinction values of the series measured after 5 min
against blanks of distilled water treated in a similar manner. The Beer-Lambert law
is obeyed over the range 0-20 pg nitrogen in the sample.
Calculation : Standard graph of absorption Vs ng of nitrogen fixed was drawn and
test readings were referred to it.