Annex 8.1
Preparation of reagents for biochemical tests
NITRATE REDUCTION TEST
Reagents and solutions
Sodium nitrate substrate in buffer pH 7
NaNO
3
(sodium nitrate, MW 85)
0.085 g
KH
2
PO
4
(potassium dihydrogen phosphate, MW 136.1)
0.117 g
Na
2
HPO
4
.
12H
2
O
(disodium hydrogen phosphate dodecahydrate, MW 358.14)
0.485 g
Distilled water
100 ml
Check pH at 7.
Sterilize by autoclaving at 121 °C for 15 minutes. When needed, aliquots of the substrate
solution are aseptically dispensed into sterile screw
capped tubes in 2
ml quantities.
Reagent 1: Hydrochloric acid solution
Concentrated HCl (36%)
10 ml
Distilled water
10 ml
Slowly add concentrated HCI to distilled water
‒
never add water to acid.
Reagent 2: Sulfanilamide solution, 0.2%
Su
lfanilamide
(C
6
H
8
N
2
O
2
S; MW 172.21)
0.2 g
Distilled water
100 ml
Dissolve sulfanilamide in distilled water and store in an amber bottle in the dark in a
refrigerator.
Reagent 3: N
naphthylethylene
diamine solution, 0.1%
N
naphthylethylene
diamine d
ihydrochloride
0.1 g
Distilled water
100 ml
Dissolve naphthylethylene
diamine in distilled water and store in an amber bottle in the dark in
a refrigerator.
Discard any reagent if the colour changes or a precipitate forms.
NITRATE REDUC
TION TEST: S
TANDARD PREPARATION
Reagents
Stock solutions
1.
0.067
mol/litre
Na
2
HPO
4.
12 H
2
O
D
issolve 23.68
g disodium h
ydrogen phosphate dodecahydrate
(MW 358.14
) in 1000 ml
distilled water.
2.
0.067
mol/litre
KH
2
PO
4
D
issolve 9.07 g potassium dihydrogen phosphate (MW 136.1)
in1000 ml distilled water