Annex 8.1
Preparation of reagents for biochemical tests
NITRATE REDUCTION TEST
Reagents and solutions
Sodium nitrate substrate in buffer pH 7
NaNO3 (sodium nitrate, MW 85)
KH2PO4 (potassium dihydrogen phosphate, MW 136.1)
Na2HPO4.12H2O (disodium hydrogen phosphate dodecahydrate, MW 358.14)
Distilled water
0.085 g
0.117 g
0.485 g
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%)
Distilled water
10 ml
10 ml
Slowly add concentrated HCI to distilled water ‒ never add water to acid.
Reagent 2: Sulfanilamide solution, 0.2%
Sulfanilamide (C6H8N2O2S; MW 172.21)
Distilled water
0.2 g
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 dihydrochloride
Distilled water
0.1 g
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 REDUCTION TEST: STANDARD PREPARATION
Reagents
Stock solutions
1. 0.067 mol/litre Na2HPO4.12 H2O
Dissolve 23.68 g disodium hydrogen phosphate dodecahydrate (MW 358.14) in 1000 ml
distilled water.
2. 0.067 mol/litre KH2PO4
Dissolve 9.07 g potassium dihydrogen phosphate (MW 136.1) in1000 ml distilled water.
3. 0.067 mol/litre Na3PO4.12H2O
Dissolve 25.47 g trisodium phosphate dodecahydrate (MW 380.12) in 1000 ml distilled
water.
4. 1% phenolphthalein: dissolve 1 g in 100 ml 95% ethyl alcohol.
5. 1% bromothymol blue: dissolve1 g in 100 ml 95% ethyl alcohol.
6. 0.01% bromothymol blue: prepare by mixing 1.0 ml of solution 5 above in 100 ml of distilled
water.
7. Mix 35 ml of stock solution 1 and 5 ml of stock solution 2 and 100 ml of solution 3.
8. To 10 ml of stock solution 7, add 0.1 ml of stock solution 4 and 0.2 ml of stock solution 6.
Procedure
1. Place eight clean test-tubes (numbers 1‒8) in a rack. Use the same size tubes as for the
nitrate reduction test.
2. Put 2 ml of stock solution 7 into tubes 2 through 8.
3. Add 2 ml of solution 8 to tube number 1. This is the 5+ colour standard.
4. To tube number 2 in the series, add 2 ml of solution 8. Mix well and transfer 2 ml to the
next tube (number 3). Continue to make serial dilutions of 2 ml, discarding 2 ml from the
8th tube.
The colour standards:
tube 1
tube 2
tube 3
tube 5
tube 6
tube 8
=
=
=
=
=
=
5+
4+
3+
2+
1+
+/-
purplish red
deep red
red
deep pink
clear pink
faint pink
Autoclave tubes, seal and store at 5 °C. These keep indefinitely and should be used whenever
nitrate tests are done.
CATALASE TEST
Reagents
0.067 mol/litre phosphate buffer, pH 7.0
Reagent 1 ‒ Dissolve 9.07 g KH2PO4 (potassium dihydrogen phosphate, MW 136.1) in 1000
ml distilled water.
Reagent 2 ‒ Dissolve 23.68 g Na2HPO4.12H2O (disodium hydrogen phosphate dodecahydrate,
MW 358.14) in 1000 ml distilled water.
Mix 37 ml of reagent 1 with 63 ml of reagent 2 to adjust to pH 7.0. Check on pH meter.
30% H2O2 (hydrogen peroxide: not the 3% H2O2 obtainable from pharmacies)
10% Tween 80
Mix 10 ml Tween 80 in 90 ml distilled water, autoclave at 121 °C for 10 minutes. Store in the
refrigerator.
Substrate ‒ Mix equal volumes of 30% H2O2 and 10% Tween 80.
Note: Always prepare the substrate fresh just before test. Wear plastic gloves and a
protective eye shield when handling H2O2