Dear Maren

Dettermination nitrifiers and denitrrifiers  usingthe most probably numbers (MPN) technique, (Cochrane 1950)

Nitrifiers:

Determined using the Stephenson’s medium for autotrophic nitrifiers

(Stephenson, 1950):

Ammonium sulphate         2.00 g

K2Hpo4                                               0.75 g

FeSo4.7H2o                        0.01 g

KH2 PO4                            0.25 g

MnSO4.4H2O                     0.01 g

MgSO4.7H2O                     0.03 g

CaCl2                                  0.002 g

Distilled water                   1000 ml.

After sterilization, a knife tip of sterile CaCO3 was added to each tube.

Incubation temperature was 30 +0.5 CO for 21 days. Diphenylamine in

concentrated sulphuric acid was used for detection of the produced nitrate.

 Denitrifiers:

Were counted using the yeast – extract– peptone- nitrate medium (El-

Saify, 1976):

Peptone             10.0 g

Yeast extract     0.5 g

KNO3                1.0 g

K2HPO4             0.5 g

Agar agar           2.0 g

Distilled water  1000 ml.

pH                     7 – 7.2

The determination tubes were equipped by inverted Durham tubes. Incubation was for 10 days at 28 + 0.5 OC. Replicates showing gas in the

Durham tubes after addition of a strong NaOH solution were considered to be denitrifiers positive.

Cochrane, W.G.(1950). Estimation of bacterial densities by means of the       «most probable number». Biometrics 6, 105-116.

Stephenson, M. (1950). Bacterial metabolism, 3rd ed., Longmans, Green&Co. London.

El-Saify,N.A. (1976).Nitrate reduction and denitrification in soils. M. Sc. Dept. Fac. of Agric. Ain. Shams univ. A.R.E.

Please do a Quantitative PCR targeting functional genes to estimate their population. Nitrifers are autotrophic in nature hence a single media is not sufficient to culture all the nitrifiers and will result in underestimation of their population.

Thank you all for your input into this.