Dear Dr. Abdel-Gawad,

To enumerate the endophytic nitrogen fixers in the root, I do not prefer to use the MPN but the plate count using the general medium for isolation of nitrogen fixers.

First, take 10 g of the root and just use the method followed to wash and sterilize the root nodules of legumes to sterilize the roots.

Secondly, use a sterilized mortar to crash the roots carefully then transfer the crushed roots quantitatively to the dilution bottle that contains 90 ml of sterile distilled water. After that follow the procedure of the total counting.

Wishing you all success   

There is a medium called NITROGEN-FREE, so there is no chance for the nitrogen scavengers to grow esp. under sterile conditions. 

Dear Abdelgawad Elsaadany

study of the paper entitled  Isolation and characterization of plant growth promoting endophyticdiazotrophic bacteria from Korean rice cultivars (Microbiological Research 169 (2014) 83– 98) by Sang Hye Ji et al. (2014) can be useful for you.