John disease or Paratuberculosis is a kind of chronic granulomatous enteritis in ruminants with the etiologic agent of Mycobacterium avium subspecies paratuberculosis(MAP). It seems optimal method of cultivation and the growth of this bacteria can be important for a better and more understanding from these bacteria. in his research isolated species Mycobacterium avium subspecies paratuberculosis (MAP) were cultured under different combinations of growth medium and after Optimizationof bacteria culture media, bacteria growth curve was drawn in selected medium. After growing the bacteria in selected culture medium and bacteria determination in culture medium with zielneelsen staining, DNA was extracted from bacteria withInnovative methodof boiling. After doing PCR andobservation of desired band in electrophoresis. Bacterial growth curve was drawn within 35 days.

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