Presently amplification of specific loci (IS900) by PCR is popular for confirming the Mycobacterium avium subspecies paratuberculosis cultures but it requires loopful of growth, which is rarely available for sheep and human isolates. In case of other animals also MAP colonies are minute due to use of old batches of certain critical chemicals, (personal observation). Two methods of DNA isolation; a non-chemical and non-enzyme physical method referred from Challans et al., (1994) (Method 1) has been compared with standard DNA isolation method described by van Soolingen et al., (1991) (Method 2). Both methods were used to recover DNA from 1 ('Single Colony PCR') to few (1-3) extremely minute and tiny MAP colonies. Using standard protocol DNA (method 2) was only recovered from 33.3% of cultures processed and only 14.6% samples gave positive amplification in PCR. Using method 1 (physical method), DNA was recovered from all the cultures processed and 90.0% of cultures processed yielded positive amplification in IS900 PCR. The new 'physical method' recovered DNA of PCR quality and helped to characterized the minute colonies cultured first time from Crohn's disease patients, dairy cattle, raw milk and pasteurized commercial milk supplies, in India.