In-Vitro Cultivation of Mycobacterium Leprae and Its Confirmation by Molecular Methods

Debaprasad Pal, Kumarjyoti Ghosh, Atanu Ray, Rajasree Mazumder, Nibedita Das.

Abstract

Mycobacterium leprae was the first bacteria to be described as the causative agent of a disease by Dr. Gerhard H.Armauer Hansen in 1873. Many organisms have since been discovered and almost all have been cultivated in artificial culture media (in-vitro cultivation) except M.leprae. Numerous attempts were made to develop a synthetic artificial media for M.leprae over several years, without any success.

The present authors describe here the development of a bi-phasic artificial media (henceforth called “PRG Media”) in which acid fast organisms with globi formation resembling M.leprae were repeatedly grown in liquid and solid phases between 18-22°C over 12-16 weeks. No growth was observed at 37°C till the end of incubation.

The organisms grown in the bi-phasic media were identified as M.leprae by molecular methods whereby gel images of RLEP gene showing amplified repetitive gene sequence of 129 base pair which is unique and 100% specific for M.leprae were detected in the isolates. Another molecular marker, pseudogene ML1545 which is also used for detection of M.leprae was also found to be positive using PCR.