In vitro cultivation of leprosy bacilli on hyaluronic acid based medium. 1. Preliminary report

In vitro cultivation is reported of Mycobacterium leprae on a medium (designated LA-3) based on hyaluronic acid with additional ingredients of yeast extract, bovine albumin and glycerin together with phosphate buffer. The medium is also incorporated with agar or agarose (designated LA-3P) to serve as culture plates. Initial growth in LA-3 in test tubes required about six weeks but subsequently this was speeded up to about two weeks utilizing large quantities of media with aeration by shaking twice a day. Growth on LA-3P yields numerous small orange-yellow colonies in two to three weeks. Facets of the emerging aspects of the life cycle of M. leprae under cultivation are given preliminary report. The bases for the allegation of M. leprae identity of the cultured bacilli are essentially the following six determinations. 1. Pathologic and experimental determined rationale for the essential M. leprae nutrient requirement. 2. Several cultures having the same characteristics have been isolated from LL patients widely separate in time and by geography. 3. Failure of culture isolates to subculture on the usual media employed in the cultivation of mycobacteria at both 37 degrees C and room temperature. 4. 1 degree cultures in liquid medium successfully transferred to 2 degrees liquid medium and to 2 degrees agar medium plates. 5. Bacillary isolates and bacilli of 1 degree and 2 degrees liquid medium cultures all stain with pooled LL serum, FITC coupled, M. leprae specific antibody with which a broad range of other mycobacteria do not react. 6. M. lepraemurium also presents good growth on this medium.