Orientation staining for the demonstration of Mycobacterium leprae in semithin sections

In Spurr-embedded biopsies for ultrastructural examinations, Mycobacterium leprae hardly differed from the surrounding tissues using the staining technique of Richardson. Also, the usual histological staining methods of Ziehl-Neelsen and Fite did not achieve positive results for the determination of M. leprae. Therefore, we applied the methylene blue-borax and basic fuchsin technique for the demonstration of the bacilli in plastic-embedded tissue of 11 patients suffering from Hansen's disease. In every patient the diagnosis was confirmed by histological examination of skin biopsies. Portions of the biopsies of nine patients were then fixed in glutaraldehyde and osmium tetroxide and embedded according to Spurr. In the other two cases, the material was first fixed in 10% Formalin and embedded in paraffin. After cutting 3-micron sections for routine histological examination, the remaining material was prepared adequately for ultrastructural examination. Using the methylene blue-borax and basic fuchsin technique, the semithin sections of plastic-embedded material presented a considerably more differentiated picture than other comparable methods. M. leprae located in foamy cells or in the tissue stained violet. These findings were corroborated in subsequent electron-microscopic examinations. The semithin sections thus prepared allow, through the clear demonstration of the microorganisms, a precise demarcation of the ultrathin area.