Koch first described the beaded forms of the tubercle bacillus and regarded the beads as evidence of spore formation. Later investigators attempted to correlate the beaded forms with the age and the virulence of the microorganism. These studies, however, have not been fully confirmed. In the present paper, data will be submitted to show that the beads exhibited by tuberele bacilli stained with earbol fuchsin are greatly influenced by the preparation of the dve used, by the presence of electrolytes in the system, and 1y subsequent washing with alcohol and other organic solvents. EFFECT OF ELECTROLYTES A four-weeks-old culture of virulent tubercle bacilli, Strain H37, grown in Long's synthetic medium, was transferred to a Buchner filter, and the bacilli freed from the medium by washing with distilled water. The washed bacilli were ground in an agate mortar and then suspended in distilled water. Twenty films of tubercle bacilli were prepared by spreading one loopful of this suspension on clean glass slides. The films were air-dried. The solution of carbol fuchsin used in this experiment was prepared as follows: To 10 ml. of a saturated solution of pararosaniline hydrochloride (Lot i 9155 from the National Aniline Company Buffalo, N. Y.) in 95 per cent ethyl alcohol, there were added 10 ml. of alcohol and 180 ml. of 5 per cent aqueous phenol. This solution of earbol fuchsin was divided into two parts: A. 95 ml. carbol fuchsin + 5 ml. distilled water. B. 95 ml. carbol fuchsin + 5 ml. 10 per cent NaCl solution. Ten films were flooded with solution A (no salt) and ten films with solution B. The slides were heated in an electric oven at 90'C. for twenty minutes. The excess dye was poured off, the slides washed with acid alcohol for thirty seconds, and then thoroughly washed with distilled water and air-dried. On microscopic examination, the preparations stained with solution A (no salt) revealed the bacilli to be solidly stained, with a slight purplish tinge; no beads could be seen in the cellular bodies (fig. 1). On the contrary, the preparations stained with solution B (i.e., in the presence of NaCl in final concentration of 0.5 per cent) showed intense beading. In many, instances the beads within the bacilli gave the appearance of a chain of cocci. The beads were of a dark purplish color, whereas the cellular body itself was of a faint pink tinge. Each bacillus exhibited from I to 5 beads, irregular in distribution and of a diameter greatly exceeding that of the bacillus itself (fig. 2). This experiment was repeated and confirmed many times.
Michaelides (1907), using a modified Gram method, simultaneously made the clai that this variant of the acid-fast rod could be stained in pathogenic tuberculous material (perlsucht-nodules of cattle and human cold abscesses) which, after staining with the Ziehl-Neelsen procedure, failed to show any acid-fast bacilli. Since the time of Much's report these gram-positive bodies have been repeatedly studied, and numerous proposals have been made relative to their composition, function, and filterability. It has been suggested that they are reproductive units composed largely of proteins (Weiss, 1909; Deycke, 1910) or nucleoproteins (Liebermeister, 1909) or that possibly they are reserve centers for lipids (Hollande and Cr6mieux, 1928). Support for a wax or fatlike component was supplied by the apparent disappearance of the granules from the cells extracted with trichloroethylene (Aronson, 1910) or with an acidified ether-alcohol mixture devised by Aronson (Krylow, 1912). The same extraction rendered the cells non-acid-fast. Such definite, minute granular components interested those who later demonstrated a filterable agent for the tubercle bacillus. Earliest of these investigators was Fontes (1910), and for him the granule of Much or some division of it became the filter-passing agent. The same proposal was later supported by Calmette, Valtis, and Saenz (1929), and others. What appears to be a most convincing
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