Factors Affecting the Beading of the Tubercle Bacillus Stained by the Ziehl-Neelsen Technique

Abstract

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.

The Nature of the Acid-fast Stain

“…In accordance with the work of Yegian and Baisden (1942) and Porter and Yegian (1945) it has been confirmed that beading occurs in the step of the Ziehl-Neelsen procedure in which the smears are washed with water. The action is sudden and independent of the species of acid-fast organism being studied.…”

“…Therefore, when equal quantities of saturated alcoholic solutions of these dye salts are used to make up the carbol-fuchsin, it is more likely that with the acetate dye salts high enough concentrations of dye will be present to cause a loss in solubility of the phenol than in the case of the chloride. In agreement with this statement is the reported observation (Yegian and Baisden, 1942) that employment of the acetate dye salts to make up carbolfuchsin gave a colloidal suspension instead of a clear solution as in the case when the chloride dye salts were used.…”

“…A critical review of the literature on the subject has been presented by Wells and Long (1932). Since then a new and major observation has been made by Yegian and Baisden (1942) and Porter and Yegian (1945), who have demonstrated that beading and Much's granules are artifacts dependent upon the staining procedure. No theory of the acid-fast stain offered to date can satisfactorily explain the origin of these artifacts.…”

“…A generalization which can be made from the work of Yegian and Baisden (1942), Yegian and Budd (1943), and Porter and Yegian (1945) is that factors which decrease the 8olubility of phenol in water are those leading to the occurrence of artifacts in the Ziehl-Neelsen procedure. Thus these investigators add sodium chloride to carbol-fuchsin to get staining solutions which will regularly yield a large percentage of stained "beaded" cells.…”

The Nature of Acid-Fastness

“…t;4 dye is removed in the course of the process, and a certain minimum amount of free dye is necessary for precipitation. Yegian and Baisden (1942) have shown that when the concentration of the dye is reduced the beads decrease in size and number but not in density. '-p -.. :.…”