Gary R. Pearson, Bob A. Freeman, William D. Hines
1963 Journal of Bacteriology  
tetrazolium chloride, 0.05 g; ferrous ammonium sulfate, 0.2 g; sodium thiosulfate, 0.2 g; and 1 liter of distilled water. After being boiled to ensure coml)lete dissolutioni, the composite is distributed in tubes, autoclaved for 15 min at 15 psi (121 C), and allowe(d to cool in an upright position. WTith semisolid media, motility is macroscolically manifested by a diffuse zone of growth spireading from the line of inoculation. The effects arc cumulative, and localized outgrowths appear when
more » ... ths appear when only a small lprol)ortion of motile cells are involved. Growtlh and diffusioni in semisolids, however, may be so slight as to necessitate comparison with a control tube for lprolper interlpretation. Kelly and Fulton (Am. J. Clin. I'atlhol. 23:512, 1953) corrected thel difficulty in discerninig growthl in semisolids by incorporating tetrazolium salts inito the test me(liuimi and, in this way, inducingo a' cellular formi.atioin of re(I pigments. Nonmotile, nonsulfide-producinog organlisms reveal a red line along the route of inoculation. Nonmotile, sulfide-producing cultures form a black line. Mfotile, sulfide-nonproducing culturies (levelol) a diffuse pink cloudc throughlout the medium. A[otile, sulfide-p)roducing cultures blacken the entire tube. This medium has been used for over 2 y-ears on numerous cultures. In all cases, there has been coml)lete agreement \-ith the sulfide response rendered witlh triple-sugar ino igar. During an investigation to (letermine the intracellular fate of Brucella, by use of thin-section electron microscopy techniques, we observed that the Brucella are enclosed in a membranelboun(d vesicle in the intracellular environment. Electron micrographs demonstrating this finding are l)resented here. Monocvte cultures wN-ere infecte(d by B. suis strain 321' by use of techniques lpreviously rel)orted (Freeman et al., J. Infect. Diseases 108: 333, 1961). The host cells wAere infected at a high multiplicity with either smooth or rough Britcella to assure that most of the monocvtes Nould be infectedl. After a 4-hr ingestion leriod, the Brucella were alloweed to multiply for another 4 hr in the intracellular environment before the infecte(d cells wN-ere fixed in osmium tetroxide (Pala(le, J. IEA'xptl. -Med. 95:285, 1952). They w-ere then dehydrate(d in ethanol and embedded in Elpon (Luft, J. Biophys. Biochem. CYtol. 9:409, 1 961). Tlhin sections N-ere stained with uranyl acetate for 1 hr and observed wN-ith an RCA electron microscol)e (model E1\W-3C). Representative electron micrographs of smooth Brucella in the cytol)lasm of the mononuclear phagocyte are shown in Fig. 1 and 2 ; rough Brat-cella are showN-n in Fig. 3 and 4 . Blotlh forms appear to be localized in a membrane-bound v-esicle in the host cell. This is an unexlpecte(l findlinug, since staine(l preparations of infected cells ba(d not given an-indlication of such a vesicle. That this membrane surrounds both indivi(dual Britcella and groups of Brucella is clearly in(licated in the electron microgral)hs of the rowbl forlmls ( Fig. 3 and 4) . The l)resence of a membrane arouin(1 lparticles in a phagocytic cell is not unusual. IKarrer (J. Biophys. B3iochem. Cyrtol. 7:357, 1960) stud(lied the process of p)hag,ocytosis of India ink lparticles with an electron microscop)e and observedl th,at these l)articles were localized in membrane-bounld vesicles in the alveolar macrophages. l7shiba (Tohoku J. Exptl. AMed. 76:133, 1962) found that, in mice andl guinea p)ig monocytes, Salmonella enteriditis was lpresent in vacuoles stirrounded byr a clear limiting membrane. Chapman, Hanks, an(d Wallace (J. Bacteriol. 77:205, 1959) reported that in mouse spleen infectedI with .Jlycobacteriium lepraemuritim the bacteria were sel)arated from the host cytoplasm by a capsuleenclosing membrane Nhich seemedl to i)e (lerived fromn the host cytoplasm. Kessel et al. (Federa-NOTES 112;3 on May 7, 2020 by guest
doi:10.1128/jb.86.5.1123-1125.1963 fatcat:c5slcygvozbe7gaevriwylyjya