Electron micrographs of Proteus vulgaris. RCA electron microscope Model EMU 2C. Cultures of 4 hr in plain broth. Figure 1. Control; not fixed; shadowed with ...
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ELECTRON MICROSCOPY OF GRANULES IN PROTEUS VULGARIS TREATED WITH TRIPHENYLTETRAZOLIUM CHLORIDE M. THIAGO DE MELLO, NIBER PAZ M. SILVA, AND HANS MUTH Section of Bacteriology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil
Received for publication October 18, 1956
Granules revealed by the reduction of tetrazolium salts to formazan inside cells of many species of bacteria have been interpreted as mitochondria by several workers (see review by Mudd, Ann.
Rev. MIicrob., 8, 1, 1954). Other opinions, however, may exist (Widra, J. Bacteriol., 71, 689, 1956; Alexander, Bacteriol. Revs., 20, 67, 1956). During experiments with Proteus vulgaris incu-
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Electron micrographs of Proteus vulgaris. RCA electron microscope Model EMU 2C. Cultures of 4 hr in plain broth. Figure 1. Control; not fixed; shadowed with chromium at an angle of 100 (X 6,500). Discrete granules in bacilli. Figure 2. Cells treated with TTC for 30 min; not fixed; not shadowed (X 8,800). Granules absent; collapsed membranes. Figure S. Cells treated with TTC for 30 min; not fixed; shadowed with chromium (100). (X 8,000). Granules absent; remains of membranes. Figure 4. Cells treated with TTC for 30 min; not fixed; shadowed with chromium (100). (X 14,400). Granules yet present in the cell, with net crystalline contour. Figure 5. Cells treated with TTC for 30 min; fixed with formaldehyde; shadowed with chromium (100). (X 7,200). Granules present altering the contour of the cells. Figure 6. Cells treated with TTC for 30 min; fixed with formaldehyde; shadowed with chromium (100); treated with acetone (X 7,100). Granules removed leaving collapsed membranes. 682
1957
683
NOTES
bated in plain broth (4 hr at 37 C) and then treated with 2,3,5-triphenyltetrazolium chloride (TTC) in a concentration of 0.1 per cent at 37 C, we observed sites of TTC reduction within the bacterial cells similar to those described by Sorouri and Mudd (Indian J. Med. Research, 41, 333, 1953). The cells were treated with TTC, for different intervals of time (up to 30 min), and then washed twice with distilled water. These cells were observed to contain granules which altered the morphology. If the exposure to TTC was for a short time the granules were scattered in the cytoplasm but after longer exposures they tended to coalesce at the poles and along the bacterial body. Normal bacilli (figure 1) show discrete granules almost at the same sites as the large granules after treatment with TT'C. The aspect of the cells was quite distinct whether these were or were not fixed with formaldehyde, after 30 min of contact with TTC. In microorganisms not fixed with formalde-
hyde, most of the granules ruptured the cells during the electron microscopy leaving remains of these, like "ghosts," in the sites where the granules had been located (figures 2 and 3). This suggested that vacuolization occurred after intense electron bombardment, as described by Mudd et al. (J. Bacteriol., 62, 459, 1951), in normal cells of Mycobacterium thamnopheos. The fate of the formazan crystals that surrounded the volatilized mitochondria (?) in unfixed material could not be determined, because pure formazan crystals, alone, did not volatilize under the same conditions. In some cases large granules were not volatilized and the cells remained deformed, with a net crystalline contour (figure 4). When the cells were fixed with formaldehyde the granules did not volatilize and remained (figure 5), their size proportional to the time of contact with TTC. They could be removed from the cells by treatment with acetone leaving empty collapsed membranes (figure 6), another evidence of their formazan contents.
ADAPTATION PATTERNS IN THE UTILIZATION OF THE STEREOISOMERS OF TARTARIC ACID BY A PSEUDOMONAD1 WILLIAM R. MARTIN2 AND J. W. FOSTER Department of Bacteriology, University of Texas, Austin, Texas Received for publication November 8, 1956
A soil isolate of Pseudontonas fluorescens displays a pattern of utilization of the stereoisomers of tartaric acid different from that of Pseudomonas putida (La Riviera, Biochem. et Biophvs. Acta, 22, 206, 1956). Cells harvested from a meso-tartrate mineral salts medium oxidized meso-tartrate at once, but they oxidized L( +)-, D (-)-, and racemic tartrates only after a definite induction period (figure 1). L(+)- or D(-)tartrate grown cells were, however, simultaneously adapted to all the isomers (figure 2).
Sonic extracts of L( +)-tartrate grown cells oxidized all the tartrate isomers (figure 3), whereas extracts of meso-tartrate cells oxidized only meso-tartrate (figure 4). Chemical tests
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M FS -TARTRATE
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D(-)-TARTRATE DL -TARTRATE
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L(.) -TARTRATE
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1 This work was supported in part by the Division of Biology and Medicine of the Atomic Energy Commission, by a grant-in-aid from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council, and by the Microbiology Branch of the Office of Naval Research. 2 Present address: Department of Bacteriology, uJniversity of Chicago, Chicago 37, Ill.
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TIME IN MINUTES
Figure 1. Oxidation of stereoisomers of tartaric acid (5 umoles) by meso-tartrate grown cells of Pseudomonas species.
