Decomposition Studies in Two Central Ontario Lakes Having Surficial pHs of 4.6 and 6.6.

The rates of cellulose breakdown, composition of detrital microflora, and density of bacterial populations were determined in the epilimnetic sediments and water columns of two poorly buffered, oligotrophic, Canadian Shield lakes having mean surficial pHs of 4.6 (Bat Lake) and 6.6 (Harp Lake). The decomposition rate was significantly lower in oxic sediment of the acidified lake than of the circumneutral lake, but water column rates were almost identical in the two lakes. These results are explained in terms of the groups of cellulolytic microorganisms which were observed by phase-contrast microscopy as being active at the different sites: fungi in Bat Lake water and Cytophaga-like bacteria in the water and sediment of Harp Lake. Cytophaga-like bacteria were also the main decomposers in Bat Lake sediment, but their activity was restricted at porewater pHs of <5.0. Acridine orange direct counts of bacteria in the top centimeter of sediment ranged from 3.7 x 10 to 1.0 x 10 per g, and counts in planktonic water samples ranged from 4.9 x 10 to 1.2 x 10 per ml. Bacterial densities at most sites decreased significantly (P < 0.001) from August to late October, but did not show a consistent pattern of differences related to pH.

Microbial decomposition of cellulose in acidifying lakes of South-central ontario.

The rate of cellulose breakdown and density of bacterial populations were measured in the epilimnetic sediments and water columns of lakes in central Ontario that differ in pH, alkalinity, and nutrient status and are particularly sensitive to acidic inputs from atmospheric decomposition. There was no significant difference in decomposition rate in either oxic or anoxic sediment when mean epilimnetic pHs were in the range 5.5 to 6.9. The importance of these findings for the breakdown of autochthonous detritus in Canadian Shield lakes is discussed. Furthermore, the results of these experiments, in which dyed strips of cellophane (regenerated cellulose) were used as substrate, were compared with results of earlier decomposition studies carried out with coarse litter (leaves, twigs). Acridine orange direct counts of bacteria in the top 1 cm of sediment ranged from 5.5 x 10 to 1.0 x 10 per g and in planktonic water samples from 1.1 x 10 to 1.8 x 10 per ml. Bacterial densities were significantly higher in both the shallow sediment (P < 0.01) and the water column (P < 0.05) of dystrophic lakes than at these sites in oligotrophic lakes.

Cell wall growth during differentiation of Proteus swarmers.

This paper describes the process by which the cell wall of Proteus mirabilis, as measured by the presence of the O antigen, develops during the differentiation of swarmers from short cells on an agar surface. The sequence was followed by fluorescent-antibody staining, with both the direct and reverse methods. When the organisms were labeled with fluorescent antibody by the direct method, they showed a progressive diminution of the marker along the cell surface and some increase in the length of the bacteria. However, the label had become completely diluted out before typical swarmers developed. When the bacteria were exposed initially to unlabeled antibody by the reverse technique, and then incubated with fluorescent antibody, they showed a progressive increase both in the intensity of the label along their entire periphery and in cellular length, culminating in the formation of swarmers. It is concluded that in P. mirabilis, as in the few other gram-negative bacteria examined so far, cell wall synthesis takes place diffusely, i.e., by intercalation of new with old components along the length of the cell wall.

Cytology of spore formation in Clostridium perfringens.

The sequential morphological events in spore formation by Clostridium perfringens type D were observed in Ellner's medium where 80 to 100% of the cells formed spores. Gross structural changes were studied with the light microscope under phase-contrast, and in fixed cells by the use of both nigrosin and Giemsa preparations. Fine structure was examined with the electron microscope in both thin sections and frozen-etched preparations. During the first 3 hr of incubation, the original rod-shaped cells became ellipsoid to ovoid in shape; by 5 to 6 hr, subterminal spores had developed within these enlarged cells. The fine structural sequence was in most respects identical to that in other Bacillaceae, although some stages were illustrated with particular clarity. A unique feature was the development of a convoluted, membranous exosporium which adhered to the outer surface of the two coats and had an unusual fine structure resembling a rectangular array of subunits.

Cytology of spore germination in Clostridium pectinovorum.

The process of spore germination in Clostridium pectinovorum has been followed by phase-contrast and electron microscopy. Unlike most other Bacillaceae, germination of this species takes place within the sporangium. Under phase-contrast, the spore darkens and swells slightly, and then the vegetative rod slips out through the end opposite the collar-like extension of the sporangium. In thin sections, a spore from an early stage in germination consists of a central protoplast, core membrane, germ cell wall, cortex, and two coats. Within a short period, the cortex disintegrates and the young cell develops. It possesses a large fibrillar nucleoplasm and several mesosomes. Subsequently, the young cell elongates, becomes somewhat deformed, and then emerges through a narrow aperture in the inflexible coats of the spore, finally rupturing the sporangium. Free vegetative cells of C. pectinovorum resemble in their structure other gram-positive rods.

A "microtubule" in a bacterium.

A study of the anchorage of the flagella in swarmers of Proteus mirabilis led to the incidental observation of microtubules. These microtubules were found in thin sections and in whole mount preparations of cells from which most of the content had been released by osmotic shock before staining negatively with potassium phosphotungstate (PTA). The microtubules are in negatively stained preparations about 200 A wide, i.e. somewhat thicker than the flagella (approximately 130 A). They are thus somewhat thinner than most microtubules recorded for other cells. They are referred to as microtubules because of their smooth cylindrical wall, or cortex, surrounding a hollow core which is readily filled with PTA when stained negatively. Since this is probably the first time that such a structure is described inside a bacterium, we do not know for certain whether it represents a normal cell constituent or an abnormality, for instance of the type of "polysheaths" (16).

Basal bodies of bacterial flagella in Proteus mirabilis. II. Electron microscopy of negatively stained material.

This paper investigates further the question of whether the flagella of Proteus mirabilis emerge from basal bodies. The bacteria were grown to the stage of swarmer differentiation, treated lightly with penicillin, and then shocked osmotically. As a result of this treatment, much of the cytoplasmic content and also part of the plasma membrane were removed from the cells. When such fragmented organisms were stained negatively with potassium phosphotungstate, the flagella were found to be anchored-often by means of a hook-in rounded structures approximately 50 mmicro wide, thus confirming Part I of our study. In these rounded structures a more brilliant dot was occasionally observed, which we interpret as being part of the basal granule. A prerequisite for the demonstration of the basal granules within the cells was, however, the removal of both the cytoplasm and the plasma membrane from their vicinity. In some experiments, the chondrioids were "stained" positively by the incorporation into them of the reduced product of potassium tellurite. The chondrioids were here observed to be more or less circular areas from which rodlike structures extended. The chondrioids adhered so firmly to the plasma membrane that they were carried away with it during its displacement by osmotic shocking, while the basal bodies were left behind. This observation disproves our previous suggestion that the flagella might terminate in the chondrioids. The basal bodies often occur in pairs, which suggest that they could be self-reproducing particles.

Basal bodies of bacterial flagella in Proteus mirabilis. I. Electron microscopy of sectioned material.

Years ago (16, 18, 19), in a study of shadowed preparations of Proteus vulgaris that had been autolyzed in the cold, the observation was made that the flagella arose from basal bodies. However, recently (3, 7, 24, 33) doubt has been cast on the conclusion that the flagella of bacteria emerge from sizable basal bodies. This problem has, therefore, been reinvestigated with actively developing cultures of Proteus mirabilis, the cell walls of which had been expanded slightly by exposure to penicillin. Two techniques were applied: ultramicrotomy, and negative staining of whole mount preparations. This paper deals with the thin sections of bacteria after the usual fixation technique had been altered slightly: the cells were embedded in agar prior to their fixation and further processing. The flagella then remained attached to the cells and were seen to extend between the cell wall and the plasma membrane. Occasionally, the flagella appeared to be anchored in the cell by means of a hook-shaped ending. In sections of cells rich in cytoplasm, the basal bodies are particularly difficult to visualize due to their small size (25 to 45 mmicro) and the lack of properties that would enable one to distinguish them from the ribonucleoprotein structures; in addition, their boundary appears to be delicate. However, when the cytoplasm is sparse in the cells, either naturally or as a result of osmotic shocking in distilled water, the flagella can be observed to emerge from rounded structures approximately 25 to 45 mmicro wide. Contrary to a previous suggestion (21), the flagella do not terminate in the peripheral sites of reduced tellurite, i.e. the chondrioids. The observations in this part of the study agree with those described in the following paper (15) dealing with negatively stained preparations.