Introduction

In July, 1970, while examining water moulds trapped on boiled hemp seed from pond water collected at Otari Plant Museum, Wellington, the attention of one of us (J.E.S.) was arrested by the activities of a number of spiral micro-organisms so large that their movement could readily be followed at a magnification of 100X in the light microscope. These organisms moved swiftly, rotating as they went, until striking an object (e.g. a fungal hypha) on which they then reversed to continue as before. Periodically they came to rest and the number of spiral turns could be counted — some 2-3. Negative staining with nigrosin showed morphology clearly (Fig. 1) and staining with Leifson's stain (1951) readily demonstrated a single flagellum at each end (Fig. 2). When stained with methylene blue conspicuous metachromatic granules (volutin) were evident (Fig. 3). Movement was studied under phase contrast: the organisms darted back and forth, rotating as they went, but the body remained rigid. When at rest the flagellum moved slowly backwards and forwards without showing any wave motion. There was nothing to suggest that the flagellum was compound. Because of the large size (15.8 mic.^* × 1.1 mic.), ease with which motility could be studied and demonstration of flagella and volutin, this organism was considered to be ideal for teaching purposes. An attempt was made, therefore, to identify it.

It obviously belonged to the family Spirillaceae of the order Pseudomonadales because the spiral axis was rigid and it possessed polar flagella. Dobell, in 1912, described a spiral organism from water from the river Granta near Cambridge, England, which he placed in the new genus Paraspirillum. It averaged 15 mic. × 1.5-2.0 mic., had a single flagellum at either end, numerous volutin granules, a definite nucleus and tapered towards the ends. It was only encountered once. Our organism resembled Dobell's organism in possessing a single flagellum at each end but it lacked a conspicuous nucleus (no bacterium possesses a true nucleus) and it did not taper towards the ends. It seemed more likely to belong to the genus Spirillum. Migula, in 1900, separated this genus from other spiral bacteria on the basis of motility by means of a tuft of polar flagella. page 104

Figure 1: Spirillum volutans. Negative stain.

He noted that sometimes the tuft was aggregated into a fascicle which appeared as a single flagellum in flagellar stained preparations. Later opinions differed regarding the value of this character but Giesberger (1936) insisted that only those with a tuft of polar flagella should be included in Spirillum. He placed those species with a single flagellum in the genus Vibrio. Williams and Rittenberg (1957) described six species of the genus Spirillum as having a single flagellum in flagellar stained preparations. In phase contrast observations of living material of the larger species only a single flagellum could be seen. Some time later Williams and Chapman (1961) made an electron microscope study of some twenty-six species and showed that in all, with one possible exception, the apparently single flagellum is in fact compound — a fascicle of many flagella. In a comparison of many flagellar staining methods they found that Leifson's method gave closest agreement with the findings obtained by electron microscopy. page 105

Figure 2: Spirillum volutans. Leifson's stain showing flagella.

Figure 3: Spirillum volutans. Methylene blue stain showing volutin granules.

It appeared desirable, therefore, that we look at our organism in the electron microscope. We had noted the presence of other spiral organisms, mostly smaller in size, in our light and phase contrast microscopic study and we also studied these by electron microscopy.

Materials and Methods

Electron Microscope Studies

Two hundred mesh grids were covered with a vacuum evaporated carbon film and allowed to dry. Specimens were pipetted off from the culture and a small drop placed on a standard microscope slide. The prepared grid was then gently lowered into the surface of the drop, withdrawn, and the adhering material allowed to dry.

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Figure 4: Spirillum volutans. Electron micrograph showing a fascicle of flagella which appears to be spirally twisted.

Negative staining as described by Brenner and Horne (1959) was used. A 2% solution of phosphotungstic acid (PTA) in water was prepared and pH adjusted to a neutral value of between 6.8 and 7.4 by adding N-KOH. A small drop of the resulting potassium phos-photungstate (KPT) being then placed on a glass microscope slide and the grid lowered on to the surface of the drop and allowed to rest for one minute. After washing in distilled water and drying, the grid is ready for use in the microscope. Electron microscopic studies were carried out with the Zeiss EM 9A electron microscope and the included electron micrographs were made with this instrument.

Results and Discussion

Five different spiral micro-organisms were studied in the electron microscope and an attempt was made to relate findings to those of the light microscope. It has been possible to identify with certainty only two or the organisms. The large organism to which our attention was first drawn is identified as Spirillum volutans (Figs. 1-5); another, not seen in the light microscope, is identified as Leptospira biflexa (Figs. 6 and 7). These are described in detail below.

Figure 5: Spirillum volutans. Electron micrograph showing individual flagella.

Figure 6: Leptospira biflexa. Electron micrograph showing characteristic hook at one end and tight spiral coil.

Figure 7: Leptospira biflexa. Electron micrograph showing axial filament.

The other three organisms comprise: (1) A large organism (29.1 mic. × 0.8 mic.) which spiralled as it moved but appeared to flex and did not have demonstrable flagella in phase contrast. However, in Leifson stained preparations a number had a flagellum at one or both ends. The flexing motion combined with spiral shape is characteristic of the spirochaetes but the possession of flagella and movement by rapid spiralling is characteristic of the Spirillaceae. Further studies are necessary before any conclusions can be reached (Fig. 8). (2) A small organism (2.3 mic. × 0.5 mic.) with a single flagellum at each end demonstrable only in the electron microscope (Fig. 9). This would be excluded from Spirillum if Giesberger's ideas are followed and may belong to the genus Vibrio. Vibrios are generally ‘comma’-shaped but the progeny may remain attached, forming spirals. Also, according to Begey's Manual (Society of American Bacteriologists, 1957) the borderline between straight rods found in Pseudomonas and curved rods found in Vibrio is not sharp. Further studies are necessary to identify this organism with confidence. (3) The small curved rod (2.5 mic. × 0.3 mic.) shown in Fig. 10 appears to be attached to a bacterial cell. Recently Stolp and Starr (1963) have described a new genus Bdellovibrio for a predatory, ectoparasitic, and bacteriolytic micro-organism. Our photograph is strongly suggestive of such activity. No flagellum can be seen.

Figure 8: Electron micrograph of unidentified spiral organism. (See text.)

Figure 9: Electron micrograph of a very small, unidentified spiral organism with a single flagellum at each end.

Spirillum volutans. In the electron micrographs the apparently single flagellum seen in light and phase contrast appears as a fascicle of some 20 individual strands. There is also the suggestion that this is spirally coiled (Fig. 4). No basal granules can be seen at the origin of the flagella. These findings agree with those of Williams and Chapman (1961).

Leptospira biflexa. Only two species of Leptospira are described in Bergery's Manual; L. icterohaemorrhagiae representative of the page 112

Figure 10: Electron micrograph of a vibrio-like organism attached to a spherical bacterial cell.

parasitic species and L. biflexa representative of the saprophytic species. Our organism shows clearly the very fine coil and hook (at one end only) which are characteristics of the genus Leptospira (Fig. 6). It measures 8.2 mic. × 0.18 mic. The single axial filament can be seen wound round the main coils of the organism. The point of attachment can be seen in Fig. 7. Holt and Canale-Parola (1968) have shown some excellent electron micrographs of Spirochaeta stenostrepta, a related organism, in which the insertion of the axial filament is clearly seen — the insertion disc. They also demonstrated, for the first time, helical elements in the protoplasmic cylinder but we have not seen these in Leptospira.

The application of electron microscopy has shown the single flagellum of Spirillum volutans, seen in light and phase contrast studies, to be compound. The axial filament of Leptospira shows up clearly. Further work is necessary before the other three organisms can be identified with certainty.

Spirillum volutans, as here described, is an excellent organism for demonstrating the spiral form, motion, flagella and volutin to students of microbiology. We have based our identification on data given in Bergey's Manual. However, there is still a need for further investigations in connection with species of the genus Spirillum, particularly page 113 in relation to isolation, cultivation and physiological characters as pointed out by Williams (1959). The picture is further complicated by the fact that there is a tendency of species of Spirillum to lose their spiral curvature, appearing as straight rods, making it difficult to distinguish the genera Pseudomonas, Vibrio and Spirillum. We have not yet grown any of our organisms in pure culture. Dobell's Paraspirillum remains a puzzle — it is an anomalous organism since no bacterium is known to have a clearly defined nucleus.

Acknowledgments

We are grateful for Professor V. B. D. Skerman's comments on electron micrographs which we sent to him.