NOTES

Morphological characteristics of a spirochete present in activated sludge

MARIA H. DEINEMA LoOorcitory oJ'Microbiology, Agr~ic~rlt~ircil University, Wogerringen, Tlre Netl~erlrir~ds

Accepted August 20, 1976 I

DEINEMA, M. H. 1976. Morphological characteristics of a spirochete present in activated sludge. Can. J. Microbiol. 22: 1768-1771.

A free-living spirochete has been found in the activated sludge of purification plants in Am5terdam. A pal-tial enrichment of the spirochete could be obtained with a passage through a thin layer of water agar. The molphology of this spirochetal strain does not fit into one of the described genera in Bergey's Manual. The cells have many regular primary coils on which broad secondary coils are superimposed and the protoplasmic cylinder is tightly wound around an axistyle.

D E I N E M A , M. H. 1976. Morphological characteristics of a spirochete present in activated sludge. Can. J. Microbiol. 22: 17611771.

Un spirochkte vit librement dans la boue activeedes usines d e purification d'Amster-dam. On a pu obtenir une accumulation partielle de cet organisme par passage sur une couche mince de gClose aqueuse. La morphologie de cette souche ne correspond pas a celle des genres decritsdans le Manuel de Bergey. Les cellules presentent plusieurs spires primaires regulikres sur lesquelles sont supelpost5es de larges spires secondaires et le cylindre de protoplaste est etroitement enroule autour d e I'axistyle.

[Traduit par le journal]

In several recent papers (2, 3, 8, 9, 10) the morphology and physiology of the smaller spiro- chetes of the genera Spbochaeta, Treponema, Borrelia, and Leptospira have been described. These bacteria consist of helical rods with either regular or irregular spirals and one or more axial fibrils inserted at each end of the protoplasmic cylinder. An external sheath encloses both axial filament and protoplasmic cylinder. The classi- fication of these smaller spirochetes is mainly based on their physiological properties. The characteristics of the larger spirochetes viz. Spirochaeta plicatilis and Crisrispira species are but scarcely known as they have not yet been cultivated in pure cultures (1, 7, 9, 1 I).

For many years a free-living spirochete has been found in relatively large numbers in the activated sludge of the water purification plants in Amsterdam. Many spirochetes were detected particularly in the interior of the sludge flocs, but the motile cells could also be seen on the surface of the flocs. It was possible to concentrate the spirochetal cells by using the following technique: sludge flocs were put on the surface of a water agar (0.3% agar in tap water) and covered with a cover slip. A small agar block was prepared by cutting around the edges of the cover slip. A

number of blocks were inverted in sterile Petri dishes and kept at room temperature and a t 10 "C. Within a few hours the spirochetes crept upwards through the soft agar and accumulated on the wet surface of the blocks. Most other bacteria were much slower in creeping through the layer of agar. Samples of the wet agar surface, containing many spirochetal cells, were transferred into hanging-drop slide cultures and so different media with low concentrations of substrates were tested. Low substrate concentra- tions were used since it has been observed that the spirochetes were particularly present in sludge flocs of underloaded purification plants. At a temperature of 10°C the spirochetes re- mained viable for more than 14 days and their cell length increased considerably, but actual growth by increase in cell numbers did not occur. Sooner or later the cells died or were overgrown by contaminants.

Samples with predominantly spirochetal cells were used to examine the morphology of this bacterium. Phase-contrast microscopy of wet mount preparations showed that the cells have many, very stable, and regular primary coils and they also exhibit broad secondary coils o n which the primary coils are superimposed (Figs. 1 and

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NOTES 1769

FIG. 1. Spirochetes and other rod-shaped bacteria from activated sludge. Phase-contrast microscopy, 1500 x . FIG. 2. Single spirochete. Phase-contrast microscopy, 1900 x . FIG. 3. Electron micrograph of the spirochete. Pd shadow, 7500 x . FIG. 4. Electron micrograph. Pd shadow. The axistyle (AS) consists of a sheath surrounding axial fibrils (AF), 35 000 x .

2). Both types of coils persist in the presence and Formvar-coated grid, dried, and shadowed with absence of movement. This strain closely re- palladium, without using a fixative. For thin- sembles the spirochete photographed by Robinow sectioning the cells, enrobed in agar, were fixed (6). in a solution of 1% OsO, in 1% Stirensen buffer,

For electron microscopy cells were placed on a pH 6.8. The specimen was embedded in a mix-

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1770 CAN. J. MICROBIOL. VOL. 22, 1976

FIG. 5. Electron micrograph. Pd shadow. The protoplasmic cylinder is wound around the axistyle (AS), arrows. The diffuse background is caused by some agar particles, 22 000 x . FIG. 6. Thin section of a spirochetal cell. The protoplasmic cylinder is surrounded by an outer membrane (OM). Nuclear material (NM) is visible. The axistyle (AS) consists of an inner membrane (IM) and two to three axial fibrils (AF), 46 500 x .

ture of styrene and n-butylmethacrylate (5) and cylinder and axial filament. A n inner membrane the thin sections were stained with uranylacetate (IM), surrounding the axial fibrils, is also visible. followed by lead citrate (4). The classification of this spirochete, according

The regular primary coils, a few secondary to Bergey's Manual (9), meets with difficulties. coils, and one axial fibril can be clearly seen in The cells always have many regular primary Fig. 3. Terminal hooks and spherical bodies have never been observed. From Figs. 4 and 5 it appears that the protoplasmic cylinder is tightly wound around an axistyle (axial filament) which consists of an inner sheath with a core of three t o five axial fibrils. The dimensions of this spiro- chete are 0.20-0.25 by 20-100 pm and the diam- eter of the axistyle is 0.10-0.13 pm. Insertion of the axistyle at the end of the protoplasmic cylinder, as present in other spirochetes, could not be ascertained here. A thin section (Fig. 6) of

coils on which broad secondary coils are super- imposed, resembling Spiroclraeta plicatilis (9). But the latter organism is much larger and has a higher number of axial fibrils. An axistyle is often present in representatives of the genus Leprospira (8), but in general the structure of leptospiral isolates differs from that of the strain described here.

Acknowledgments Thanks are due to Mr. H. G. Elerie and Elsbeth

a spirochetal cell demonstrated the presence of an van Lohuizen of the Technical and Physical outer membrane (OM) consisting of more than Engineering Research Service at Wageningen for one layer and surrounding both protoplasmic help in taking the electron micrographs.

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NOTES 1771

1 . BLAKEMORE, R. P., and E. CANALE-PAROLA. 1973. Morphological and ecological chal-acteristics of Spirochaetcrpliccrtilis. Arch. Mikrobiol. 89: 273-289.

2. BREZNXK, J . A. 1973. Biology of nonpathogenic, host-associated spirochetes. Clit. Rev. Microbiol. 2: 457-490.

3. CANALE-PAROLA, E., 2. UDRIS, and M. MANDEL. 1968. The classification of free-living spirochetes. Arch. Mikrobiol. 63: 385-397.

4. ECHLIN, P. 1964. Intra-cytoplasmic membranous in- clusions in the blue green alga, Anncystis nidrrlrrtrs. Arch. Mikrobiol. 49: 267-274.

5. KUSHIDA, H. 1961. Styrene-methacrylate resin, em- bedding method for ultrathin sectioning. J . Electron Micros. 10: 16-19.

6. ROBINOW, C. F. 1958. Irr Genelal Microbiology. Edited by R. Y . Stanier, M. Doudoroff and E. A. Adelberg. MacMillan & Co., Ltd., London.

7. RYTER, A , , and J . PILLOT. 1965. Structure des spirochetes. 11: Etude du genre Cristispircr au micro- scope optique et au microscope electronique. Ann. Inst. Pasteur, 109: 552-562.

8. SMIBERT, R . M. 1973. Spirvcl~netoles, a review. Crit. Kev. Microbiol. 2: 491-552.

9. SMIBERT, R . M., E. CANALE-PAROLA, D. A. KUHN, 0. FELSENFELD, and L. H. TURNER. 1974. The spirochetes. 111 Bergey's Manual of determinative bacteriology, 8th ed. Williams and Wilkins Company, Baltimore, Md. pp. 167-193.

10. STALHEIM, 0. H. V . 1973. Chemical aspects of lep- tospirosis. Crit. Rev. Microbiol. 2: 423-456.

11. ZUELZER, M. 1912. UeberSpirucl~oetcrplicc~tiIis Ehbg und deren Verwandtschaftsbeziehungen. Arch. Pro- tistenkd. 24: 1-59.

Survival value of chemotaxis in mixed cultures

WENDY K. PILGRAM~ A N D FRED D. WILLIAMS^ Deporlmetlt ofBcrcterivlogy, I V I I ~ Strite Ut1i1'ersity. Atnes, IA, U.S.A. 50011

Accepted September 21, 1976

PILCRAM, W. K., and F. D. WILLIAMS. 1976. Su l~ iva l value of chemotaxis in mixed cultures. Can. J . Microbiol. 22: 177 1- 1773.

A motile, chemotactic strain of Prote~rs rnirol~ilis outgrew a motile. non-chemotactic mutant in a semisolid, amino acid medium, although the two strainsgrew equally well in broth.

We assume that bacteria capable of a chemo- tactic response will find themselves at a selective advantage in an environment that allows the establishment of fairly stable gradients of nutri- ents or waste products, but we know of n o studies that have experimentally tested this assumption. The study of Smith and Doetsch (2) presented evidence that a motile, chemotactic strain of Pseudomonas j!uorescer~s outgrew a non-motile straln in stationary broth culture, but they presented no evldence that the selective advantage was due to chemotaxis and not ran- dom motility. Thus, they carefully limlted the interpretation of their studies t o the survival value of motility in mixed cultures. As part of our studies on the mechanism of swarming of Proreus we isolated several motile, non-chemo- tactic mutants of Proteus mirabilis, and decided to test this hypothesis using one of these mutants and its motile, chemotactic wild type.

'Present address: Iowa Beef Processors, South Sioux City, Nebraska 68776.

2 A ~ t h o r to whom correspondence should be sent.

The mutant is designated Nsw203 and was derived from our wild-type P. n~irabilis IM47 after mutagenesis with N-methyl-N1-nitro-N- nitrosoguanidine (3). It has lost its chemotactic response to all amino acids (and presumably to all other attractants as well), but is still motile. In a soft agar medium a central inoculum will gradually increase in size until reaching the edge of a plate, but n o chemotactic bands are formed. The mutant displays more frequent tumbling than the chemotactic wild type.

T o determine viable cell number of both the wild type (IM47) and the non-chemotactic mutant (Nsw203) in mixed culture, we selected for spontaneous antibiotic-resistant strains of each. We used a strain of 1M47 that was resistant to 50 pg/ml nalidixic acid and sensitive to chloramphenicol, and a strain of Nsw203 that was resistant to 5 pg/ml chloramphenicol but sensitive to nalidixate. These strains were used for the pure culture studies as well as in the mixed cultures.

Quantitative growth studies were conducted

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