Factors Affecting Filamentous Growth of Sphaerotilus natanst

GAUDY, ELIZABETH (University of Illinois, Urbana), AND R. S. WOLFE. Factors affecting filamentous growth of Sphaerotilus natans. Appi. Microbiol. 9:580-584. 1961. Filamentous growth in cultures of Sphaerotilus natans can be measured and compared with total growth by a standardized procedure of winding filaments around an inoculating needle. Filaments and residual growth are then separately washed on Millipore filters, dried, and weighed. This method has been used to study changes in the growth habit of S. natans elicited by changes in the concentration of nutrients in the medium. The concentration of peptone, in a medium containing a sugar, phosphate buffer, and inorganic salts, has a much greater effect on the proportion of filamentous growth than does the nature or concentration of the carbon source or the concentration of phosphate buffer. Filament formation is significantly inhibited by concentrations of peptone greater than 0.25 %; further increases in peptone concentration stimulate the production of large amounts of capsular material. Increasing the concentration of phosphate buffer to 0.05 M almost completely inhibits growth of S. natans. Sphaerotilus natans is a common inhabitant of polluted streams and may seasonally become an important nuisance, especially in commercial fishing areas. It has been suggested that the sudden appearance of the filamentous form of Sphaerotilus in large amounts implies the prior existence of the organism in the stream in the form of Cladothrix dichotoma (Naumann, 1933). Bahr I Supported by U. S. Public Health Service grant 6430. (1953) has reported the conversion of a culture of Cladothrix, grown in 0.05 % meat extract medium, to the Sphaerotilus form after addition of 0.3 % asparagine to the medium. The morphological variations of Sphaerotilus have been carefully defined by Pringsheim (1949a, b). Lackey and Wattie (1940), Stokes (1954), and Hohnl (1955) have estimated relative amounts of growth in various media differing in carbon source, as well as in organic and inorganic nitrogen sources. Scheuring and Hohnl (1956) made extensive studies of the ability of S. natans to utilize selected carbon and nitrogen compounds as well as sulfite pulping wastes, measuring growth by visual estimation or by total dry weight. In the present study, a method of estimating filamentous growth was developed and used in studying the growth habit of S. natans elicited by the variationi in concentration of nutrients in the medium. Under certain conditions a large amount of slime is produced by the cells. The isolation, purification, and chemical analysis of a polysaccharide obtained from the slime layer will be described in a future communication. MATERIALS AND METHODS Isolation. Filamentous material clinging to leaves taken from a local polluted stream was streaked on a medium composed of 0.05 % beef extract (Difco)' and 2 % agar in tap water. Plates were dried for 48 hr at 25 C before use. Standard aseptic techniques were used throughout this study. After approximately 18 hr of incubation at 25 C, the typical, curling filamentous colonies of S. natans were visible under a dissecting microscope. Well-isolated colonies were transferred with 2Difco Laboratories, Inc., Detroit, Mich. 580 [VOL. 9 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom FILAMIENTOUS GROWTH OF1 S. NA TANS a fine, curved capillary pipette to plates containing the same medium, streaked, and incubated at 25 C. Usually this procedure was sufficient to separate S. natans from contaminating organisms. In our hands this direct method was more successful than that recommended by Stokes (1954). Culture conditions. The medium used for maintaining stock cultures had the following composition (values in per cent); dextrose, 0.1; peptone, 0.025; agar, 0.2; M\gSO4*7H20, 0.005; CaCl2, 0.00125; FeCl3 6H20, 0.00025; potassium phosphate buffer, pH 7.1, 0.01 M; deionized water. Cultures were transferred weekly. Inocula for experiments were prepared by transferring the stock culture to an agar slant of the following composition: yeast extract, 0.2%; agar, 1.5%. After incubation for 24 hr at 30 C, the cells were scraped from the surface of the slant and suspended in 10 ml of sterile, deionized water. Liquid medium. The basal medium contained 0.02 % MgSO4 77H20, 0.0050% CaCl2, and 0.001 % FeCl3 616H20 in deionized water (Stokes, 1954). Concentrations of glucose and peptone, except where varied, were 0.5% and 0.05%, respectively. Potassium phosphate buffer was maintained at pH 7.1 at all concentrations used and was sterilized separately and added to the flasks before inoculation. Each sugar was also sterilized separately. The liquid medium, 100 ml in 500-ml Erlenmeyer flasks, was inoculated with 0.25 ml of cell suspension and incubated at 30 C without shaking. Maximal growth was reached in approximately 48 hr. Measurement of total growth and filament formation. After 48 hr, the contents of each flask were divided equally among three test tubes (22 by 200 mm). Removal of filaments was accomplished by winding them on a long inoculating wire, using the following standard procedure. The contents of each tube were stirred with the wire 60 times, approximately twice per second, the collected filaments pushed off the wire onto a previously weighed Millipore3 filter (SM 5 ,u pore size), and the procedure repeated. The combined filaments from the three tubes were washed while on the filter with 10 ml of deionized water and the filter placed in an aluminum weighing dish. The dish and filter were dried together, before use, at 103 C for 2 hr, cooled, and weighed. The winding procedure removed all growth from the medium except single cells and short filaments, approximately 3 mm or less in length. The remaining culture fluid from each tube was then poured onto a second tared filter and the tubes were rinsed with deionized water onto the filter. The residue was washed while on the filter with 10 ml of deionized water and the filter placed in its dish. All filters with deposited organisms were dried in an oven at 103 C to constant weight (approximately 5 hr). Mlicroscopic examination. Cell morphology and sheath 3Millipore Filter Corporation, Watertown, Mass. formation in various cultures were examined in wet mount preparations under oil immersion, using a phase contrast microscope. Observations of the form and relative amount of capsular material were made using the India ink method recommended by Duguid (1951).