Growth in citrate broth was turbid, rusty brown in color with some slime formation. The glucose broth had a milky white color with pronounced slime accumulation. Cells grown on the citrate medium were uniformly spaced within the sheath and ranged from 1 to 4 μ in length and 1 to 2 μ in width. The sheath was uniform in shape and ranged from 2.0 to 2.5 μ in width in young cultures. Sheath irregularities began to appear by the third day and were the greatest, 1 to 12 μ, by the fifth day when they gave the appearance of disintegrating. Cells grown on the glucose medium had clear intracellular structures and ranged from 2.5 to 6.0 μ in length, 1.0 to 2.5 μ in width with the sheath ranging from 1.5 to 3.5 μ

Goss, Robert C.

Tjaden, David H.

Name not available

Sheath and Cellular Irregularities of Sphaerotilus natans Caused by Ferric Ammonium Citrate and Glucose

University of Northern Iowa

Proceedings of the Iowa Academy of Science Volume 76 Annual Issue Article 7 1969 Sheath and Cellular Irregularities of Sphaerotilus natans Caused by Ferric Ammonium Citrate and Glucose David H. Tjaden University of Northern Iowa Robert C. Goss Let us know how access to this document benefits you Copyright ©1969 Iowa Academy of Science, Inc. Follow this and additional works at: https://scholarworks.uni.edu/pias Recommended Citation Tjaden, David H. and Goss, Robert C. (1969) "Sheath and Cellular Irregularities of Sphaerotilus natans Caused by Ferric Ammonium Citrate and Glucose," Proceedings of the Iowa Academy of Science, 76(1), 38-44. Available at: https://scholarworks.uni.edu/pias/vol76/iss1/7 This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact scholarworks@uni.edu. Sheath and Cellular Irregularities of S phaerotilus natans Caused by Ferric Ammonium Citrate and Glucose DAVID H. TJADEN1 AND RoBERT C. Goss Abstract. Growth in citrate broth was turbid, ru_sty brown in color with some slime formation. The glucose broth had a milky white color with pro-nounced slime accumulation. Cells grown on the citrate medium were uni-formly spaced within the sheath and ranged from 1 to 4 µ in length and 1 to 2 µ in width. The sheath was uniform in shape and ranged from 2.0 to 2.5 µ in width in young cultures. Sheath irregularities began to appear by the third day and were the greatest, 1 to 12 µ, by the fifth day when they gave the appearance of disintegrating. Cells grown on the glucose medium had clear intracellular structures and ranged from 2.5 to 6.0 µ in length, 1.0 to 2.5 µin width with the sheath ranging from 1.5 to 3.5 µ. Some confusion has existed in the nomenclature of Sphaerotilus natans. One reason for this is that S. natans can assume two or possibly three different physiological forms, which are classified as S. natans, Cladothrix dichotoma, and Leptothrix ochracea (8). These three organisms can give cise to similar cultures by appro-priate treatment ( 4, 9). Deoxyribonucleic acid base composition, estimated from the buoyant density in ceasium chloride, of S. natans and S. discophorus indicate that these two organisms are closely related (7). Identification of S. natans is based on its most characteristic structure, the filaments. These consist of chains of rod-shaped cells with rounded ends which are enclosed in closely-fitting sheaths ( 11). The sheath is slimy and optically invisible but can be demon-strated in India ink mounts (6). Various stains have been used in studying the sheath but all seem to be ineffective unless stainabl1e material is present in the culture and is absorbed by the sheath (2). The diameter of the cells has been reported to vary from 1.2-1.8 p. ( 11) and 2-3 p. (3). The differences probably are due to difficulty in differentiating between the cell and surrounding sheath. The length of the cell varies with age, habitat and nutrients. Butcher ( 1) reported cells ranging from 3-6 p. in length but Lac-key and Wattie (6) indicated 2.5-16 p. and Hohnl (4) reported lengths of 5-11 /J.· S. natans displays two basic colony shapes when cultured on a solid medium. Fresh isolates from natural habitats normally grow with a filamentous colonial form which is compared to the R-type of colony found among some bacteria ( 11) . With continued lab-oratory culturing, the R-type dissociates and an S-type develops. 1 Former graduate student, Department of Biology, University of North-ern Iowa. 38 1Tjaden and Goss: Sheath and Cellular Irregularities of Sphaerotilus natans CausedPublished by UNI ScholarWorks, 19691,969] SHEATH AND CELLULAR IRREGULARITIES 39 The colonies are strikingly different in that they are smooth, glisten-ing, and often hemispherical with a regular edge. Intermediate forms between the R- and S-colony types occur. The factors that determine colony type in Sph.aerotilus are not clear, but glucose can stimulate S-colony formation of S. discophorus. Glucose and citrate are among the carbon sources that can be utilized by S. natans (3, 11). When S. discophorus and S. natans are grown in a medium containing glucose, they exhibit a very lim-ited capacity to oxidize sugars, amino acids, and compounds of the tricarboxylic acid cycle ( 12). However, if grown in a medium deficient in glucose, they oxidize these compounds rapidly and extensively. A review of the literature indicates that S. natans is a highly variable organism. Possible explanation for the discrepandes ob-tained by different investigators may be found in the use of dif-ferent basal media, different substrate concentrations or genetic variants. The object of this study was to compare the growth and morphology of the cells and sheath of S. natans when glucose is substituted for ferric ammonium citrate in the medium. PROCEDURES S phaerotilus natans was isolated from a riffle area in the Yellow River at Volney, Iowa, using the method of Stokes ( 11) . Stock cultures were maintained on a base medium consisting of 5 g pep-tone, 0.5 g ferric ammonium citrate, 0.2 g magnesium sulfate, 0.5 g calcium chloride, 0.01 g ferric chloride and 12.5 g agar in 1 liter of distilled water. The pH was adjusted to 6.5-7.0 with 0.1 N sodium hydroxide. The glucose medium was made by eliminating the ferric ammonium citrate from the base medium and substitut-ing 0.5 g glucose. Two hundred fifty ml of the base broth were put into 500 ml, 4-baffle bottom flasks and sterilized. The flasks were inoculated with agar plugs cut wi:th a sterile :f:/:4 cork borer from 48 hour cultures grown at ambient conditions. The flasks were incubated at 35°0 in a water bath shaker. Dry weights were determined by placing the contents of the flask in a sterile blender and blending for two minutes. Two, 50 ml samples were centrifuged for 20 minutes, washed with distilled water and centrifuged a second time. The pellets were resuspended in 25 ml of distilled water and dried at 105°0 for 24 hours. Microscopic slides were made by transferring the organism to a drop of SO% India ink located at one end of the slide. The mate-rial was mixed and a thin film prepared by drawing the mixture across the slide with another slide heid at a 45° angle. Slides were 2Proceedings of the Iowa Academy of Science, Vol. 76 [1969], No. 1, Art. 7https://scholarworks.uni.edu/pias/vol76/iss1/740 IOWA ACADEMY OF SCIENCE [Vol. 76 air dried and stained with 1 % aqueous crystal violet for 2 minutes. The stain was rinsed off and the slides air dried. DrscussroN Citrate medium changes from a clear, light yellow color to a turbid rusty brown. This color remained constant after 24 hours. The glucose medium changed from a clear, light yellow to a milky white. With increased incubation the glucose medium took on a light brown color. Differences in color of the two media probably were due to the amount of ferric oxide produced or the presence of glucose. The citrate medium had a greater potential for forma-tion of ferric oxide, either chemically or by S. natans. The method of formation and deposition of ferric oxide by S. natans is not def-initely known but appears to be enzymatic (5). On the other hand, glucose represses synthesis of many enzymes ( 12) . No visible floes formed in the citrate medium. There was an even dispersion of small, suspended masses of material at all times after 24 hours. Slight slime accumulation occurred on the sides of the flasks. The glucose medium contained large amounts of white flocculent material after 24 hours. The slime characteristic was most pronounced after 24 hours and then decreased with increased incubation. At-test was used to test differences between the mean dry weights of the two cultures. The amount of growth for the first day was significantly greater in the glucose medium, Table 1. The differ-Table 1: Mean dry weight comparisons of S. natans grown on citrate and glucose media (p=.01, t>3.169). MEAN DRY WEIGHT (mg) DAY glucose citrate t-VALUE 1 26.9 17.3 5.30 2 32.3 27 .. 2 3.76 3 30.2 31.2· .31 4 24.5 38.9 1.48 5 24.5 32.0 5.77 6 28.4 27.7 .94 7 21.2 30.0 8.54 ... ence between the means were not significant ·on days 3, 4, and 6. Ori days 5 and 7 the growth on th~ citrate medium was significant-ly greater. 3Tjaden and Goss: Sheath and Cellular Irregularities of Sphaerotilus natans CausedPublished by UNI ScholarWorks, 19691969] SHEATH AND CELLULAR IRREGULARITIES 41 Cells grown on the glucose medium appeared to have nonstain-ing vacuoles or granular material (Fig. 1) . S. natans contains Figure 1: G lucose grown cells with vacuoles and granules. prominent and numerous refractive bodies of various sizes. These bodies are present in young and old cultures ( 11 ) and are poly-,B -hydroxybutryic acid (10) . Some of the cells contained two larger, terminal nonstaining spores. Cells were difficult to distinguish in 24 hour cultures and appeared to be packed very close together and completely filled the sheath, but as the culture aged it became easier to distinguish individual cells. The cell s ranged from 2.5-6.0 /J, in length, depending on age, with the longest cells appearing on day three. The width of the cells ranged from 1.0-2.5 µ, with the width of the sheath ranging from 1.5-3.5 µ, . Cell s grown on the citrate medium were uniformly spaced with- 4Proceedings of the Iowa Academy of Science, Vol. 76 [1969], No. 1, Art. 7https://scholarwo ks.uni.edu/pias/vol76/iss1/742 Figure 2: IOWA ACADEMY OF SCIENCE ·. \ ~ \ \ \ ,, \ \ \ \ \ ,, ' Ferric ammonium citrate cells uniformly stained. [Vol. 76 in the sheath, did not develop intracellular bodies and stained evenly (Fig. 2) . The cells ranged from 1-4 /.l in length and 1-2 /.l in width. The sheath, in young cultures, was uniform in shape and ranged from 2.0-2.5 /.l in width. Sheath irregularities began to ap-pear by day 3 and were the greatest, 1 to 12 /J., by day 5 when they gave the appearance of disintegrating. The cells within the sheath became smaller and took on a coccus-shape. Some false dichotomous branching was observed. Large sample techniques were used to test for differences be-tween the mean cell size produced on the two media. Differences were significant at the 0.01 level depending on the age of the organism (Table 2) . The length of the glucose grown cells was 5Tjaden and Goss: Sheath and Cellular Irregularities of Sphaerotilus natans CausedPublished by UNI ScholarWorks, 19691969] SHEATH AND OELLULAR IRR.EGULAR:ITIES 43 Table 2: Z-values for cell and sheath sizes obtained from growth on citrate and glucose media (p-.01, Z>2.58). DAY Cell Cell Sheath LENGTH WIDTH WIDTH l l.30* 8.70* 4.69* 3 9.75* 13.16* 2.44** 5 4.04* ].{)]* 6.55** 7 10.80* 3.43* 1.03** * glucose medium ** citrate medium significantly greater at all ages than the citrate cells and the width of the glucose cells was significantly greater for days 1, 3, and 7. The sheath of the glucose cells was significantly wider for the first day, but by day five the citrate sheaths were significantly wider. SUMMARY Various aspects of the growth of S. natans on glucose medium and citrate medium differ. Glucose caused greater cell length than citrate at all stages of growth tested. Cell width is greater with glucose in early stag.es (days 1 and 3) and late stages (day 7) of incubation with the width being approximately the same during the intermediate stage (day 5). The glucose medium produced greater width and abnormalities from days 3 to 7. Glucose stimulated slime formation. Glucose stimulated greater total growth than citrate for days 1 and 2. Dry weight determinations indicated greater growth on the citrate medium as the cultures aged. Further study is needed to determine whether the citrate actually stimulated greater total growth in older cultures or if the increase is a result of chemical reactions with the medium. LITERATURE CITED 1. BUTCHER, R. W. 1932. Contributions to our knowledge of the ecology of sewage fungus. Brit. Mycol. Soc., Trans. 17: 112. 2. DONDERO, N. C. 1961. Sphaerotilus, its nature and economic signif-icance. Adv. Appl. Microbial. 3: 77-107. 3. HARRISON, M. E. & H. HEUKELEKIAN. 1958. Stream pollution. Slime infestation-literature review. Sewage and Industrial Wastes 30 ( 10) : 1278-1301. 4-. HoHNL, G. 1955. Investigation into the physiology and nutrition of Sphaerotilus natans. Arch. Mikrobiol. 23:207. 5. JOHNSON, A. H. & J. L. STOKES. 1966. Manganese oxidation by S phaerotilus disco phorus. J. Bacterial. 94 ( 4) : 1543-154 7. 6. LACKEY, J. B. & E. WATTIE. J 940. Studies on sewage purification. XIII. The biology of Sphaerotilus natans Kutzing in relation to bulk-ing of activated sludge. Sewage Works J. 12(4) :669-684. 7. MANDEL, M. A. JOHNSON & J. STOKES. VTFF. Deoxyribonucleic acid base composition of Sphaerotilus natans and Sphaerotilus discophorus. J. Bacterial. 91(4):1657-1658. 6Proceedings of the Iowa Academy of Science, Vol. 76 [1969], No. 1, Art. 7ttps://scholarworks.uni.edu/pias/vol76/iss1/744 IOWA ACADEMY OF SCIENCE [Vol. 76 8. McKEE, ]. E. & H. W. WoLF, (ed.). 1963. Water quality cirteria, 2nd ed. The Resources Agency of California, State Water Quality Control Board. Pub!. No. 3-A. Pp. 333-335. 9. PRINGSHEIM, E. G. 1949. The filamentous bacteria Sphaerotilus, Lep-tothrix, Cladothrix and their relation to iron and manganese. Phil. Trans. Roy. Soc. (London), Ser. B. 605: 233-453. 10. RouF, M. A. 1964. Culture and physiology of Sphaerotilus discophorus and S phaerotilus natans. Diss. Abstr. 25 ( 3) : 1496. 11. STOKES, ]. 1954. Studies on the filamentous sheathed iron bacterium Sphaerotilus natans.]. Bacteriol. 67: 278-291. 12. STOKES, ]. & M. T. PowERS. 1967. Glucose repression of oxidation of organic compounds by SjJhaerotilus Can. J. MicrobioL 13: 557-563. 7Tjaden and Goss: Sheath and Cellular Irregularities of Sphaerotilus natans CausedPublished by UNI ScholarWorks, 1969

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Sheath and Cellular Irregularities of Sphaerotilus natans Caused by Ferric Ammonium Citrate and Glucose

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