Standard Methods for the Examination of Water and Wastewater 3. Sulfur Bacteria 4a, Sulfate-reducing bacteria 1) To enumerate sulfate-reducing bacteria such as Desulfovibrio, use a sulfate-reducing medium.!8 noculate tubes and fill completely with sterile medium to create anaerobic conditions. For comparative purposes, incubate one or two uninoculated controls with each set of inoculated tubes. To sample volumes greater than 10 mL, pass sample through a 0.45-im membrane filter and transfer filter to screw-cap test tube with medium. If sulfate-reducing bacteria are present, tubes will show blackening within 4 to 21 d of incubation at 20 to 30°C. 2) An agar medium suitable for growth and enumeration of sulfate-reducing bacteria also is available,!° The medium consists of trypticase soy agar, 4.0%, fortified with additional agar, 0.5%, to which is added 60% sodium lactate (0.4% viv), hydrated magnesium sulfate, 0.2%, and ferrous ammonium sulfate, 0.2%. Adjust pH to 7.2 to 7.4 and sterilize. Medium should be clear and free from precipitate. Inoculate all plates within 1 or at most 4 h after agar hardens to prevent saturation with oxygen. To prevent moisture condensation on petri dish covers, replace covers with sterile absorbent tops until 10 to 15 min after agar hardens, Place uninverted plates in desiccator or Brewer jars and replace atmosphere with tank hydrogen or nitrogen by suecessive evacuation and gas replacement. Altemnatively, use a disposable anaerobic generating system.*#(51) Incubate at room temperature (21 to 24°C) or at 28 to 30°C, the optimum temperature for these organisms, Growth and blackening around the colonies is typical of sulfate-reducing bacteria and may occur at any time between 2 and 21 d, although the usual time is2to7d. 3) Media suitable for enumeration or isolation of various species of sulfate-reducing bacteria are available.20-22 b. Photosynthetic purple and green sulfur bacteria: Because these organisms are so specialized and rarely cause problems in water and wastewater treatment processes, methods for their isolation and enumeration are not included here. In certain instances they can be beneficial because of their ability to oxidize hydrogen sulfide and thus reduce odor. An excellent review is available. Also, media formulations and methods for cultivating specific members of this group of bacteria are available. ¢. Thiobacillus spp.: The growth and physiology of different species of the single-celled sulfur-oxidizing bacteria of the genus Thiobacillus have been evaluated carefully.24?5 Media’ suitable for enumeration of Thiobacitlus thioparus and Thiobacillus thiooxidans by an MPN technique are listed in Section 9240D.1. Inoculate medium and incubate for 4 to 5 d at 25 to 30°C. Growth of thiobacilli produces elemental sulfur, which sinks to the bottom with a coincident decrease in pH and turbidity of the medium. Chemical tests for formation of sulfate are necessary to confirm presence of Thiobacillus. d. Filamentous sulfur-oxidizing bacteria: 1) Beggiatoa—Beggiaioa exist in most aquatic habitats where sulfide and oxygen are both © Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment FederationStandard Methods for the Examination of Water and Wastewater present,2” including fresh and marine water, sediments, and wastewater systems. Because Beggiatoa is a multicellular bacterium there is no accurate method of determining the number of viable cells in a sample. To determine the population of Beggiatoa make a direct microscopic count of the number of filaments. Marine beggiatoas may be quite large, up to 100 um or more in diameter.28 They have not been grown in the laboratory. However, small marine species (up to 5 um diam) have been isolated.2? The media for isolating freshwater and marine beggiatoas differ slightly. a) Enrichment—Inoculate a tube of extracted hay, {| 1k1) above, with enough water and a little mud from the sample site to fill the tube to a depth of at least 8 em, Incubate for at least 1 week and examine for the presence of “tufts” or “puff-balls” consisting of tangled filaments of Beggiatoa, Examine by phase-contrast microscopy for the presence of individual filaments.3° If no “‘puff-balls”” are found, continue incubation for another week and repeat the examination. Continue examining the enrichment for up to 4 weeks before discarding it. b) Isolation—With sterilized fine-tipped forceps and using a dissecting microscope, transfer tufts of Beggiatoa from the enrichments to a small petri dish containing sterile basal medium,° 12) above. Shake the tufts with the forceps to remove adherent bacteria and transfer tufts to a new petri dish with sterile basal medium. Continue until tufts have been washed at least five times. Transfer tufts to a ‘drying plate’? containing basal medium and 1.6% agar for about | min to remove excess fluid, then transfer the tufts to the center of separate plates of either MY or MP medium,33! qs 143) and 4) above. Incubate plates at room temperature or below and examine with a dissecting microscope every 5 to 10 h for the presence of gliding filaments of Beggiatoa. Select filaments that have glided well away from the other filaments and appear to be uncontaminated and transfer them to separate plates of the same medium with a sterile, flattened wire, inoculating needle, or toothpick, Take a little agar with the filament to avoid drying the filament during transfer. Examine the first transfer plates every 5 to 10 h as before and transfer pure filaments to fresh media. 2) Thiothrix—Obligately myxotrophic Thiothrix have been isolated and characterized from sulfur springs and other bodies of flowing water that contain sulfide.31-52 Heterotrophic strains have been isolated and characterized from activated sludge wastewater treatment plants.7-3 While the techniques for the isolation of the myxotrophic and heterotrophic strains are similar, the isolation media differ. a) Isolation of myxotrophic Thiothrix—Collect tufts of the bacterium from rocks, water pipes, or other substrates. Using a dissecting microscope, pick up filaments with fine-tipped forceps, shake to remove contaminating bacteria, and transfer to sterile basal medium, § 142) above. Repeat at least five times to try to obtain filaments with little or no contamination, With a sterile Pasteur pipet transfer separate drops to the edge of either MP or MY agar [{s 143) and 4), above} petri dishes and tip the dishes so that the drops run from one side of the dish to the other. Draw off excess moisture with the pipet and incubate plates at room temperature or © Copyriaht 1999 by American Public Health Associaton, American Water Works Assocation, Water Environment FederationStandard Methods for the Examination of Water and Wastewater below for about 48 h. Examine plates under a dissecting microscope for the appearance of typical filamentous colonies. With sterile toothpicks, pick colonies that are widely separated and transfer individually to fresh plates of the same medium, Streak transferred material with a wire loop. After about 48 h incubation, examine plates and restreak colonies that appear to be pure. b) Isolation of heterotrophic Thiothrix—There are two different procedures to use, depending on the concentration and size of the Thiothrix filaments in the sample.5+ In one procedure. wash individual filaments or rosettes of large strains of Thiothrix several times in MSV broth, § 1/ 1) above, by transfer with a Pasteur pipet while observing with a dissecting microscope. Afier several washings transfer filaments to a small amount of MSV (1 to 3 mL) and plate on one or more of the following solid media, fs 1! 3)-9) above. If the filaments are small or scarce concentrate the sample by centrifuging. Then dilute 1:5 in MSY, sonicate at 30 W for 10 s, and wash three times by centrifugation at 1900 * g for 2 to 5 min. The supernatant contains free filaments that are used to inoculate one or more of the solid media, {s 1!3}-9) above. 4. References 1, GHIORSE, W.C. 1984, Biology of iron- and manganese-depositing bacteria. Annu, Rev Microbiol. 38:515. 2. HALLBRECK, EL. & K. PEDERSON, 1986. The biology of Gallionella, In D.R. Cullimore, ed. Proc. International Symposium on Biofouled Aquifers: Prevention and Restoration, American Water Works Assoc., Denver, Colo. 3. STARR, MP,, H. STOLP, H.G. TRUPER, A. BALOWS & H.C. SCHLEGE. 1981. The Prokaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria, Volume 1. Springer-Verlag, New York, N.Y. 4. STALEY, I-T., MP. BRYANT, N, PFENNIG & J.G. HOLT, eds. 1989. Bergey’s Manual of Systematic Bacteriology, Volume 3. Williams & Wilkins, Baltimore, Md. 5. ARMBRUSTER, E.H. 1969. Improved technique for isolation and identific: Sphaerotilus. Appl. Microbiol. 17:320. 6. FARQUHAR, GJ. & W.C BOYLE, 1971. Identification of filamentous microorganisms in activated sludge. J. Water Pollut. Control Fed. 43:604. 7. VANVEEN, W.L. 1973. Bacteriology of activated sludge, in particular the filamentous bacteria, Antonie van Leeuwenhoek (Holland) 39: 189. 8, DONDERO, N.C.,R.A. PHILIPS & H. HEUKELEKIAN, 1961. Isolation and preservation of cultures of Sphaerotilus. Appl. Microbiol. 9:219. 9. MULDER, E.G. & W.L. VANVEEN. 1963. Investigations on the Sphaerotilus-Leptothrix group. Antonie van Leeuwenhoek (Holland) 29:121 10. UNZ, RF. & D.G, LUNDGREN. 1961. A comparative nutritional study of three chemoautotrophic bacteria: Ferrobacillus ferrooxidans, Thiobacillus ferrooxidans, and Thiobacillus thiooxidans. Soil Sci. 92: 302. of © Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation