Enrichment and Isolation Procedures

Chromatium okenii and C. weissei have so far been found only in sulfide-containing freshwater habitats, which is of primary importance for the design of enrichment experiments. Because of the selectivity of any enrichment condition, even species and strains that were not detected in the original sample from the natural habitat may become dominant in liquid enrichment cultures. Therefore, enrichment cultures are only indicated when a certain species should be enriched and isolated from a natural sample, and selective conditions for this bacterium are known. When information on the diversity of culturable species in a natural sample is desired, enrichment cultures should be avoided and dilution series in agar deeps should be prepared directly from the natural sample. Agar cultures should be incubated under conditions similar to those used for liquid cultures of the desired bacteria. The defined medium1 proved to be relatively nonspecific and may be used for the cultivation of not only Chro-matium species, but also most of the freshwater and, with the addition of appropriate concentrations of NaCl, marine Chro-

matiaceae species. A somewhat simpler, but less widely applicable, culture medium was described by Pfennig and Triiper (1981). In agar shake dilution cultures, growth may be enhanced by the addition of 3 mM acetate to the defined medium.

Chromatium species have a selective advantage when cultures with relatively low sulfide concentrations (1-2 mM) are incubated at about 20°C, at low light intensities of 50-300 lux with diurnal light and dark phases (e.g., 16 h light, 8 h dark). They keep swarming throughout the whole bottle and can be further enriched by using cell suspensions from the upper part of the enrichment culture as inoculum for subsequent enrichments (Pfennig, 1962).

The sulfide concentration initially provided in fresh culture medium does not support much growth. To achieve reasonably high population densities in enrichments or pure cultures, repeated additions of neutralized sodium sulfide solution are necessary. Additions are made when the previously added sulfide is consumed and the transiently stored S0 is nearly oxidized. The sulfide solution for feeding of cultures can be prepared by neutralizing a stirred sodium sulfide solution (60 mM) with a certain amount of sterile 2 M sulfuric acid to a pH of ~7.5. This neutralized solution has to be applied immediately. Preferably, a special device for preparation of sterile sulfide solution, neutralized and stored under CO2-pressure, would be used (Siefert and Pfennig, 1984).

Pure cultures are obtained by repeated application of the agar shake dilution method. Water agar is prepared with 1.8% (w/v) of agar. Before the solution is prepared, the agar should be washed several times in distilled water. Depending on the salinity of the sample from nature or the enrichment culture medium, appropriate amounts of NaCl and MgCl2-6H2O are added. The agar solution is dispensed in 3-ml amounts into test tubes, which are stoppered with cotton plugs and autoclaved. The agar tubes are kept molten in a water bath at 50°C. Ready-prepared, defined

1. Defined medium for Chromatium and other freshwater and marine Chromatiaceae species after Pfennig (Pfennig, 1965, 1989b; Pfennig and TrUper, 1981, 1992): The medium is prepared in an Erlenmeyer flask with an outlet near the bottom at one side. Connected to the outlet is a silicon rubber tube with a pinchcock, and a bell for aseptic distribution of the medium into bottles or tubes. The flask is closed by a silicon rubber stopper with an inlet and an outlet for gas, and a screw-capped glass tube through which additions can be made or samplings taken.

The defined basal medium has the following composition (per liter of distilled water): KH2PO4, 0.34 g; NH4Cl, 0.34 g; KCl, 0.34 g; CaCl2-2H20, 0.25 g; NaCl (only for seawater medium), 20.0 g; MgSO4-7H2O (3.0 g for seawater medium), 0.5 g; and trace element solution, 1 ml. After the medium has been autoclaved and cooled under an atmosphere of N2, the following components are added aseptically per liter of medium from sterile stock solutions, while access of air is prevented by continuous flushing with N2: 15 ml of a 10% (w/v) solution of NaHCO3 (saturated with CO2 and autoclaved under a CO2 atmosphere), 4 ml of a 10% (w/v) solution of Na2S-9^0 (autoclaved under an N2 atmosphere), and 1 ml of a vitamin Bl2 solution containing 2 mg vitamin B^ in 100 ml of distilled water. The pH of the medium is adjusted to pH 7.2 by stirring under an atmosphere of CO2 (0.5 bar pressure) for approx. 40 min. The medium is then dispensed aseptically under pressure of N2 into sterile 100-ml bottles with metal screw caps containing auto-clavable rubber seals. A small pea-sized air bubble is left in each bottle to meet possible pressure changes. Trace element solution (SL12 of Pfennig and Tmper, 1992) contains (per liter of distilled water): ethylenediaminetetraacetate-Na2, 3.0 g; FeSO4-7H20, 1.1 g; CoCl2-6H20, 190 mg; MnCl2-4H20, 40 mg; ZnCl2, 42 mg; Na2MoO4-2H20, 18 mg; NiCl2-6H20, 24 mg; H3BO3, 300 mg; and CuCl2-2H20, 2 mg. The EDTA-Na2 is dissolved first, followed by the other components. More information on preparation of media and on isolation procedures is described in Pfennig and Tmper (1992).

culture medium is prewarmed to 50°C, and 6-ml amounts are added to the tubes of liquefied agar. Exposure to air is minimized by dipping the tip of the pipette into the agar medium. Starting with a few drops of sample from nature or an enrichment culture as inoculum and using 6-8 tubes, serial dilutions are made. All tubes are then hardened in cold water and immediately sealed with a sterile overlay consisting of 1 part paraffin wax and 3 parts paraffin oil. The overlay should be 2 cm thick. Alternatively, the tubes are finally flushed with N2/CO2 (90:10) and sealed with butyl rubber stoppers. The tubes are kept in the dark for 6-12 h and subsequently incubated at the desired light intensity and temperature. During the first 2 days of incubation, the paraffin overlay is gently reheated to achieve a complete sealing effect. Well-separated pink, purple-red, purple-violet, or orange-brown colonies that develop in the higher dilutions can be removed for microscopic examination and further purification with sterile Pasteur pipettes, without breaking the tube. The cells are suspended in 0.5-1.0 ml of anoxic medium and used as the inoculum for subsequent agar shake cultures. The process is repeated until a pure culture is achieved.

When pure agar cultures are obtained, individual colonies are isolated and inoculated into liquid medium. It is advisable to start with small-sized bottles or screw-capped tubes (10 or 25 ml), and then to scale up to the regularly used sizes. Purity is checked both microscopically and by use of AC medium (Difco), adjusted to the appropriate salinity.

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