Add pond water to 20 ml BIM in a 25ml Balch tube to the top of the neck of the tube so no air is left.
Put a syringe needle into a bung with the tip just through to the inside.
Put a paper towel over the base of the needle (to absorb the liquid displaced from the tube by the bung), then use it to stopper the bottle. The idea is to get the bung in trapping as little air in the tube as possible. The needle allows the displaced air (and fluid) to escape when you push the bung in, and allows the escape of gas generated during the incubation. If you can't press the bung in, it probably means the needle isn't quite far enough in. Leave the needle in place during incubation to allow gas to escape.
Incubate for 2-3 weeks in the 30C lighted incubator.
sulfur bacteria is indicated by red, purple or brown color. Growth of sulfur reducing organisms is indicated by an opaque black color (metal sulfides).
When the tube is brightly colored , mix and draw a sample of the enrichment from the tube by syringe and streak onto a BIM plate. Seal plates in anaerobe jars with an active CO2/H2 generater and Paladium catalyst, and incubate as before for 1 week.
Streak out a fresh plate with a well-isolated pigmented colony. Incubate 1 week as before. Examine individual colonies carefully and make wet mounts for microscopic morphology.
Can you identify the genus of your isolate? The purple non-sulfur Bacteria can be generally classified by gross microscopic morphology into 4 groups: Rhodomicrobium, Rhodobacter, Rhodopseudomonas and Rhodospirillum. Another group is Rhodocyclus, which looks like a lockwasher, but we have yet to isolate this specie. In previous semesters, the majority of isolates have been Rhodomicrobium, but Rhodospirillum is also common.