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The anoxygenic photosynthetic bacteria comprise a large and heterogenous group of organisms, brought together primarily because they all use light as an energy source. These bacteria are mainly anaerobic organisms, and require a reduced compound as electron donor, such as H₂S, H₂ or an organic compound. They do not produce oxygen, whereas the cyanobacteria use H₂O as electron donor and produce molecular oxygen (O₂), carrying out a typical green plant type of photosynthesis.

The anoxygenic photosynthetic bacteria can be divided into 7 subgroups, separated on the basis of their photopigments, organization of their photosynthetic structures, and whether they accumulate sulfur granules. For this experiment we will be interested in the "purple" bacteria, placed in Subgroups 1-3 and 7. Bacteria from subgroups 4,5, and 6 had previously been grouped together as green bacteria, but recent taxonomic advances has lead to a rethinking of this categorization.

The purple bacteria, ranging in color from purple-violet, purple-red, to rose-red, have bacteriochlorophyll a or b. Subgroup #1 accumulates sulfur granules inside their cells, subgroup #2 accumulate sulfur globules outside their cells. Both subgroups 1 and 2 are referred to as purple sulfur bacteria. Organisms from sub-group #3 do not accumulate sulfur and therefore are referred to as the purple non-sulfur bacteria.

The purple non-sulfur bacteria, are facultative phototrophs and facultative anaerobes. When oxygen is present, they can grow as simple aerobic chemoheterotrophs, not differing especially from morphologically related nonphotosynthetic heterotrophs. In the absence of oxygen, and with suitable electron donors (primarily organic compounds or H₂, but in some cases H₂S at very low levels), they grow photosynthetically.

Enrichment of photosynthetic bacteria is based on their ability to grow photosynthetically under anaerobic conditions, using H₂S or organic compounds as electron donor. Inoculum sources are mud, anaerobic lake waters, or blooms from lakes or sulfur springs.

Anoxygenic photosynthetic bacteria are found in nature in aquatic environments wherever conditions are anaerobic (or reducing) and light is present. These photosynthetic bacteria can be selectively isolated by a combination of culture techniques either directly from nature or from an enrichment culture established in the laboratory.

Period 1

Materials

Pond or stream water samples from one or more midwestern sites will be available for those who didn't bring in their own sample.

Pipettes for dispensing the water samples

Flask of Succinate Broth which consists of a mineral salts solution plus the supplements indicated above: ammonium chloride (0.1%), yeast extract (0.1%) and sodium succinate (1%)

glass-stoppered bottle (approx. 60 ml)

1. Add about 5 ml of water sample to the glass-stoppered bottle. Be sure to record the details about the sample you are using (source, date of collection). Fill the bottle to the top with the Succinate Broth such that it nearly overflows. Stopper the bottle so that no air bubbles are trapped.

How do you expect that anaerobic conditions will be achieved after the bottle is stoppered, as the medium probably contains much dissolved oxygen?

2. Place the bottle 1-2 feet away from the lamp in the 30°C incubator and let it incubate for 4-7 days.

Period 2

Materials

2 plates of Succinate Agar which consists of Succinate Broth (see Period 1 materials) plus 1.5% agar

A enrichment bottle after incubation

phase scope pictures of several microbes

1. Check the enrichment bottles for the appearance of pigment (a bloom) which indicates growth of photosynthetic organisms. Mix the contents of the bottle and make a wet mount using the phase microscope (40X objective lens in place and no immersion oil; refer to the directions for using this microscope if necessary). You will probably see a variety of different types of cells as the enrichment is certainly not a pure culture. The most recognizable purple non-sulfur photosynthetic bacteria (but unfortunately the least-often isolated) will be Rhodospirillum and Rhodomicrobium (note descriptions below).
2. Streak 2 plates of Succinate Agar for isolated colonies from the enrichment.
3. Place your plates (inverted - as always!) in the anaerobic jar at the front of the lab. This jar will be sealed and the air within it replaced with an oxygen-free gas mixture (95% nitrogen, 5% carbon dioxide). The jar will be placed in the presence of light for 4 to 7 days at 30°C.

Period 3

Materials

Phase microscope demonstrations of the four major genera of purple non-sulfur bacteria

2 tubes (per isolate) of melted Succinate Agar - in 50°C water bath

A photosynthetic plates after incubation

An anaerobe jar with plates

CAUTION: When you pick up your plates (which are inverted), do not flip them right-side up until you have shaken out condensed water, if any, from the top lid.

1. Phase-scope demonstrations of at least the first two genera are available:
• Rhodospirillum: Note the spiral-shaped cells. If the cells are motile, test for phototaxis by placing your hand over the light source for about a half-second; many of the cells will reverse the direction of their movement. Keep repeating this procedure and watch one or more of the cells become fatigued, only to spring back into action a moment later.
• Rhodomicrobium: Note the oval-shaped cells connected by thin filaments.
• Rhodopseudomonas: Note the slightly curved rods which divide by budding. Rosettes of cells are produced by some species and may be evident. Microscopic differentiation of this genus from Rhodobacter can be difficult.
• Rhodobacter: Note the oval to long, straight rods which divide by binary fission.
2. Observe your plates for pigmented colonies. Ignore all white colonies, as these would not be what we are interested in. (These microbes grow as chemoheteroorganotrophs, how are they able to grow in this medium?) Pick out as many different, well-isolated, pigmented colonies as you can, and label them by numbers or letters.
3. Begin to tabulate your observations of these colonies by making note of the colonial characteristics (color, relative size, etc.). When you begin to make wet mounts (step b), you will note the consistency of the colonies (hard, soft, gummy, etc.).
4. Prepare a wet mount for each of the numbered colonies. (Save your plates in the refrigerator or at room temperature until you are satisfied with your microscopic observations.) Observe the wet mount with the phase microscope. Do not expect motility to be seen, as many of these organisms will not be motile after having grown on a solid medium for several days.
5. From your observations, determine the most probable genus for each of your isolates. If the rod-shaped organisms are difficult to determine, you can indicate either Rhodopseudomonas or Rhodobacter. Do not expect each different colony type to yield a different genus; you could have several species of one genus on your plates.
6. For each isolate (which had better be a pure culture!), inoculate two tubes of melted Succinate Agar by the method used to inoculate Thioglycollate Medium in Experiment 5.1 (page 18). Incubate one tube in your 30°C tray, and incubate the other near the lamp in the 30°C incubator.

Four major genera of purple bacteria

Succinate agar after incubation

Period 4

1. For each isolate, check the relative amounts of growth and pigment production between each of the two tubes of Succinate Agar.
2. As the original colonies on the isolation plates (which had incubated anaerobically and in the light) were pigmented, do you see pigmentation? Which tube? Aerobic or anaerobic region?
3. Do you see aerobic growth in the tubes? What physiological process would be responsible?

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