11-2 Isolation of photosynthetics

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The purple non-sulfur photosynthetic bacteria constitute a group of versatile organisms in which most can grow as photoheterotrophs, photoautotrophs or chemoheterotrophs - switching from one mode to another depending on conditions available such as the degree of anaerobiosis, availability of carbon source (CO2 for autotrophic growth, organic compounds for heterotrophic growth), and availability of light (needed for phototrophic growth).

Originally it was thought that the purple non-sulfur bacteria could not use hydrogen sulfide as an electron donor for the reduction of carbon dioxide when growing photoautotrophically, hence the use of non-sulfur in their group name. However, sulfide can be used if present in a low concentra-tion. Higher concentrations of hydrogen sulfide (in which the purple and green sulfur bacteria can thrive) are toxic to the purple non-sulfur bacteria.

Chemotrophic growth for the purple non-sulfur bacteria is achieved by respiration, although there are some exceptional ones which can ferment.

Purple non-sulfur bacteria thrive in anaerobic habitats which receive sunlight, such as mud at the bottom of shallow lakes and ponds. They can migrate from these areas and/or be swept into currents and thus can be found at any depth in lakes and streams. In the isolation of these organisms, we find it most advantageous to set up conditions for photoheterotrophic growth: utilizing a source of light, anaerobic conditions (needed for phototrophic growth by these organisms), no hydrogen sulfide, and an organic carbon source not generally utilized by other bacteria under these conditions such as sodium succinate or malate. Not only will most other types of organisms be restricted from growing, but the purple non-sulfur photosynthetic bacteria will be easily recognized by the presence of photosynthetic pigments - causing a characteristic bloom in the enrichments and (usually) reddish coloration of the colonies. Other medium considerations include yeast extract (a source of growth factors required by some photosynthetic bacteria) and ammonium chloride (the nitrogen source).

One must appreciate the fact that the purple non-sulfur bacteria would probably be overrun (crowded out) by various respiring chemotrophs from the sample if our enrichments and plates were to be incubated under aerobic conditions. Aerobically, the photosynthetic pigments would not be as visible (if at all), and picking likely colonies of these organisms would be difficult or impossible. Likewise, they would probably be overrun by fermenting chemotrophs if our enrichments and plates were to be incubated under anaerobic conditions with a popular carbon source such as glucose in the medium.

Period 1

Materials

Lake, 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 (page 135) 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.
  2. How do you expect that anaerobic conditions will be achieved after the bottle is stoppered, as the medium probably contains much dissolved oxygen?
  3. 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 plus 1.5% agar

Figure 11-1 Growth in succinate enrichment bottles

The top image shows the collection of enrichment bottles in front of an incandescent light bulb. The other figures show closeups of two enrichment bottles.

  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

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.

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.

Figure 11-2 Streak plates from succinate enrichment

A loopful of the succinate enrichment broth was streaked onto a plate of succinate agar. (The same medium as the succinate broth except 15 g/L of agar was added). The photosynthetic microbes form pigmented colonies.

Figure 11-3 Wet mounts of enrichments

Two wet mounts of photosynthetic bacteria. The top photomicrograph is of Rhodobacter sphaeroides. The bottom photomicrograph is a wet mount of the initial enrichment.

  1. Observe your plates for pigmented colonies. Ignore all white colonies, as these would not be what we are interested in. (What might they be doing on our plates?) Pick out as many different, well-isolated, pigmented colonies as you can, and label them by numbers or letters.
  2. 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.).
  3. 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.
  4. 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.
  5. 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. Incubate one tube in your 30°C tray, and incubate the other near the lamp in the 30°C incubator.

Period 4

Figure 11-4 Reactions in succinate agar deeps

Typical reactions after incubation of succinate agar deeps. Note the extensive growth in the tubes that were incubated in the light, while the more limited growth of the tube incubated in the dark. Why does growth only appear on the top of the tube incubated in the darkness?

  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?

Recall that we used Thioglycollate Medium to test for oxygen relationships of various organisms. Realize that Thioglycollate Medium does not allow for anaerobic growth that is due to (1) anaerobic respiration (where electron acceptors other than oxygen are used) or (2) light (which allows for anaerobic growth of the purple non-sulfur photosynthetic bacteria). We do not determine oxygen relationship in the above test with the Succinate Agar tubes but we are able to characterize our isolates as being either strictly or facultatively phototrophic. Recall the discussion given in the introduction to Exp. 5.

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