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.
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)
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.
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.
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?
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.[Prev] | [Next]