Microbio Lab 102 Report

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Enrichment and Isolation of
Purple Non-Sulfur Photosynthetic Bacteria
Heather Clark
December 7, 2012
Microbiology 102

Introduction
The purpose of this experiment is to isolate and enrich purple non-sulfur photosynthetic bacteria (PNSB) for observation reasons. The purple non-sulfur bacteria are of the Proteobacteria phylum, mainly in class Alphaproteobacteria with some species within the class Betaproteobacteria, (2) and important genera include Rhodospirillum, Rhodomicrobium, Rhodospeudomonas and Rhodobacter (1). These Gram-negative phototrophic bacteria are very environmentally diverse, thriving in many types of aqueous environments and sediments, as long as those areas receive enough light and provide a mostly anoxygenic environment (3). They can also be carried away via environmental conditions, such as rain, snow and wind, and transplanted outside of their normal environment (1). PNSB are physiologically and metabolically adaptable, preferring to grow in light conditions but able to sustain growth in dark conditions (facultative phototrophs), and changing their modes of metabolism depending upon their environmental surroundings. They are able to grow as photoheterotrophs, photoautotrophs or as chemoautotrophs (1).The term “purple non-sulfur bacteria” can be somewhat misleading, since the colors of all phototrophs range from purple-red through yellow and brown (3). The dramatic colors produced by this species are a result of their combinations of different carotenoid and bacteriochlorophyll pigments used during photosynthesis (3). One of their defining characteristics is anoxygenic photosynthesis, which does not produce oxygen during photosynthesis. This occurs because the bacteria do not use H2O as an electron donor, instead obtaining their electrons for use in photosynthesis via the oxidation of organic molecules (photoorganoheterotrophy) or by oxidizing inorganic molecules other than water (photolithoautrophy) for the generation of NADH and NADPH (2). By culturing them under anaerobic conditions, we will utilize the ability of PNSB to grow in the absence of oxygen to selectively isolate them from the water samples. Another characteristic of the bacteria we will exploit to select them is their preferential use of organic carbon source, providing them a source that encourages their growth but is not normally used as a substrate for other organisms that may also be found within the sample, such as sodium succinate or malate (1). Further selection will include a light source, as this is a phototrophic species. Since PNSB can only tolerate low levels of hydrogen sulfide, the final method for selection will be providing them an environment free of H2S.

Materials and Methods
Procedural methods followed for Experiment 11B are listed on pages 50-53 of the General Microbiology Lab Manual for Fall Semester, 2012. Water samples offered were obtained from: A. A pond near Hayward, Wisconsin, collected 9/9/12

B. A “mystery sample” of unknown origin and unknown date of collection C. The Yahara River, Monona, Wisconsin, collected 10/2/12 We chose samples from A and B. The only deviation from the lab instructions was the omission of a wet mount

Results
Observations of succinate broth in enrichment after seven days of incubation in the light: | Sample A| Sample B|
Macroscopic Appearance| Mainly yellowish with reddish brown sediment on the bottom| Mainly yellowish with dark red sediment on the bottom| Microscopic Appearance| Numerous coccus-shaped cells| Numerous rod-shaped cells|

Observations of Isolates in Period 3 and 4:
| Sample A| Sample B|
Colony Characteristics | Color| Reddish brown| Venous blood red, almost purple| | Other Features| Edges lack defined borders, somewhat amorphous. When touched with inoculation needle, substance was pliable.| Edges had defined borders. When touched with inoculation needle, substance was pliable.| Microscopic Shape| Round| Rods...
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