|Gram-positive Bacteria as a group are common soil organisms. Bacillus species are very common mesophilic, aerobic heterotrophs that produce heat-resistant endosopores. The enrichment and isolation of Bacillus is straightforward - a sample of soil (rich in Bacillus) is heated to kill non-spore-forming mesophiles, and then plated on rich media and incubated aerobically at 30C. Thermophiles will not grow at this temperature, and anaerobic spore-formers (e.g. Clostridium) will not grow aerobically. Other mesophilic aerobic endospore-formers (e.g. Heliospirillum) are phototrophic, scarce, and require lots of light for growth.|
|Although the focus of this course is on Bacteria & Archaea, most eukaryotes are also microbial. One of the most important of the microbial eukaryotes to humans has been the unicellular fungus Saccharomyces cerevisiae, the brewers & bakers yeast. In this lab, we will attempt to isolate yeast from rotten fruit using two general properties: preference for acidic environments and resistance to broad-spectrum antibiotics. We often also get filamentous fungi in this isolation. In addition, we get ampicillin-resistant Bacteria, usually members of the Sphingobacteriales, that are inherently resistant to penicillin-type antibiotics.|
|Purple non-sulfur Bacteria are anaerobic phototrophs. Enrichment of these organisms therefore relies on providing an anaerobic environment without a fermentable carbon source and plenty of light. The anerobic environment is generated microbiologically - aerobes use glycerol until the oxygen is depleted, and then are unable ferment it and so stop growing. Not many organisms can grow on glycerol anerobically, but photosynthetic anaerobes can grow either autotrophically (getting carbon from CO2) or photochemotrophically (using glycerol for carbon but not energy). The tubes turn dark brown to bright red, or sometimes green, because of the organisms photopigments.|
|In this enrichment, we rely on a simple artificial sea water to provide basic mineral/ion requirements, and add agar as the only carbon and energy source. Agar degraders will begin to break down these substrates as they grow. However, other organisms can also then grow on the sugars released by this degradation. Separation of the degraders from the organisms living on leftovers occurs when the organisms are plated out and forced to make a living by themselves. Most often, we get one of two classes of bacteria in this enrichment; white colonies of the family Cytophaga and yellow colonies of the family Flavobacteria.|
Although most will think of environments like Yellowstone hot springs or deep-sea smokers when they think of thermophiles, there are lots of microenvironments that are hot at least periodically. It might also be that cooler environment might contin some numbers of thermophiles from other envirnments "chillin' out" on the chance that it might get hotter. The goal is to isolate organisms that can grow heterotrophically at high temperatures (55°C). The media is just standard LB, made in mineral water instead of distilled water because many thermophiles have higher than usual requirements for some metal ions.
|Sergei Winogradsky was the pioneer in the investigation of microbial ecology. One of the methods he developed for the study of microbial nutrient cycling in the environment is the Windogradsky column. These can be set up in an amazing variety of ways to study sulfur, nitrogen, carbon, phosphorus, or other nutrients. In our case, we set the columns up looking for conspicuous sulfur-cycling organisms and photosynthesizers.|
|Since the early days of microbiology, it has been known that cell counts of environmental samples obtained by cultivation are much lower, by many orders of magnitude, than direct microscopic cell counts. Some of this discrepency is attributable to differing requirements of organisms. In other cases, organisms are known to enter a noncultivatable resting state, and many organisms rely on each other for any of a variety of reasons and cannot be cultivated in isolation. Imagine mixing all of the nutritional requirements of a rabbit (carrots, water, air) in a huge fermentor, innoculating with a big chunk of forest, and hoping to culture rabbits!|
|Most Microbiology labs involve an "unknown". Rather than identify some boring standard domesticated teaching lab bacterium from a pure culture or simple mixture, in this experiment you will take something, probably several somethings, who knows what, that you've isolated in the experiments above, and then identify them phylogenetically from the sequence of their small subunit ribosomal RNAs. The experiment has two components: 1) Some molecular biology that results in ssu-rRNA sequences from these organisms, and 2)The molecular phylogenetic analysis of these sequence (this is the Term Project).|