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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 isolate something, probably several somethings, who knows what, from environmental
samples you bring in, and then identify them phylogenetically from the
sequence of their small subunit ribosomal RNAs. The experiment has three components: 1) The isolation of some organisms, 2) Some molecular biology that results in ssu-rRNA sequences
from these organisms, and 3)The molecular phylogenetic analysis of these sequence (this
is the Term Project). |
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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. |
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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. |
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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. |
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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. |
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The goal is to isolate organisms that can grow heterotrophically using caffeine as their sole carbon and nitrogen source. If Graduate students can do it, why not Bacteria? The media is therefore just a basal salts medium with caffeine. There are reports in the literature of caffeine degradation in wastewater by Pseudomonas putida, so it seems likely that we'll get some Pseudomonads. What else? Who knows? |
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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. |
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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! |
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