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Classroom Notes
In order for this to be successful, enough time has to be given for the columns to develop. They will have growth in a week but they won’t fully
form and stabilize for about four weeks. This investigation is designed as an ongoing investigation in a unit on ecology, microbiology, biodiversity, evolution or any number of other biological themes.
The ideal situation would be for students to grow and investigate them over the course of the year.
Isolating these bacteria can be tricky and, like the developing column, many of them, especially the anaerobes, take time to develop. The recipes and isolation techniques given below will give you the
basic bacteria that are listed in the chart but these are only the “standard” species and there may be others that develop in the column. There are books listed in the references that will help you to identify
other bacteria.
Table 1.
Physical Properties of Bacteria
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Bacteria
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Shape
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Gram stain
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Classification
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Beggiatoa
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Filamentous, long, gliding
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-
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Non-photosynthetic Chemolithotropic
sulfur oxidizing
|
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Thiobacillus
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Rods
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-
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Colorless sulfur aerobic
Chemolithotropic
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Rhodospirillum
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Spirals, polarly flagellated
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-
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Purple, non-sulfur
Anoxygenic photosynthetic
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Rhodopseudomonas
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Rods, polarly flagellated; divide by budding
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-
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Purple, non-sulfur
Anoxygenic photosynthetic
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Chromatium
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Ovals or rod, polarly flagellated
Sulfur deposits internally
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-
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Purple sulfur anoxygenic photosynthetic
|
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Chlorobium
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Straight or curved rods; nonmotile
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-
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Green sulfur anoxygenic photosynthetic
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|
Clostridium
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Rod
|
+
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Endospore forming anerobic
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Desulfovibrio
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Vibrio
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-
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Sulfur Reducing Anaerobic
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Isolation Techniques
Unlike many of the bacteria that are used in the classroom, the above eight
bacteria, which represent the most common species found in the column, have isolation techniques that are a little more complex.
Rhodospirilium and Rhodopseudomonas – both of these organisms are non-sulfur purple bacteria and are cultured using Pfennig’s medium (Brock,
1988). The concentration of the sulfide should be reduced to .01 to .02% Na2S·9H2O or eliminated and an organic substance added to provide Carbon.
Chromatium and Chlorobium – both Chromatium (Kingdom I: Proteobacteria) and Chlorobium (Kingdom VII: Green Sulfur) are both photosynthetic anoxygenic bacteria. Though they are unrelated they have the
same function. They each use a specialized medium to isolate them. The basic procedure is to add mud to a jar and enough of the respective medium covers
the mud to a depth of .5 cm. It is then incubated for 7 days at room temperature while exposed to light. Then .1ml of the medium is then transferred
to enriched agar shake deeps and incubated and additional 4-7 in the same conditions. Slides can then be made from the isolated colonies. The media
recipes and more detailed procedure can be found in a number of microbiology laboratory manuals some of which are listed in the reference section.
Beggiatoa-(Bej je-ah to’ah) these are extremely interesting organisms. They were the initial bacteria that Winogradsky used to study the role of
microorganisms in the cycling of sulfur. Beggiatoa, when isolated under the microscope, can be observed to have sulfur granules stored in its
cells. Beggiatoa also produce filaments and can migrate. The organism is cultivated by placing them in a sulfide agar closed tube and overlayed with
initially sulfide-free mineral agar. Beggiatoa grow at a well defined interface between O2 and the H2S which migrates upward.
Clostridium-these are endospore forming bacteria. They don’t reduce sulfates and are fermentative. They have strong industrial use as producers of
ethanol, acetone and butanol. Their main habitat is soil and can have harmful effects on humans causing botulism and gangrene.
Thiobacillus-Thiobacillus are chemoautotrophs and require an inorganic source of energy. They are found under aerobic conditions that contain sulfur
or sulfides. The predominant method to isolate them is a mixture of Starkey’s Medium, Thiosulfate Medium and a coal dust innoculant. This does take time
but is effective. Another method is to take the soil sample and cook it to 80 C. Thiobacillus will sporolate and they can then be isolated and grown.
Desulfovibrio-is a sulfate reducer obligate anaerobe. They may also reduce nitrates and just use sulfate in place of the nitrates. Desulfovibrio uses a
specialized Desulfovibrio Medium to isolate them.
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