11-3 Isolation of Bacillus
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A wide variety of microorganisms can be isolated from soil. In rich, moist soil, where many nutrients are available, vegetative cells of many genera of bacteria and fungi can flourish. Bacteria such as Bacillus, Streptomyces, Pseudomonas, Micrococcus, coliforms, lactic acid bacteria and (in the anaerobic pockets) Clostridium can actively metabolize decomposing plant and animal matter and various inorganic nutrients. Fungi such as molds are also active in such an environment.
As nutrients become depleted or are made less available by the drying out of the soil, vegetative cells of some of these organisms such as Streptomyces and molds can produce reproductive spores which can withstand a considerable degree of dryness and be carried in wind and water currents to a new habitat. Vegetative cells of certain other organisms, notably Bacillus and Clostridium, produce endospores (one per vegetative cell) which can withstand a wider variety of deleterious conditions such as radiation, abrasion, extremes of heat and cold, and lack of nutrients and water. Like reproductive spores, endospores will germinate when growth conditions return, and generations of vegetative cells will again thrive as long as appropriate nutrients are available. The endospore cycle, including the sporulation, germination and outgrowth events, will be covered briefly in a lab lecture, and more information can be obtained from the lecture course and a good textbook.
In this experiment, we will isolate Bacillus from soil by the use of a procedure involving extreme heat which is designed to eliminate vegetative cells (including those of Bacillus which have not already formed endospores) and reproductive spores from consideration. Therefore, the only colony-forming units we expect will be endospores when a heated soil sample is plated. (Think about why we don't have to worry about thermophiles in this experiment.) The variety of different types of colonies resulting after aerobic incubation will represent many different species of Bacillus which had saved themselves from extinction by having already formed endospores in their soil habitat prior to the heat treatment. The endospores seen microscopically when these colonies are stained will show how the endospore cycle continues on in the artificial habitat of our petri dishes. Think about what this means regarding nutrient depletion and endospore formation wherever these organisms may be growing.
Note that we use the term heat-shocking for the heating/cooling process applied to the soil suspensions. This term is actually better applied to another area of bacteriology which the instructor can explain.
Consider the fact that two oxygen relationships are represented within the genus Bacillus: Some species are strictly aerobic while others are facultatively anaerobic.
How should we incubate our plates to maximize isolation of as many species of Bacillus as possible - aerobically or anaerobically? Were we to incubate our plates under the opposite condition, what two genera could we isolate?
Soil samples from one or more midwestern sites will be available for those who didn't bring in their own sample.
One screw-capped tube containing about 12-15 ml of saline
Water bath set at 80°C
Eight 9 ml saline dilution blanks
Pipettors and sterile tips
Eight plates of Nutrient Agar
Three tubes of Glucose Fermentation Broth (with Durham tubes)
One plate of Starch Agar
Dropper bottle of malachite green (5% aqueous solution, filtered)
Figure 11-5 Dilution plates of soil sample, no heat treatment
A soil sample was diluted in saline and then plated onto nutrient agar.
Figure 11-6 Dilution plates of soil sample, with heat treatment
A soil sample was heated at 80Â°C for 10 minutes, diluted and then plated on nutrient agar.
Figure 11-7 Examples of Bacillus isolates
Colonies from the Bacillus isolation plates were picked and run through the endospore stain. Vegetative cells stain red, while spores stain green.
Dropper bottle of 3% hydrogen peroxide
Empty plastic petri dishes for the slide catalase test
Figure 11-8 Reactions of Bacillus in glucose fermentation broth
Positive (left) and negative (right) reactions of Bacillus isolates in glucose fermentation broth.
Figure 11-9 Starch test of Bacillus isolates
Reactions of Bacillus isolates iin the starch test. The isolate on the right side of the plate has a zone a clearing around it, where the starch has been degraded by amylase, and is positive in the test. The other two isolates are starch (-).
Figure 11-14 Catalase test for Bacillus isolates
A bit of growth was tested for catalase activity. Microbes that have catalase will break down added H2O2 to O2 and H2O. The O2 evolves as bubbles from the culture.