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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?

Period 1

Materials

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

1. Place about half a teaspoonful of soil in the screw-capped tube of saline and mix well. Record the details about the sample you are using (source, date of collection).
2. Prepare four, serial decimal dilutions of the soil suspension and inoculate 0.1 ml from each of the four dilutions onto a separate spread plate of Nutrient Agar. Label the plates NOT HEAT-SHOCKED.
3. Screw the cap of the tube on tightly and label the cap top with an identifying mark. Completely immerse the tube in the 80°C water bath for 10 minutes.
4. Remove the tube (with forceps) and cool it in a glass of cold tap water for a few minutes. This heating and cooling constitute the heat-shocking procedure. (What may this procedure do besides eliminating vegetative cells and reproductive spores?)
5. After mixing the suspension, prepare plates from dilutions as you did in step 2. Label the plates HEAT-SHOCKED.
6. Incubate the plates at 30°C for 2 or more days.

Period 2

Materials

Three tubes of Glucose Fermentation Broth (with Durham tubes)

One plate of Starch Agar

Dropper bottle of malachite green (5% aqueous solution, filtered)

1. Compare the two sets of plates (labeled heat-shocked and not heat-shocked)
2. Did the heat-shocking cause any noticeable effect in the variety of different types of colonies? As Bacillus colonies tend to be light or tan-colored, any brightly-pigmented colonies would be of other genera, and fuzzy, filamentous colonies would probably be molds. (Any such colonies on the heat-shocked plates may indicate faulty aseptic tech-nique!) Of the various cell types found in soil (vegetative cells, endospores, reproductive spores), what type(s) of cells could serve as colony-forming units before and after heat-shocking?
3. Determine the number of CFUs per ml of the suspension for both sets of plates and note any difference. When counting colonies, remember to choose one plate (having between 30 and 300 colonies) to determine the CFU/ml count. (Don't count all the plates!) Any large, spreading colony - such as the spiral-colony-forming Bacillus mycoides which can cover an entire plate in time - should be counted as one colony.
4. On the plates marked heat-shocked, proceed as follows, recording your observations in table form. (Save your plates until you are satisfied with your microscopic observations in step 3. You may wish to continue incubating them.)
5. Pick out at least three different, well-isolated colonies. Label them by number or letter and record their colonial appearances.
6. Then, prepare heat-fixed smears from each numbered colony for subsequent staining (step 3, below). For one of the larger colonies, prepare two smears - one from the center of the colony and one from the edge.
7. From each numbered colony, inoculate a tube of Glucose Fermentation Broth, and also spot-inoculate a sector of the plate of Starch Agar. (A separate plate should be obtained for any wide-spreading colony.) Incubate at 30°C.
8. When time permits (this period or next), stain the smears by the endospore-staining procedure
9. Look for the presence of rod-shaped vegetative cells (red) and circular or oval endospores (green). The endospores may be found within the vegetative cells and/or free. (If no endospores are seen, what may this mean? Consider time of incubation so far, and re-incubate your plates if necessary.)
10. For the smears you made from the edge and center of the same colony, explain any noticeable difference in the apparent ratio of endospores to vegetative cells between the two sites.

Period 3

Materials

Dropper bottle of 3% hydrogen peroxide

Empty plastic petri dishes for the slide catalase test

1. From each of the isolates growing on the Starch Agar plates, perform the slide catalase test (method 2 on page 144; keeping the plate covered as directed!) and record the results. Discard the slide into the disinfectant and the plastic petri dish in the usual place.
2. For each of the isolates, note the reaction(s) of the Glucose Fermentation Broth and the amylase reaction. (Recall how you did this in Experiment 7.)
3. Were the overall results as expected? Between your isolates and those of your neighbors, does amylase production appear to be 100% positive? Considering the results of the glucose broth and the catalase test, was more than one oxygen relationship seen?

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