Antibiotics in Action

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    Biology Activity
    Name That Actinomycete
    Isolating Soil Organisms

    Introduction

    This exercise illustrates the general procedures used to isolate and test for fungi and actinomycetes, organisms that might have antibiotic properties, in soil samples. The activity allows you to isolate soil organisms that have the ability to decompose polysaccharides such as starch and cellulose rather than producing an antibiotic. A simple chemical test is performed to detect this decomposition action by the soil microbe.
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    Introduction
    Purpose
    General Safety Guidelines
    Microbiology Safety Guidelines
    Procedure
    Data Analysis
    Starch-Agar and Gram's Iodine
    Stain Recipes

    Purpose

    To illustrate scientific investigation based on historically important work in the search for antibiotics from soil organisms.

    Procedure

    1. Be sure to read the General Safety Guidelines and the Microbiology Guidelines linked at the top of this page.

    2. Weigh 1.0 gram of a soil sample to be used as a source of microbial decomposers. Several different sources of soil should be used including forest soil and soil with a high clay content.

    3. Add the 1.0 gram sample to 99 cm3 of STERILE DISTILLED water in a sterile bottle with a cap. Shake vigorously, allow to settle, then transfer 1 cm3 of the mixture to 9 cm3 of sterile distilled water in a sterile test tube, using a sterile pipette and transfer bulb.

    4. Obtain a minimum of two starch agar plates for each soil sample to be tested. (If preparing the starch agar plates, refer to the recipe that follows). Transfer of the soil cultures requires a sterile pipette capable of accurately measuring 0.1 cm3 of solution. Using this pipette, transfer a 0.1 cm3 sample to a starch agar plate. Spread the solution uniformly on the agar surface using a glass loop spreader that has been first dipped in 95% ethanol, briefly flamed, cooled in air, then use for spreading.

    5. Cover the agar plate and INVERT, leaving overnight at room temperature.

    6. The following day, check the plates for decomposition activity. To do this, hold the plate up to a diffuse light source and look for the presence of any clear areas.

    7. If any clear areas appear on the agar, add enough Gram's iodine stain to completely cover the agar surface. Pour off excess iodine after 1 minute.

    Data Analysis

    1. Compare the colors on the surface of the plate tested for the presence of soil organisms with the color of a starch agar plate that has been tested with iodine solution but that does not contain any soil organisms (a REFERENCE plate). What are the differences? What caused the differences?

    2. If the soil organism has chemically changed the starch, what is(are) the product(s)?

    3. What are the results of iodine reacting with starch?

    4. What are the breakdown products of starch decomposition?

    5. Can we decompose starch? Do we use bacteria?

    Starch-Agar Recipe

    1. Add 2.0 grams of soluble starch to 400 cm3 of distilled water.

    2. Add 9.2 grams of nutrient agar to the solution from Step #1, then autoclave 15 minutes at 121ºC (15 psi of steam pressure). Glassware and water to be used in the experiments also can be sterilized with the same procedure.

    3. This amount of agar will make 20 plates.

    Gram's Iodine Stain Recipe

    1. Add 1.0 gram of iodine (I2) and 2.0 grams of potassium iodide (KI) to 100 ml of distilled water.

     

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