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Antibiotic Production by Soil Actinomycetes

By John William Goudie



Abstract:

Students attempt to identify Streptomycetes with antibiotic properties in local soil samples.

Type of Activity:

  • Hands-on
  • Open ended
  • Group/cooperative learning

Target audience:

  • Biology
  • Advanced biology
  • Biotechnology
  • Microbiology


Introduction

This lab shows how basic science can lead to discovery. Once classified as a Fungi, the order Actinomycetales, which means ray fungus, includes many soil bacteria. A few are normal inhabitants of the mouth and several species are pathogenic. The group of Actinomycetes that interests us most are the Streptomycetes. None of the Streptomycetes are pathogenic but many are medically significant because of the antibiotics that they produce. The purpose of this investigation is to try and discover Actinomycetes from local soil samples that have antibiotic properties. The use of known species of Streptomycetes that produce antibiotics can be easily seen as they produce zones of inhibition on a lawn of bacteria; the antibiotic activity from local soil samples is variable and takes many samples to find a few that produce zones of inhibition.

Notes for teacher:

  1. Fungi do not grow overnight; they may take a few days to a week to grow.

  2. Known Species of Streptomycetes can be ordered from:
    American Type Culture
    12301 Parklawn Drive
    Rockville, Maryland 20852
    Toll Free: Call 1-800-638-6597
    Fax 1-301-231-5826

  3. Use media that is selective for Actinomycetes like Hickney-Tresner Agar from Gibco. Without selection media, the plates become over-grown with other bacteria. Best to acquire a copy of the Difco Manual from Difco, Inc. Detroit, Michigan 48232. This manual describes dehydrated medias and reagents for microbiology.

  4. Plugging straws can be made from plastic straws about 3 inches in length. These needed to be sterilized.

Materials needed:

  • Cultures Streptomycetes such as S. steffisburgenis and S. griseus
  • Soil samples
  • Cultures of Micrococcus luteus
  • Petri dishes
  • Thin layer chromatography plates
  • Methylene chloride: methanol solvent (8:1)
  • Plugging straws
  • Incubator or room temperature
  • Toothpicks (sterile)
  • Q-tips
  • Baggies
  • Nutrient agar plates


Procedure

Part I. Cultivation of organisms
  1. Be sure to use a selective agar for Actinomycetes.
  2. With a sterile toothpick or cotton swab, remove spores from a colony and spread them across a fresh agar plate. Use Streptomyces steffisburgenesis as a control culture; it produces a bright yellow antibiotic called steffimycin.
  3. Incubate at 28 degrees C for 1 week.
  4. Note different colors, colony surface shapes.

Part II. Thin layer chromatography

  1. Using silica gel chromatography plates, score each plate about 0.5" from the bottom. This line serves as the origin.
  2. Be sure to handle the plates by the edges.
  3. Use a plugging straw to create a 4mm agar plug. Use sterile forceps to remove the plug from the agar plate. Place the plug on the origin line of the TLC plate. Students could run unknown soil plug along side known culture plug.
  4. Allow the plug to sit on the origin for 15 minutes.
  5. Add 3 drops acetone to the top of each plug.
  6. Allow the plug to sit on the origin for an additional 15 minutes.
  7. Remove the plug and discard.
  8. Place the plate in a TLC chamber (Beaker with watch glass) previously equilibrated with methylene chloride: methanol (8:1). This should be done in a hood.
  9. Allow the chromatography to run until the solvent line is within 1/8 inch from the top of the plate.
  10. Mark location of solvent front.
  11. Remove the plate from the chamber and allow to dry in a fume hood.

Part III. Bioautography

  1. Inoculate 50ml of nutrient broth with Micrococcus luteus and allow to grow overnight, shaking at 28 degrees C.
  2. Using a sterile cotton swab, spread a lawn of M. luteus over the nutrient agar plates.
  3. Carefully lay the TLC plate on the same nutrient agar plate. Sterile forceps or spatulas may help.
  4. Allow the plates to sit in the refrigerator for 30 minutes.
  5. Mark where the corners of the TLC plate sit on the agar plate for orientation.
  6. Remove the TLC plate using sterile forceps.
  7. Incubate overnight at 28 degrees C.

Part IV. Interpretation

  1. Look for growth of M. luteus on the indicator plate; it should be a yellow lawn.
  2. The lawn should be interrupted by clear zones of inhibition where antibiotic-producing Actinomycetes (hopefully TLC zones will correlate with zones around plugs.
  3. To determine Rf values, measure the ORIGIN (not the bottom) of the TLC plate to the middle of each inhibition zone; the formula for Rf value is: Rf = Distance from the origin to the center of the zone Distance from origin to the solvent line

Part V. Experimental

  1. Have students collect soil samples in baggies from a variety of environments.
  2. Dilute the soil samples by mixing 1 gram of soil in 99 grams of sterile water.
  3. Pipet 0.5ml of the solution onto a selective agar plate for Actinomycetes and spread over the surface with a sterile cotton swab or Q-tip.
  4. Grow for about 1 week at 28 degrees C or room temperature.
  5. Examine for fungi.
  6. Repeat the procedure above to determine if any fungi samples have antibiotic properties.

Part VI. Evaluation

  1. Did you observe any unknowns with antibiotic properties?
  2. How do you go from discovery of antibiotic properties to isolation of the active factor?
  3. How do you get a new compound to market?

Part VII. Extensions

  1. Do we have enough new antibiotics to fight the new resistant bacterial strains? Teachers should discuss this problem of resistant strains of bacteria with students. Check newspapers and magazines for articles dealing with these new resistant strains and what antibiotics are needed.
  2. Investigate how antibodies work. Use sensitivity discs to demonstrate the effects of antibiotics on different lawns of bacteria. These discs are available from most supply companies.

Good Luck!


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