Antibiotics in Action

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    Biology Activity
    Medicine from Dirt
    Isolating Actinomycetes

    Major Concept

    Techniques for isolating antibiotic-producing soil organisms such as actinomycetes are based on standard microbiological procedures. In an optional activity, students can attempt to isolate the active antibiotic compound(s) through a microscale fermentation technique used by pharmaceutical companies.
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      General Safety Guidelines
      Microbiology Safety Guidelines
      Major Concept
      Level and Expected Student Background
      Time
      Safety
      Materials and Apparatus
      Making a Soil Extract
      Soil Extract Agar Recipe
      Procedure 3 (Optional)
      Relevant National Science Education
      Standards
      Relevant New Jersey State Science
      Education Standards

    Level and Expected Student Background

    This activity is best done with students who can follow detailed instructions and respect safety precautions when working with bacteria. They should be skilled in the use of the microscope, including oil-immersion lenses. The labs require a series of days for experimentation and follow-through. Students must be responsible for accurate note-taking while in lab.

    Time

    Each procedure would benefit from students having double periods. There are intervals of up to 7 days in between procedures to allow cultures to grow sufficiently. Total time that elapses is a minimum of 15 days (not including the optional microbe fermentation activity) with a minimum of three separate lab periods for the activities. For summation of results, allow an additional two class periods.

    Safety

    General Safety Guidelines
    Microbiology Safety Guidelines

    1. Make sure the students understand the safety precautions peculiar to working with microbes. A separate list of precautions and procedures are listed elsewhere in this module.

    2. Students should wear lab coats (inexpensive paper coats that are durable and reusable are available from a variety of biological supply companies) and be provided with inexpensive lab gloves (rolls of these “uni-hand” gloves are available from the supply companies).

    3. Provide disinfectant clean-up materials.

    4. Provide a way by which bacterial media and contaminated materials are kept separate from normal trash.

    Materials and Apparatus

    Procedure 1

    • Centigram balance or equivalent with precision to 1.0 gram and a maximum capacity of 500 grams (if preparing in advance the soil extract for agar plates)
    • 10 pipettes (10-ml capacity), 1 transfer bulb
    • 5 test tubes (minimum capacity, 10 ml), 19 mm x 150 mm
    • 2 petri dishes (sterile)
    • 60 ml sterile distilled water
    • 30 ml of soil extract agar at 450ºC (prepared in advance—see recipe)
    • Incubator, preset to 280ºC

    Procedure 2

    • Transfer loop
    • Bunsen burner
    • 5 agar slant test tubes (prepared in advance using 19mm x 150 mm tubes; agar in tube, both sterilized)
    • Incubator, preset to 280ºC
    • 30 ml of sterile water
    • 5 sterile nutrient agar plates (see recipe for Soil-Extract Agar)
    • Commercial bacteria of Sarcina lutea, Serratia marcescens, Saccharomyces cerevisiae
    • 5 sterile test tubes (19 x 150 mm)

    Making a Soil Extract

    To make a soil extract for use in agar planting:

    1. Obtain 500 grams of a rich or fertile soil, preferably that produced from decomposition of organic matter as in a forest or highly productive garden to which has been added much organic debris over the years.

    2. To this soil sample, add 1500 cm3 of tap water; heat the suspension in an autoclave or pressure cooker at 121ºC (15 pounds pressure or 15 psi) for 30 minutes.

    3. Filter the suspension first through cloth, then filter paper. Refilter if cloudy.

    4. If cloudiness persists, add 0.5 g of calcium carbonate (CaCO3) to the suspension, allow to react for 5 minutes, then filter.

    5. Adjust the final volume to 1000 cm3 with tap water. The extract should be as free as possible from turbidity.

    Soil Extract Agar Recipe

    To allow for the growth of soil organisms (bacteria, fungi, protozoans), soil dilutions are added to special soil agar (sterile) in petri dishes and incubated for specified times and temperatures. To prepare the soil agar, the following is combined:

    Soil extract (procedure given previously) 500 cm3
    Tap water 500 cm3
    Glucose 1.0 g
    Yeast extract 0.5 g
    K2HPO4 0.5 g
    Agar 15.0 g

    Procedure 3 (Optional)

    This procedure is a study of antibiotic production in liquid medium—a model of modern antibiotic production techniques known as fermentation.

    1. Using the actinomycetes culture that demonstrated antibiotic activity on the solid agar plates, transfer a sterile loop of the culture to a 125-cm3 Erlenmyer flask that contains 50 cm3 of nutrient broth (not nutrient agar). A second flask containing 50 cm3 of nutrient broth will not be inoculated and serve as a control.

    2. Both flasks should be loosely plugged with a wad of sterile cotton and incubated at 28ºC anywhere from 7 to 10 days. Ideally a shaking machine, if available, would give the added advantage of producing the actinomycetes in small pellets that sink to the bottom of the flask, leaving a clear solution or supernatant. If the solution is not clear, then the cloudiness or turbidity indicates contamination of the culture that would have to be discarded.

    3. Assuming good growth in the culture, the growth is removed by paper filtration.

    4. The filtrate (the liquid that passes through the filter paper) is heated to 60ºC for 15 minutes (this is considered to be pasteurization rather than sterilization).

    5. To test the filtrate (that contains the antibiotic produced by the actinomycetes) for effectiveness against bacteria, the antibiotic will be absorbed by sterile disks of filter paper and placed on agar plates inoculated with bacteria. The plates to be used for this testing must first be prepared as follows:

      1. For each dish, add 10 cm3 of sterile nutrient agar. A second layer of agar is added that contains the cells of a bacterium that was previously tested and found to be sensitive to the antibiotic. To prepare this agar that contains the bacteria, take 100 cm3 of nutrient agar and sterilize. Allow the liquid to cool to 45ºC and add a loop of the bacteria from the water culture used previously. From this, pour 10 cm3 of the warm, inoculated agar onto the surface of the agar already present in each of the plates previously prepared.

      2. After the agar has solidified, add the filter paper disks that have been immersed in the filtered liquid containing antibiotic from the actinomycetes. Disks also should be immersed in the control liquid as well. Four disks, two from the actinomycetes and two from the control, are placed in a circle on top of the agar. Incubate the plates at 28ºC for 48 hours.

      3. Examine the plates after the incubation period has finished. If the liquid into which the disks were immersed did contain an antibiotic produced by the actinomycetes, then there should be a clear area or halo around any one of the disks containing antibiotic. The clear area is due to inhibited growth of the bacteria. Were halos produced around those disks immersed in the control solution?

    Relevant National Science Education Standards

      Unifying Concepts and Processes — The activity studies the interaction of different components of a biological system, depends on drawing conclusions based on evidence, and involves measurement to detect change or constancy in the systems examined.

      Science as Inquiry — The activity involves conducting systematic observations, interpreting and analyzing data, drawing conclusions, and communicating results.

      Physical Science — The activity involves chemical reactions.

      Life Science — The activity requires an understanding of the cellular nature of life, examines the behavior of organisms (both actinomycetes and the bacteria targeted by actinomycetes' antibiotic compounds), and the role of matter (antibiotic compounds) in living systems.

      Science and Technology — The activity explores the technology of antibiotic isolation and identification.

      Science in Personal and Social Perspectives — The activity shows science harnessed to address a very practical health issue.

      History and Nature of Science — The activity demonstrates the nature of scientific knowledge.

    Relevant New Jersey State Science Education Standards

      5.1 The activity involves conducting systematic observations, interpreting and analyzing data, drawing conclusions, and communicating results.
      5.3 The activity involves mathematics.
      5.4 The activity examines the science behind the technology of antibiotics.
      5.5 The activity requires understanding of the basic characteristics of living organisms and examines a rather curious life form, the actinomycetes.
      5.6 The activity investigates the chemical behavior of matter (antibiotics) in biological systems.

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