Isolation of Antibiotic-Producing Organisms from Soil

Betsy Barnard

Abstract

This is a lab experiment in which students attempt to isolate antibiotic-producing bacteria (of the genus Streptomyces) from soil. Even though antibiotics have gotten a bad rap in the press lately because many disease-causing organisms are multiply resistant, it is still important to discover new antibiotics. This experiment replicates the first step in the search and screen research that is currently underway in many labs worldwide. This three-part experiment has three objectives: Students will

1. Isolate an antibiotic-producing organism from soil
2. Determine the number of colony forming units (CFU's) per gram of soil sample
   
3. Become proficient in the following techniques:
   • aseptic technique
   • dilution series and plating
   • plate counting
   • treak plating
   • two assay methods
   • simple staining
   • ing oil immersion microscope

Target Audience:

Advanced Biology

Credits

This particular protocol was developed by Dr. Jerry Ensign, Emeritus Professor, Bacteriology Dept., University of Wisconsin-Madison. Classroom tested by Betsy Barnard, West High, Madison, Wis.

Time Requirements

This lab takes 7 separate class days (times listed below) over about a 4 week period: (Seems like a lot of prep and class time but it's worth it!) Drying: 10 min Explanation and Dilution plating: 2 class periods (50 min each) Observation, calculation and pure culture preparation: 1 class period single streak for assay: 10 min Assay setup: 1 class period Observation of assay: less than 1 class period

Part 1: Determine the number of colony forming units (CFU's) of the genus Streptomyces per gram of soil

Materials

• 2 plates of isolation media
  
• 1 plate of transfer media
  
• Bunsen burner, wire loop or glass spreader & alcohol
  
• marking pen
  
• 5 sterile test tubes filled with 9 ml sterile distilled water (these are the dilution blanks)
  
• 1 g. soil brought in by student
  
• sterile 1 ml pipettes, or 1 ml micropipettors with sterile tips
  

Allow soil to dry at room temperature for about a week (or for less time in a drying oven) This is done to kill off some of the many bacteria in soil that compete with those of the genus Streptomyces. Streptomyces will form spores in the dry soil and will grow on media.

B - DILUTION PLATING

1. Label 4 dilution blanks 1-4
2. Put 1 gram of soil into test tube #1 and mix well. (The volume of soil solution should be 10 ml., if it isn't add more sterile water to a line that marks 10 ml on the test tube)
3. Use a sterile pipette to transfer 1 ml of soil solution from tube #1 to blank tube #2. Mix well.
4. Use a new sterile pipette to transfer 1 ml of soil solution from tube #2 to blank tube #3. Mix well.
5. Use a new sterile pipette to transfer 1 ml of soil solution from tube #3 to blank tube #4 Mix well. 6. Use a marking pen to divide 2 petri plates of isolation medium in half. Label each half 1,2,3 and 4 to correspond to the test tubes numbered #1-4.
6. Starting with tube 4, place 0.1 ml (100 microliters) of soil solution onto the half-plate labeled 4. Immediately spread with flamed loop or glass spreader, being careful not to cross the line drawn to separate the halves. (Be sure to cool the loop on the side of the agar before spreading)
7. You may use the same pipette to continue transfers from tube #3 to half-plate #3, then continue with tubes #2 and #1 to half-plates #2 and #1, respectively. (As long as you go from less to more concentrated, you can use the same pipette)
8. Label plate with name and incubate at room temperature for 4-7 days. (I incubate them in the dark, since this mimics natural conditions of the soil.)

Part 2: Identification and pure culture of Streptomyces

Materials (per pair of students)

• 1-2 plates transfer media
• wire loop
• Bunsen burder

Procedure

1. Observe plates. Streptomyces are round, chalky colonies. Different species are white, greenish brown, gray, pink or other colors.
2. Using a flamed loop, carefully scrape a well isolated colony of Streptomyces and streak it onto a plate of transfer media. You may divide this plate in half and select 2 or more different colonies, or use another plate. This will be your pure culture.
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3. Incubate for 3-5 days at room temp. (Again, I do this in the dark)
4. Make a qualitative observation of the original dilution plates. Does it look like the number of colonies on plate 1 is 10 times more than plate 2, which is 10 times more than plate 3 etc.?
5. Choose a dilution plate that has between 30 and 300 colonies and count these colonies.
6. Calculate how many colony forming units of Streptomyces were in your original 1 g. sample.

Optional Activity

Do a simple stain of a selected colony and observe microscopic characteristics of Streptomyces.

1. Using a flamed loop, scrape part of a colony onto a drop of water on a slide. Let dry.
2. Heat fix by gently passing the slide thru the flame 3 or 4 times until it is hot to the touch.
3. Flood slide with crystal violet or methylene blue for 1 min.
4. Gently rinse and pat dry.
5. View under oil immersion lens.

Part 3: Assay

Materials

Pure cultures of test organisms (on solid or liquid media). These can be other organisms from the soil, or any other culture such as those of the genus Bacillus, Micrococcus, etc. (Any non-pathogen is acceptable for classroom use)

Procedure

1. Make a single streak of Streptomyces (from your pure culture) down the middle of a plate of assay media (same as transfer media)
2. Incubate 5 days to allow for any antibiotics to be produced.
3. Put a single streak of each test organism perpendicular to the Streptomyces streak. You can usually place 4 or five test organisms perpendicular to streptomyces streak.
4. Incubate 2-3 days at room temp or 24 hr at 30 deg. C.
5. Observe inhibition by Streptomyces.

This is a little more complicated but provides replicate information of the other assay.

Materials

• 12-24 hr. broth cultures of test organisms
• 4 petri plates of growth (assay agar)
• sterile straws (drinking straws work fine, cut into about 5 cm lengths and sterilized in foil)
• 200 microl micropipettor with sterile tips, or sterile transfer pipettes
• 95% ethanol, Bunsen burner

Procedure

1. Transfer approx. 200 microl each broth culture onto each petri plate and spread with glass rod that has been flame sterilized using 95% ethanol.
2. Using a sterile straw, transfer a "plug" of Streptomyces onto each plate of test organisms. Do this by carefully inserting the straw over the selected colony thru the agar to the bottom of the plate. Gently transfer onto the test plate. If the plug is stuck, use a sterile dowel or other device to poke the plug out. Several different plugs from different lab groups may be placed on one test plate. Careful labeling of plates is critical!
3. Incubate 2-3 days at room temp or 24 hours at 30 deg. C.
4. Observe circular zones of inhibition around susceptible test organisms.

Media Recipes

NOTE: Local labs may donate some of these ingredients.

Isolation Medium

• 0.4 g Casein
• 1.0 g Starch
• 0.5 g potassium nitrate
• 0.2 g potassium monohydrogen phophate
• 0.1g magnesium phosphate
• 0.1 g calcium carbonate
• 15 g agar
• Sterilized distilled water to final volume of 1 liter (Sterilize 15 psi for 15 minutes. Just before pouring plates, add 1 ml cycloheximide, if available, to inhibit fungal growth)

  NOTE: Pour plates fairly thick so they don't dry out . (about 45 plates per liter)

Transfer (growth) medium

• 10 g glucose
• 1 g yeast extract
• 1 g potassium nitrate
• 0.1 g potassium monohydrogen phosphate
• 15 g Agar
• Sterilized distilled water to final volume of 1 liter (Sterilize 15 psi for 15 minutes. Just before pouring plates, add 1 ml cycloheximide, if available, to inhibit fungal growth)

Assay agar

Transfer medium may be used, or use the following recipe.

• 10 g peptone
• 1 g glucose
• 15 g agar
• Sterilized distilled water to final volume of 1 liter (Sterilize 15 psi for 15 minutes. Just before pouring plates, add 1 ml cycloheximide, if available, to inhibit fungal growth)

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