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04/20/09 Name_______________________________ Section______________________________ Introductory Microbiology Pre-lab Assignment Isolation of an Antibiotic Producer from Soil 1. (1pt) Name four genera of microbes and their microbial type that produces a vast number of antibiotics. 2. (1pt) Name two ways industrial microbiologists may chemically modify antibiotics. 3. (1pt) What are the advantages of modifying antibiotics? 4. (1pt) True or False. Actinomycetes are categorized as fungus. 5. (1pt) Define antibiosis. 1 Lab Exercise ISOLATION OF AN ANTIBIOTIC PRODUCER FROM SOIL Soil is the major repository of microorganisms that produce antibiotics capable of inhibiting the growth of other microorganisms. Clinically useful antibiotics have been isolated from several groups of soil microorganisms, including bacteria (Streptomyces sp., Bacillus sp.) and fungi ( Penicillium sp. Cephalosporium sp.). Although many organisms in soil produce antibiotics, only a small portion of new antibiotics is suitable for medical use. Also, industrial microbiology is directing its energies toward chemical modification of existing antibiotic substances. This is accomplished by adding or replacing chemical side chains, by reorganizing intramolecular bonding, or by producing mutant microbial strains capable of excreting a more potent form of the antibiotic. These chemically modified drugs have been responsible for the prevention of antibiotic resistance, minimizing adverse side effects in the host, and increasing the effective spectrum of a given antibiotic. In this experiment, an attempt will be made to isolate antibiotic-producing actinomycetes from soil. These spore forming bacteria are abundant in most soils and account for much of the breakdown of organic matter that occurs there. In fact, the odor of freshly turned soil comes from the volatile compounds these bacteria produce. However, the greatest impact actinomycetes have comes from the ability of some of them to produce antibiotics. Most antibiotics in current use are produced by species of Streptomyces. On an agar plate inoculated with soil, numerous Streptomyces colonies develop. The colonies are easy to recognize by their pastel colors, characteristic texture (a hard mass that extends into the agar), and soil-like odor. Some of the filaments grow into the agar. The rest extend up from the agar and form chains of spores at the tips. The spores give the colony its characteristic pastel color. Although their general growth pattern is fungus-like, actinomycetes are distinctly prokaryotic in size and structure. In cross section an actinomycete mycelium looks like a typical Gram positive bacterial cell. 2 Day 1 – Primary Isolation Unless the organisms in a soil sample are thinned out sufficiently, the isolation of potential antibiotic producers is nearly impossible. As indicated in the diagram, it will be necessary to use a series of six dilution tubes to produce a final soil dilution of 10-6. Procedure Overview 3 Day 1 - continue Materials per group of 2 students: soil sample (will share with bench) 6 test tubes 0.9% sodium chloride (physiological saline) in flask 3 glycerol yeast extract agar plates 6 - 1ml pipets & pump 1 - 10ml pipet & pump bactispreader 95% ethyl alcohol in screw top jar Bunsen burner striker wax pencil spatula Materials for entire class: scale weigh boats vortex scissors parafilm Procedure: 1. Label six test tubes #1 through #6 with a wax pencil. Using a 10ml pipet, dispense 9ml of saline into each tube. 2. Weigh out 1g of soil and deposit it into tube 1. Cover with parafilm. 3. Vortex mix tube 1 until all soil is well dispersed throughout the tube. Mix for at least 1 minute. 4. Make a tenfold dilution from tube 1 through tube 6 by transferring 1ml from tube to tube. Use a fresh pipet for each transfer and be sure to pipet mix thoroughly before each transfer. 5. Label three glycerol yeast extract agar plates with your initials, lab section, and the following dilutions: 1:10,000, 1:100,000, and 1:1,000,000. 6. Transfer 1ml from the soil mixture in tubes 4, 5, and 6 to the appropriate plate. 7. Spread the organisms over the agar using the bactispreader. 8. Incubate the plates at 300C for 5 – 7 days. 4 Day 2 – Colony Selection and Inoculation The objective in this laboratory period will be to select actinomycete colonies that may be antibiotic producers. The organisms will be streaked on TSA plates that have been seeded with Staphylococcus epidermidis. After incubation, students will look for evidence of antibiosis. Antibiosis is an association between two or more organisms that is detrimental to at least one of them. In this experiment, if the soil organism is an antibiotic producer, the growth of S. epidermidis may be inhibited. Fourth period is optional 5 Day 2 – continue Materials per group of 2 students: primary isolation plates from previous lab session 4 TSA deeps 4 sterile petri plates TSB culture of Staphylococcus epidermidis 1 – 1ml pipet pipet pump Bunsen burner striker inoculating needle positive control- culture plate of soil antibiotic producer (see step 8 below) Materials for entire class: hot plate with 500ml beaker half filled with water Procedure 1. Place 4 TSA deeps in boiling water until the agar melts (about 5 minutes). While the agar is melting, go to step 2. 2. Label Petri plates #1 through #4 and with your initials and lab section. Plate 4 will be the positive control. 3. Remove the deeps from the water and allow them to cool until you can comfortably hold the tubes in your hand (about 8-10 minutes). 4. After TSA deeps have cooled, inoculate each one with 1ml of S. epidermidis 5. Pour the contents of each inoculated tube into separate Petri plates. Allow the agar to solidify (about 10 minutes). 6. Examine the three primary isolation plates for the presence of actinomycete colonies. They have a dusty appearance due to the presence of spores. They may be white or colored. Select three different suspect colonies 7. Using a sterile inoculating needle, scrape spores from actinomycete colonies on the primary isolation plates to inoculate the seeded TSA plates labeled #1 through #3 (see diagram on previous page). Use inoculum from a different colony for each of the three plates. 8. Repeat Step 7 using the positive control culture for plate #4. 9. Incubate the plates at 300C until the next laboratory session. 6 Day 3 – Evidence of Antibiosis Students will examine the four plates from the previous laboratory session for evidence of antibiosis (inhibition of S. epidermidis growth) and confirm results. Materials per 2 students: plates from previous laboratory session Procedure: 1. Observe plate for presence of antibiosis (clear zone around actinomycete colony). 2. Record your results in the lab report. 3. Make a smear and simple stain of your soil isolate(s), control, and Streptomyces species. 7 Name____________________________ Section_________________ Introductory Microbiology Lab Report Isolation of an Antibiotic Producer from Soil 1. (1pt) How are Streptomyces colonies recognized on agar plates?__________________ ________________________________________________________________________ ________________________________________________________________________ 2. (1pt) Evidence of antibiosis would be observed if Staphylococcus epidermidis growth is ________________________________________________. 3. (1pt) Describe the procedure used to detect evidence of antibiosis. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 4. (1pt) Results: Evidence of Antibiosis Plate # Antibiosis (+ or -) 1 2 3 4 5. (1pt) Microscopic observations under 100X magnification after smear and simple stain. Soil isolate Control Streptomyces sp. Choose a soil isolate sample that was positive 8 for antibiosis. Student Microscope Checklist When finished using the microscope, complete the following. Check off each step when completed. Name: Lab Section: Date: Microscope Number: HCC Bring the stage to its lowest level. Click the 4X objective into place. Remove the slide and dispose of appropriately. Wipe prepared slides until ALL oil/dirt is removed (top and bottom). Clean objective lenses with swabs, and, if using oil, liquid lens cleaner. Clean eyepieces with dry swabs. Clean condenser lens with dry swabs. If necessary, clean the stage with a damp Kimwipe. Turn the light switch OFF. Do not move the microscope for a few minutes before putting it away. Do NOT wrap the cord around the base. Fold the cord and secure with velcro. In order to fit back in the microscope cabinet cubicle, the head must be rotated so that the eyepieces are facing away from the stage. Replace the dust cover. Return the microscope to the appropriate location in the cabinet. 9