Microbial Antibiotic Isolation Lab

I believe that it is most likely that life originated in the aqueous fraction of soil. The combination of clay particles with surfaces capable of serving as molecular templates, protection from ultraviolet radiation and oxygen, barriers to diffusion, and the great thermal inertia of deep soils, probably met the needs of postulated early life forms better than any other environment.  On the present earth there is a tremendous variety of life in soils... the dominant forms there are microorganisms, especially prokaryotes.  The small plastic bag of soil your group will collect from under the oaks will contain hundreds of billions of bacteria alone.  The microclimate and soil qualities such as particle size distribution, depth, chemical composition, and organic matter content are major determinants of which organism are found in a particular location.  The soil you collect will contain:

In this lab, we are just going to 'scratch the surface' of geomicrobiology.  We have three objectives, estimating active microbial populations through the concept of "Colony Forming Units (CFU's)," observing the effect of using selective media when estimating CFU's, and isolating actinomycetes. While a wide variety of microorganisms can be isolated from the soil environment we are especially interested in actinomycetes as they are adapted for chemical attack on their ecological competitors and important producers of the xenobiotics we are studying, especially antibacterial compounds.

Different media encourage the growth of different types of microbes through the use of inhibiting substances such as antibiotics, specialized nutrients, or media with very limited nutrient contents. For each dilution we make, we will count the number of microbes which are capable of growing on a specific media (CFU's).

One group of organisms we hope to isolate in this lab is the Actinomycetes. Actinomycetes are a large group of Gram-positive bacteria that usually grow by filament formation, or at least show a tendency towards branching and filament formation. Under the microscope they look like fungi, but the diameters of the filaments are about one-tenth to one-fifth as wide as those eukaryotes. Many Actinomycetes can form resting structures called spores, but they are not the same as endospores. Branched forms superficially resemble molds and are a striking example of convergent evolution of a prokaryote and a eukaryote together in the soil habitat. Actinomycetes such as Streptomyces have a world-wide distribution in soils. They are important in aerobic decomposition of organic compounds and have an important role in biodegradation and the carbon cycle. Products of their metabolism, called geosmins, impart a characteristic earthy odor to soils. Actinomycetes are the main producers of antibiotics in industrial settings, being the source of most tetracyclines, macrolides (e.g. erythromycin), and aminoglycosides (e.g. streptomycin, gentamicin, etc.).

Week One - Culturing Microorganisms from Soil

Preparing the soil extract and dilutions.

• Note the weight of your 250 mL beaker using the Mettler scale.
• Collect about 100 mL of soil from under the oaks to the east of the Chapman Science Center and bring it back to the lab. Use caution to avoid the concertina wire, collapsing ground squirrel holes, UXO, and poison oak. 
• When you are inside, tap out about 1/10th of your sample into a petri dish and set it aside for later inspection.
• Weigh the beaker again.  The difference between this weight and the tare will be the weight of the soil you collected (in other words, the petri soil weight doesn't matter). Note the weight of the soil you collected, where you collected it from, how deep, etc. When there is time you will also inspect the petri soil under a dissecting scope and describe its characteristics.
• Using a spatula and a weigh boat, measure out about 50 grams of your soil. Note the exact amount. Save the rest of your labelled soil sample under aluminum foil.  We will need to dry it down and determine the dry weight.
• Pour the ~50 grams of soil from the weigh boat into a new 250 mL beaker. Add 2x sterile distilled water (v/w) to your sample.  If your sample was 49.70 grams, then add 99.4 mL of sterile distilled water.  Using a magnetic stirrer allow your sample to stir at low speed for 10 minutes.  Meanwhile, inspect the petri dish containing your ~1/10th sample. Take this time to record characteristics of your soil. Were there any arthropods or nematodes? Was there floating organic matter in your stirring beaker?
• After ten minutes, swirl your sample vigorously by hand maintaining the sediment in suspension as you quickly pour the contents of your beaker through four layers of cheesecloth supported by a funnel into a second clean 250 mL beaker.  Using a spatula scrape the remaining debris out of the first beaker. Allow to drain for ten minutes.
• During this time, set up your serial dilutions. You will need to label (1, 2, 3) three sterile glass test tubes capable of holding 10 mL.  Into each, carefully pipet 9 mL of sterile distilled water using a 10 mL pipette.
• When the cheesecloth has drained ten minutes, remove the funnel and retrieve the stir bar.  From near the top of the tea-colored soil extract in the beaker, remove 1 mL and add it to the first test tube.  Cover the top of the test tube with parafilm and your thumb, and invert the tube at least three times. The side of the parafilm that is against the no-stick paper film is sterile enough for this lab, so let it be the surface that is exposed to the solution.
• Using a new sterile blue tip, remove 1 mL from the first test tube, add it to the second test tube, cover with parafilm and your thumb, and invert three times.
• Using a new sterile blue tip, remove 1 mL from the second test tube, add it to the third test tube, cover with parafilm and your thumb, and invert three times.
• These are the three suspensions you will plate onto the various selective media.

Culturing the soil microorganisms.

You are being supplied five types of agar plates for culturing microbes. 

Please do not expose the agar in your plates to the air for any longer than absolutely necessary!

Using a new sterile yellow tip, remove 100 uL from test tube #3 and add it to plate #3,   then #2, then #1, working from most dilute to most concentrated. Spread as instructed in class using a sterilized loop or spreader such that the 100 uL is more or less evenly spread on the plate, and then cover the plate. Again, be sure to work in the 3-2-1 order to save time.  Using a parafilm strip, seal the edges of the plates.

1:2 x 1:10 = 1:20
1:20 x 1:10 = 1:200
1:200 x 1:10 = 1:2,000
100 uL is equivalent to another 1:10 dilution.  So if you find 35 colonies on plate #3 next week you would have to multiply by 20,000 to get the number of Colony Forming Units per weight of soil.

Let them stand at least 30 minutes. Then invert and incubate these plates at 22° C in the dark, checking on them Thursday (48 hours) and Tuesday (seven days).

Thursday

• Take photographs of your plates for data analysis. Save the images where you and your group members can get at them again, such as on your world folder.

Tuesday

• Prepare a data sheet to record the counts from all your plates.
• Take photographs again to compare to Thursday's. Have any new colonies become visible?
• You will want to count bacteria, fungi, and actinomycetes separately and calculate the number of CFU's per gram of soil for each type and media.  This is a good group exercise.  If you are not sure what a colony is, pick a small piece with a sterile toothpick and suspend it in a drop of water on a microscope slide.  I will be able to help you decide which type it is when you have prepared the slide.
• Actinomycetes are gram positive... so we will check possible filamentous candidates using the gram stain.
• Look carefully for any signs of antibiotic production.
• Isolate any antibiotic-producing bacteria and attempt to get a pure culture using instructions in class.