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BISC209/F13: Lab2: Difference between revisions
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→RICHNESS OF A MICROBIAL SOIL COMMUNITY: Structural Diversity in Cultured Bacteria from a Soil Community
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Now that we have some sense of the abundance of microbes in a soil community we can move on to investigating the richness (diversity) and the co-operative and competitive behaviors that maintain it. | Now that we have some sense of the abundance of microbes in a soil community we can move on to investigating the richness (diversity) and the co-operative and competitive behaviors that maintain it. | ||
=='''RICHNESS OF A MICROBIAL SOIL COMMUNITY: <BR> | =='''RICHNESS OF A MICROBIAL SOIL COMMUNITY: <BR>Culturing a few Bacteria from a Soil Community to examine structural diversity'''== | ||
<font size="+1">Using General Purpose Media for Isolation of Soil Bacteria in a Mixed Population</font size="+1"><BR> | <font size="+1">Using General Purpose Media for Isolation of Soil Bacteria in a Mixed Population</font size="+1"><BR> | ||
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Directions for [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] are found in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. <BR> | Directions for [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] are found in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. <BR> | ||
We hope that you have thousands of bacterial colonies on the | We hope that you have thousands of bacterial colonies on the dilute NA plates that you set up last week for your colony enumeration. You and your partners should share these plates to find interesting bacteria that you would like to study. Your goal is that each student ends up with two pure cultures of bacteria that are different from your classmates. Look for colonies that are different in color, texture, size, or other characteristics. We want to avoid isolating fungi so avoid black, or fuzzy growth that looks like mold. Check with your instructor after you and your partners have each selected a few colonies to attempt to isolate. You can circle the colony and put your initials and an id code on the plastic bottom on the plate to indicate the colonies you've chosen. Draw a simple sketch of the plate and indicate the location of the colony you selected in the sketch. Describe the selected colony in your lab notebook. Use descriptive words for the color (white, cream, yellow, pink), consistency (wet, milky, buttery, powdery, etc.), and shape (round with smooth edges, flat with spreading edges) to describe each colony. When you are ready to sub-culture them, get NA plates from the supply area (one per colony) and follow the directions for streaking for isolation described below. You cannot assume that any of these colonies are from the growth of only one organism, your job over the next few weeks is to separate one organism from the others until you have a pure culture of only one bacterium. | ||
'''Streaking for Isolation from Solid Medium to Solid Medium:'''<BR> | '''Streaking for Isolation from Solid Medium to Solid Medium:'''<BR> | ||
You can find the steps for [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. | You can find the steps for [[BISC209/F13: Streaking for Isolation| Streaking for Isolation]] in the [[BISC209/F13:Protocols| Protocols]] section of this wiki. | ||
<ul> | <ul> | ||
<LI>Flame your loop, let it cool a few seconds. Remove or tilt the lid of the donor agar plate with your non-dominant hand but keep the lid in your hand (don't put the lid down on the bench!). With your dominant hand holding the | <LI>Your instructor will demonstrate this technique. Practice it carefully and you will not contaminate your plates with organisms found on your hands. <BR> | ||
<Li> TIlt the lid of a new sterile plate of solid medium (in this case it will be Nutrient Agar) with your non-dominant hand so that it is partially open. Do not put the lid down on the bench; keep it in your hand! Touch the loop with the bacteria to the surface of the agar in an area near the periphery of the plate and gently glide the loop over the innoculum to spread it over a small area of the plate called Section 1 as shown in the illustration below. Do not gouge the agar! Replace the lid. | |||
Flame your loop, let it cool a few seconds. Remove or tilt the lid of the donor agar plate with your non-dominant hand but keep the lid in your hand (don't put the lid down on the bench!). With your dominant hand holding the flamed and cooled loop, touch the loop to an interesting colony of bacteria that you want to study and pick up a TINY amount of the colony. Avoid touching other bacterial colonies. Close the lid of the donor plate. | |||
<Li>Label the recipient plate on the bottom (NOT the lid!). | |||
<Li>TIlt the lid of a new sterile plate of solid medium (in this case it will be Nutrient Agar) with your non-dominant hand so that it is partially open. Do not put the lid down on the bench; keep it in your hand! Touch the loop with the bacteria to the surface of the agar in an area near the periphery of the plate and gently glide the loop over the innoculum to spread it over a small area of the plate called Section 1 as shown in the illustration below. Do not gouge the agar! Replace the lid. | |||
<LI>Flame your loop and let it cool for a few seconds. | <LI>Flame your loop and let it cool for a few seconds. | ||
<li>Tilt the lid of the plate so it is partially open and drag your loop once or twice through the area you have just innoculated (section 1) and spread the innoculum you picked up across the surface of a new section of the plate. This section is called Section 2. | <li>Tilt the lid of the plate so it is partially open and drag your loop once or twice through the area you have just innoculated (section 1) and spread the innoculum you picked up across the surface of a new section of the plate. This section is called Section 2. | ||
<li> Flame and cool your loop. | <li> Flame and cool your loop. | ||
<li>Repeat to streak sections 3 and 4 by dragging your loop through the last section innoculated once or twice and then to a new area of the plate. Replace the lid. | <li>Repeat to streak sections 3 and 4 by dragging your loop through the last section innoculated once or twice and then to a new area of the plate. Replace the lid. | ||
<li> | <li>Invert and incubate the plate at RT. | ||
<li>Check your plate for colonies | <li>Check your plate for colonies frequently and record your descriptions of the texture and shape of the colonies that appear. Look for changes from the original description of the colony you selected and notice the time (24 hours, 48 hours, 72 hours) needed for you to see visible growth. | ||
</ul> | </ul> | ||
[[Image:Fig A-2-2inch.jpg]] <br> | [[Image:Fig A-2-2inch.jpg]] <br> | ||
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Some bacteria often form tough leathery colonies, so transfer of these bacteria to new media to start a sub-culture is sometimes difficult. The powdery area may be spores, which would be interesting to visualize later. To isolate spore formers try to "break off" a piece of the colony with your sterile loop or with a sterile toothpick and transfer that whole piece of a colony onto zone one of the new plate. Then use your loop for streaking out the other zones. The tiny spores on the surface of the colony are likely to transfer to the next plate or tube when you work with it. (That's a good thing this time.) <BR><BR> | Some bacteria often form tough leathery colonies, so transfer of these bacteria to new media to start a sub-culture is sometimes difficult. The powdery area may be spores, which would be interesting to visualize later. To isolate spore formers try to "break off" a piece of the colony with your sterile loop or with a sterile toothpick and transfer that whole piece of a colony onto zone one of the new plate. Then use your loop for streaking out the other zones. The tiny spores on the surface of the colony are likely to transfer to the next plate or tube when you work with it. (That's a good thing this time.) <BR><BR> | ||
This process of transferring a single well isolated colony to new solid medium will continue | This process of transferring a single well isolated colony to new solid medium will continue until you succeed in isolating two different bacteria to pure culture. Our goal as a course is to find a diverse group of interesting soil bacteria . <BR><BR> | ||
==CLEAN UP== | ==CLEAN UP== | ||
