BISC209: Lab5

LAB 5: Con't. Project: Soil Microbial Communities & Diversity

Preparing your clones for sequencing analysis When you examine your transformation plates after an overnight incubation, you should see hundreds of identical looking well isolated colonies, all containg the vector which expresses the kanamycin resistance gene and allows the clones to grow on this media with kanamycin that normally would disrupt bacterial protein synthesis and kill the cells. We also know that each of the vectors imparting Kan resistance contains a 16S rDNA insert from one of your soil flora. We know that insert is in the vector because it is responsible for disrupting expression of the ccdB (control of cell death) gene which, if not disrupted, expresses the ccdB protein that poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death. We hope there are hundreds of 16s rDNA gene fragments from DIFFERENT soil bacteria.

You and your partner will be given a 96 well sterile plate to fill with a bit of each of the clones you want to send away for sequencing.
Preparing Glycerol Stocks to send out for sequencing

1. Streak the original colonies out on LB plates containing 50 μg/ml kanamycin-labeling each with your team color initial, eg. Tues red= TR-1, R-2, R-3, etc up to 96. Incubate at 37C overnight.
2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50 μg/ml kanamycin. Keep your labeling clear.
3. Grow with shaking to log phase (OD600 = ~0.5)
4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer it to a well in the 96 well plate. Keep the numbering straight!!! Make a template in your lab notebook.
5. Give the plate to your instructor to seal and send away on dry ice.
The sequences should come back in a week or two.

Culturable Bacteria Identification continued

Activity 1

• Use your newest stock slant or a colony from your newest isolation streak plate to make a replacement stock slant.
• Complete or repeat any work from the prior weeks' lab to ensure your cultures are pure and growing satisfactorily.
• Check and record any important observations on your newest isolation streak plates and stocks.
• Examine and record the results of your PEA and ENDO plates. Is your Gram stain data supported?

Activity 2: Morphologic tests
At this point, based on your Gram stain data, the morphologic observations of colony growth, and reference to The Prokaryotes and/or Bergey's Manual, you may have a tentative idea about your organisms taxonomic grouping. Use the appropriate protocols Special Stains: Endospore, Acid fast, Capsule, and Flagella to continue to learn more.
This week check for the presence of spores and a capsule.
Set up the tubes needed to examine motility Motility Tests.

Activity 3Check for the presence or absence of the following enzymes:
Catalase and oxidase. Enzyme tests

Activity 4
Use tubes of OF-Glucose medium to check for the ability of an organism to catabolize glucose only in the presence of oxygen (oxidative catabolism of glucose) or to ferment it with or without oxygen (fermentative catabolism).
Use Starch agar plates to test for the ability of an organism to digest starch.

Activity 5
Today you will set up and begin the experiment to examine the ability of your isolates to digest leaf cellulose following the Protocols for determining the roles of soil isolates in the soil
Protocol:
Prepare the plates needed to examine leaf cellulose digestion and inoculate your organisms. Be sure you use melted pours of the appropriate medium for each of your samples and leaves collected from the research site in the greenhouse. These plates will be examined multiple times over the next several weeks. To prevent the medium from drying out seal the edges of the plates with parafilm and place these plates (upside down) in a resealable plastic bag as suggested in the protocol. You may also add sterile water after 2 weeks if you see any evidence that the agar is drying up.

Activity 6
Actively begin to research and develop a Student Plan for identification based on The Prokaryotes and Bergey's Manual.

Links to Labs in the Soil Microbes Project

Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9