BISC209/S12: Lab7

LAB 7: Examples of Co-operation and Competition in a Soil Community: Bacterial Interactions, Quorum Sensing, Functional roles in the Nitrogen Cycle

Confirmation of Gram stain results by Selective/Differential Media:

Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Did you confirm your Gram stain findings?

Testing for Examples of Co-operation and Competition Among your Cultured Isolates

Antagonistic and Mutualistic Interactions

• NOTE: You must remember to set up fresh nutrient broth cultures for your isolates 1-3 days before lab to do this test!
  

The microbial community living in soil is a complex one with many different microorganisms. As is true of any environment, these microbes interact with each other - both functionally and physically. Today, you will be using your cultured isolates to test for possible examples of mutualism or antagonism (co-operation or competition). Do selected bacteria from your community help each other or harm each other while trying to find a niche in your soil community? You will culture them in controlled communities to attempt to detect positive or negative interactions. Some of these bacteria may prevent the growth of others through the production of chemical inhibitors; others might promote the growth of their neighbors by producing metabolites that are needed.

Interaction Assay


1. Relabel the 8 wells in the top first row using the letters A-H in place of 1-8. You will only be using 64 of the wells on a 96 well plate for this exercise. Each pair will use 8 unique isolates (4 from each student) to test for interactions. Use the Excel template provided Media:template.xls to record the identifying codes. You will inoculate the top row of wells and the first column of wells with the 8 isolates (see image below). For example if you inoculate isolate 101 into well B-B, that isolate will also be inoculated into the first column well B-B. Likewise, the isolate placed into top row well C-C is also placed into the first column well C-C, etc. FOLLOW THE TEMPLATE CAREFULLY!!!!!! It is easy to get this inoculation messed up, but don't!

2. Using a sterile pipet tip and your P200, inoculate 50 µl of the appropriate isolate grown in fresh nutrient broth culture into the assigned top row and first column well(s).

3. Use the P200 to add another 100 µl of sterile nutrient broth to the wells that were inoculated in step 2. This will both dilute the cultures and provide enough volume for the next steps.

4. When you are finished loading the top and side wells, use the P20 micropipette to move 10 µl from the top row wells AB - AH (not AA) into wells BB - BH moving top to bottom, reloading your pipette from the top row each time. Continue loading AB-AH to CB-CH, etc. DO NOT TRANSFER ANYTHING TO THE WELLS AA, BA, etc.) You do not NEED to change the tip as you fill each empty well in the column (e.g. BB-BH) unless you think you might have contaminated your tip. Repeat this transfer until all the rows have 10 µl of the inoculum from AB-AH. (Note: if an eight tip multichannel 10 μL pipet is available, you could use it to fill the wells as long as you remove the tip on the first channel before pipetting).


4. Now, using the P20, we will transfer 10µl beginning with wells AA - HA and moving left to right across the columns . Mix by pipetting up and down 1 time very gently and change tips after each mix. NOTE: if a multichannel pipet is available, you could use it to inoculate the wells (using all 8 channels). Repeat this process until you reach A8-H8 and have 20 μL in each well (except the top and side wells.



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7. Each of your wells should now have isolates growing by themselves (AA-HA and in the diagonal wells), as well as isolates mixed together in all the other wells.

8. NEXT, we will inoculate a square tray (NUNC) containing nutrient agar medium using the contents of the wells we just prepared so they will grow on solid medium. For this step we will use either a tool called a "frogger" or a multichannel micropipette. If using the frogger, dip the tips into 96 wells to attract a drop of inoculum onto the end of each steel tip and then touch the those tips to the surface of the sterile NA square NUNC plate. Do not break the surface of the agar but make sure your pressure is even so every steel tip has touched the agar surface and deposited the same inoculum. Be sure to disinfect the frogger by dipping it into a series of disinfectant and rinse solutions that you will find at the cleaning station prepared for you .

If the frogger is not available, use a multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Repeat this until you have completed depositing the full array in the same orientation as the 48 wells.

7. Wait for your inoculated spots to dry, seal or cover the NUNC square tray, and incubate at Room Temp for a week. The 96 well plate was used only to mix the cultures so you can discard this in the appropriate manner. You will check on your assay and note any differences in the appearance of the colony growth of each isolate, alone vs mixed next lab. Remember that the inoculum in the diagonal spots should match the appearance of the colonies in the first column. Notice if there is an "edge" effect, a difference in the appearance of the colony growth in the spots along the perimeter of the plate as opposed to those growing in a more protected locations (the diagonal control colonies).

Continue Antibiotic Production test started last week

Week 2
Need fresh control cultures of Eschericia coli (Gram negative), Staphylococcus epidermidis (Gram positive) and Micrococcus luteus (Gram positive) grown in nutrient broth to the same turbidity standard used last week for isolate cultures.

PROTOCOL

Use the cultures of your isolates set up last week on NA.
Use a sterile swab to aseptically apply parallel lines of inoculation of each of the control broth cultures of : E. coli, Micrococcus, and S. epidermidis as shown in the illustration below. Use a different sterile swab for each culture. These parallel inoculation lines should be made perpendicular to the putative antibiotic producer's (your isolate's) colony growth. (See the illustration.) Be careful not to touch the putative antibiotic producer's growth with the control cultures, but come as close. Make a template in your lab notebook and label the plate to indicate where each control culture is streaked. Incubate these culture plates at RT in a place that where your instructor can monitor their development. One person/lab should also set up viability controls by swabbing each the three broth cultures onto separate areas of another NA plate. If you see growth of each of the controls next week, we will be sure that any inhibition of growth is due to sensitivity to a diffused antibiotic rather than lack of growth occurring because one or more of the control broth cultures you used today lacked enough viable cells to form colonies on your test plate.

Other Physical & Functional Capabilities:

Every isolate should be inoculated into a nutrient agar (NA) soft agar deep (NA with only 0.35% agar instead of 1.5%) and into mannitol nitrate motility medium (NMM: 1% casein peptone; 0.75% mannitol; 0.1% potassium nitrate; 0.004% phenol red; 0.35% agar) as another soft agar deep. The growth pattern in these semi-solid media gives information about motility and about other metabolic capabilities: ability to use mannitol as the sole carbon source and the ability of a microorganism to complete part of the nitrogen cycle (ability to reduce nitrate to nitrite).
A full description of these tests can be found in the protocols section: Motility Tests.
If your motility test is positive when we analyze the results in Lab 8, you could try to confirm motility by trying a flagella stain.

PROTOCOL:
Inoculating a soft agar deep involves a technique you have not yet practiced. You will use an inoculating needle: the wire extending from the handle of the needle will not have a loop on the end.

1) You will need a tube of each medium for each of your isolates and each pair needs to add 1 extra tube of each medium so they can inoculate it with E. coli, an organism that is motile.

2). Flame sterilize an inoculating needle, cool it for a few seconds, and pick up a barely visible amount on the tip of the needle. You may start with either an isolate to be tested or the control organism (E. coli).

3) Stab the needle with the inoculum deeply into the center of the NA soft agar tube, stopping just before the bottom of the tube or, if you are running out of needle, stab it until the you are almost to the end of the needle.

4) Withdraw the needle through the same inoculation channel. (This procedure is also known as "making a soft agar deep".)

5) Inoculate each of your other isolates into both NA and NMN soft agar tubes using the same technique.

6) Make sure you have inoculated a control tube of E. coli into both media.

7) Incubate all tubes for 24-72 hours at room temp. Check your cultures daily.

8) After sufficient growth is observed in the MNM tube, look for a color change from red to yellow 9) Check for motility in both the NA soft agar deep and in the MNM tube by looking for diffuse growth radiating from the stab line of inoculation. Compare the motility of each of your isolates to that of the E. coli control tube. E. coli is a motile bacterium.

9) Develop the nitrate to nitrite test in the MNM tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. Nitrate-negative organisms are unable to reduce nitrates and they yield no color after adding the reagent. Nitrate-positive: The appearance of a pink or red coloration indicates that the nitrates have been reduced to nitrites.

Gries reagent consists of solutions:
Solution A
Sulfanilic Acid 0.8% (v/v) in Acetic Acid 5N
Solution B
Alpha-Naphthylamine (0.001% v/v) in Acetic Acid 5N

Control Organisms:

Organism	ATCC	Motility	Mannitol as sole C source	Nitrate to Nitrite
Escherichia coli	25922	+	+	+
Klebsiella pneumoniae	13883	-	+	+
Proteus mirabilis	25933	+	-	+
Acinobacter anitrartum	17924	-	-	-

Microbiologists of previous generations had to do their bacterial identification exclusively from physical and metabolic tests. The tests we are performing in the course of our investigation are a tiny subset of all the morphologic, metabolic, and other tests that you could perform on your isolates to try to identify them through a battery of tests for different metabolic capabilities and characteristics. Be very glad that you are training as a microbiologist in the era of genomics!

CLEAN UP

1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.

2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.

3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.

4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.

5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.

6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.

7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.

8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.

9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.

10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.

11. Take off your lab coat and store it in the blue cabinet with your microscope.

12. Wash your hands.

Assignment

Create an Annotated Bibliography of appropriate outside sources (review articles and primary studies and a preliminary Graphical Abstract summarizing your investigation. A description and examples of Graphical Abstracts can be found at http://www.elsevier.com/wps/find/authorsview.authors/graphicalabstracts [1]. Pay particular attention to those that are less molecular and more topically ecological. There is a folder in Resources in Sakai, called Images. Your instructor has uploaded images of the Wellesley Greenhouses including the Tropical room that you may use if you wish. NOTE that these images are available as an OPTION. It is not required to use them or even suggested that any be part of your graphical abstract!
There are more instructions for this assignment found at: Assignment: Annotated Bibliography & Graphical Abstract .