View source for Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria

==Goal==
*To plate several producer dots (S-18, D39, and D39 Δblp) and D39 Δblp target lawns on TSA plates to find producer-resistant target colonies.

==Protocol==

'''Day 1: Grow Overnight Cultures of S. pneumoniae (S-18, D39, and D39 Δblp)'''

1. Grab a frozen cozy from the freezer in the MEE Lab and go to the -80°C freezer next to the MEE Lab.

2. Grab the containers of producer and target strains from the freezer and place them in the cozy

3. While under the hood:

:a. Have a beaker of Virkon ready for disposing inoculating loops
:b. Label three TSB agar blood plates “S-18,” “D39,” and “D39 Δblp”
:c. Open tube of one strain and grab a chunk of ice containing the strain using an inoculating loop
:d. Grab the corresponding plate and streak the loop horizontally, from top to bottom, across the top-third of the agar
:e. Put the inoculating loop in Virkon
:f. Grab a new inoculating loop and start streaking from the right side of the last streak and proceed to streak the bacteria downwards across the right half of the plate
:g. Put the inoculating loop in Virkon
:h. Grab a new inoculating loop and start streaking from the bottom side of the last streak and proceed to streak the bacteria upwards across the left half of the plate
:i. Put the inoculating loop in Virkon
:j. Repeat Steps c-i for the other strains on their respective plates.

4. Put plates in 37°C 5% CO2 incubator overnight.


'''Day 2: Making the Producer and Control Plates'''

*Set the small water bath to 55 degrees C (this is needed for making TSB/THY soft agar)

1. Gather four TSA plates from the fridge.

2. Label one “S-18 producer,” one “D39 producer,” one “D39 Δblp producer,” and one “D39 Δblp control”

3. Move plates to 37°C 5% CO2 incubator to warm them up. Take them out when you're ready to plate producer dots.

''Growing Producer and Target Strains''

1. Grab the strain plates from the 37°C 5% CO2 incubator.

2. Fill test tubes with 4 mL TSB broth (or Todd-Hewitt 0.5% Yeast Extract (THY) broth) and 100 ul catalase. Label one tube “S-18,” one “D39”, one “D39 Δblp,” and one tube “-” (for negative control).

3. Using a plastic inoculating loop (or, for faster growth, a cotton swab), grab several colonies from the S-18 plate and inoculate the test tube labeled “S-18.”

4. Vortex the tube using a vortex in the BSL-2 cabinet.

5. Repeat Steps 4 and 5 for the remaining strains.

6. Place the tubes in a tube rack and incubate them in the 37°C 5% CO2 incubator.


''Plating the Producer Strains''

1. Wait for the bacteria from “Growing Producer and Target Strains” to reach an OD of 0.3 (Absorbance of 0.39)

2. Plate 10 uL dots of S-18 onto the S-18 producer plate at the reference dots

3. Repeat Step 2 for the D39 producer plate and the D39 Δblp producer plate

4. While the dots are drying (this takes about 10 minutes), make a tube of TSB (or THY) soft agar:

:a. Melt a small bottle of TSB soft agar (or THY soft agar) in the microwave, using “Power Level 5”. Place in the small water bath.
:b. As the soft agar is melting in the microwave, set up the heat block in the BSL-2 hood. Place a sterile 13mm test tube in the heat block and turn on at 42 degrees. 
:c. After the heat block has reached 42 degrees, add 4 ml soft agar to the test tube.
:d. Add 100 ul catalase (at 30,000 Units / ml; found in refrigerator) and 40 ul TTC (50mg/ml; also found in refrigerator, with extra aliquots in the -20 freezer).

5. Once the dots are dry, cover each producer dot with a 20 uL TSB (or THY) soft agar dot from the tube you just made.

6. Wait a few minutes for the soft agar dots to dry.

7. Place the plates in the 37°C 5% CO2 incubator overnight.

Note: Plate out D39 Δblp again and incubate at 37°C 5% CO2. This will be for Day 3.


'''Day 3: Adding Target Lawns'''

*Set the small water bath to 55 degrees C (this is needed for making TSB/THY soft agar)
*Keep the TSB plates in the 37°C 5% CO2 incubator to keep them warm. Only take them out when you're ready to plate the target layer.

''Growing Target Strain''

1. Grab the D39 Δblp plate from Day 2

2. Fill two test tubes with 4 mL of TSB broth (or Todd-Hewitt 0.5% Yeast Extract (THY) broth) and 100 ul catalase. Label them “D39 Δblp,” and “-” (for negative control).

3. Using a plastic inoculating loop (or, for faster growth, a cotton swab), grab several colonies from the D39 Δblp plate and inoculate the test tube labeled “D39 Δblp.”

4. Vortex the tube using a vortex in the BSL-2 cabinet.

5. Place the tubes in a tube rack and incubate them in the 37°C 5% CO2 incubator until the target strain reaches an OD of 0.3 (Absorbance of 0.39).


''Plating the Target Strain''

1. Melt a small bottle of TSB soft agar (or THY soft agar) in the microwave, using “Power Level 5”. Place in the small water bath at 55 degrees C.

2. Set up the heat block in the BSL-2 hood. Place sterile 13mm test tubes in the dry bath and turn on at 42 degrees.

3. How many test tubes do you need? You will need one test tube per plate (4 mL per plate).

4. After the heat block has reached 42 degrees, add 4 ml soft agar per test tube. Each test tube can cover one plate if the contents are swirled around the plate right when the contents are poured.

5. Add 100 ul catalase (at 30,000 Units / ml; found in refrigerator) and 40 ul TTC (50mg/ml; also found in refrigerator, with extra aliquots in the -20 freezer) to each tube. This can be done to all soft agar tubes at the same time.

6. Once the target (D39 Δblp) strain has reached an OD of 0.3 (Absorbance of 0.39), inoculate one soft agar tube with 20 uL of the target strain and immediately vortex.

7. Immediately pour the contents of this tube onto one of the plates (S-18, D39, D39 Δblp, or the control) and swirl the contents around immediately to cover the entire plate.

8. Repeat Steps 6 and 7 for the rest of the plates.

9. Incubate the plates at 37°C 5% CO2 overnight.