Streptococcus Bacteriocin (Dual Layer) Assays - Light and Normal Target Lawns

Goal

Set up two assays with different amounts of target strain to test a bacteriocin's killing ability.

Protocol

Day 1: Plating S. pneumoniae

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 two TSB agar blood plates “<producer name>” and “<target name>”

c. Open tube of producer strain and grab a chunk of ice containing the strain using an inoculating loop

d. Grab the “<producer name>” 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 target strain on the “<target name>” plate.

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

Day 2

Set the small water bath to 55 degrees C.

Growing Producer and Target Strains

1. Grab producer and target 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 however many tubes “<producer name>,” however many tubes “<target name>,” and one tube “-” (for negative control)

a. The number of tubes depends on how many producer strains and target strains you are growing.

3. Using a plastic inoculating loop, grab several colonies from the producer plate and inoculate the test tube labeled “<producer name>”

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

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

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

Making First Layer: Hard Agar

1. The first layer in this assay is always TSB hard agar (which refers to ‘normal’ plate agar — 1.5% agar). Melt a bottle of agar completely in the microwave, using “Power Level 5”. Place in the small water bath.

2. At some point in advance of the assay (or even a few days before the assay): Using the BSL-2 hoods, pipet 3ml of TSB hard agar into each well of a 6-well plate. Leave the lids to the plates open, to prevent excess condensation on the lid. After these plates solidify, they can be wrapped in parafilm to prevent evaporation and stored in the refrigerator.

Making Soft Agar Target Layer

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.

2. As the soft agar is melting in the microwave, 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: (# of target lawns / 2) + 1. The (+1) is an extra tube in case of making a mistake. Additional tubes might be needed, if you are using method A (Target lawn on bottom) below.

4. After the heat block has reached 42 degrees, add 4 ml soft agar per test tube. Each test tube can make 2 lawns, using the same target strain.

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). This can be done to all soft agar tubes at the same time.

Making the Normal (100 uL) Target Lawn Plate

1. Get a 6-well plate containing hard agar.

2. Grab the three test tubes; the producer and target strains should be at an OD of 0.3 (dilute if necessary).

3. In each well that should contain producer cells (look at image), add a 10 ul dot of the producer cells (OD 0.3) on top of the hard agar, in the center of the well. Wait for the dot to dry (~10 minutes).

4. Add a 20 ul dot of soft agar + catalase + TTC on top of the producer dot (also add this dot to the wells that do not have producer dots). There should be no bacteria in this 20 ul dot. A new pipet tip is needed for each well.

5. After the dot has dried, pipette 100 ul of target cells into a 4 ml soft agar tube from “Making Soft Agar Target Layer,” and immediately vortex.

6. Immediately pipet 1.5 ml of this target layer, using 2 x 750 ul, into a well that should have a target layer (see image). Repeat for a second well, using the same tube, if needed.

7. Continue to pipette this target layer into all the wells that should have the target layer.

8. Place this plate in the 37°C 5% CO2 incubator overnight.

Making the Light (10 uL) Target Lawn Plate

1. Get another 6-well plate containing hard agar.