Streptococcus Bacteriocin (Dual Layer) Assays

Goal

• Set up three different assays 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

Repeat Steps c-i for the target strain on the “<target name>” plate Put plates in 37°C 5% CO2 incubator overnight.

Day 2

• Set the small water bath to 60 degrees C.

Growing Producer and Target Strains

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

2. Fill three test tubes with 4 mL TSB broth and 100 ul catalase. Label one “<producer name>,” one “<target name>,” and the other “-” (for negative control)

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 target strain.

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 (0.8% 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 4ml soft agar per test tube. Each test tube can make 2 lawns, using the same target strain.

5. Add 100ul catalase (at 30,000 Units / ml; found in refrigerator) and 40ul 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.

6. Ensure the OD of the target strain is ~0.3 (Absorbance ~0.39). If the absorbance is significantly over this amount, culture can be removed and TSB added to bring the absorbance to 0.39. If the OD is over 0.7 (Absorbance over 0.91), do not use the culture, as the cell might start to lyse due to reaching late log phase.

7. Pipette 100ul of target cells into the 4ml tube, and immediately vortex.

8. Immediately pipet 1.5ml of this soft agar, using 2 x 750ul, into a well. Repeat for a second well, using the same tube, if needed.

9. Allow to solidify (1-2 minutes) before adding to this well.

Setting Up the Three Assays

1. Target lawn on the bottom

a. In a well that contains 3ml TSB hard agar, add a 1.5ml layer of the target cells (OD 0.3 / Absorbance = 0.39, as explained above).

b. After this layer has solidified, add a 10ul dot of the producer cells at OD 0.5 (Absorbance = 0.65) on top of the lawn, in the center of the well. The target lawn can remain at room temperature while waiting for the producer cells to grow, if needed.

c. Add a 20ul dot of soft agar + catalase + TTC on top of the producer dot. There should be no bacteria in this 20ul dot; you can use the ‘spare’ soft agar test tube for this. A new pipet tip is needed for each well.

d. After 1-2 minutes for the 20ul dot to solidify, add 1.5ml soft agar + catalase + TTC on top, as a layer of agar. There is no bacteria in this layer. You can use the ‘spare’ soft agar test tube for this.

e. After 1-2 minutes, place the plate in 37 degrees C, 5% CO2 incubator right-side up.

2. Target lawn on top

a. In a well that contains 3ml TSB hard agar, add a 10ul dot of the producer cells at OD 0.5 (Absorbance = 0.65) on top of the lawn, in the center of the well.

b. Add a 20ul dot of soft agar + catalase + TTC on top of the producer dot. There should be no bacteria in this 20ul dot; you can use the ‘spare’ soft agar test tube for this. A new pipet tip is needed for each well.

c. Add a 1.5ml layer of the target cells (OD 0.3 / Absorbance = 0.39, as explained above). This may require diluting back a culture to this absorbance, which is OK.

d. Place the plate in 37 degrees C, 5% CO2 incubator right-side up.

3. Target lawn on top, 24 hour producer incubation

a. In a well that contains 3ml TSB hard agar, add a 10ul dot of the producer cells at OD 0.5 (Absorbance = 0.65) on top of the lawn, in the center of the well.

b. Add a 20ul dot of soft agar + catalase + TTC on top of the producer dot. There should be no bacteria in this 20ul dot; you can use the ‘spare’ soft agar test tube for this. A new pipet tip is needed for each well.

c. Place plate in 37 degree, 5% CO2 incubator right-side up. This will allow the producer strain to grow for 24 hours.

d. The next day, add a 1.5ml layer of the target cells (OD 0.3 / Absorbance = 0.39, as explained above).

e. Place the plate in 37 degrees C, 5% CO2 incubator right-side up.