Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria (6-well plate version)

Goal

To screen a larger number of colonies for resistant colonies against producer bacteriocin killing in target strain.

Protocol

Day 1: Grow Overnight Cultures of S. pneumoniae (producer and target strains)

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 (Note: Steps c-i is for streaking one plate with one strain)

a. Have a beaker of Virkon ready for disposing inoculating loops

b. Label as many TSB agar blood plates as the number of strains you will use for the experiment with the strain names

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.

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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)

Making First Layer: Hard Agar (check immediately if there are plates available, or, ideally, check before the day of experiment)

Note: if the hard agar is already in the wells, warm up the plates by leaving it at room temperature and skip this section about making hard agar layer.

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.

Tips:

1. Draw 10 mL of the agar with the serological pipette so that each draw can cover 3 wells. DO NOT EXHAUST the agar in the bubbles will IRREVERSIBLY damage the plate!!

2. It takes about two stripes of parafilm, each the length of about 3.5 small pieces, to cover the whole 6-well plate.

Logistical preparations

1. Gather n+1 6-well plates (n = number of strains you are using) from under the counter where the spectrophotometer sits.

2. Label n plates as producer plates and the last one as a control plate (e.g. “D39 Δblp producer” and “D39 Δblp control”)

For instance, n=2 and 2+1=3 plates are needed if using D39 as producer and D39Δblp as target & control.

3. Draw reference dots on the bottom of each well of the producer plate (not the control) to know where you will place each producer dot

Growing Producer 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 the producer (n as mentioned earlier) accordingly and one tube “-” (for negative control).

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

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 Strains” to reach an OD of 0.3 (Absorbance of 0.39). While waiting, make a tube of TSB (or THY) soft agar (the catalase comes in later):

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.

2. Once the reading reaches an OD of 0.3 (Absorbance of 0.39), 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 the soft agar tube (NOT the tubes in the incubator).

3. Plate 5 uL dots from the incubated tubes to each well of their corresponding plates at the reference dots. Wait for the dots to dry. Normally it takes ~10 min. Keep lids off the wells to speed up drying.

4. Once the dots are dry, cover each producer dot with a 15 uL TSB (or THY) soft agar dot from the tube you made in step 1.

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

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

IMPORTANT: Plate out the target strain plate (TSB agar blood plate) again and incubate at 37°C 5% CO2. This will be for Day 3.

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Day 3: Adding Target Lawns

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

Growing Target Strain

1. Grab the target strain TSB agar blood 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 “<target name>” and “-” (for negative control).

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

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 (# of target lawns / 2) + 1. The (+1) is an extra tube in case of making a mistake.

4. When the heat block reaches 42 degrees, add 4 ml soft agar to each of the tubes and wait about 20-30 minutes for the agar to cool down to 42 degrees (each test tube can cover two target lawns with the same target strain).

5. After the agar cools down, add 100 uL catalase and 40 uL TTC.

6. When the target strain reaches an OD of 0.3 (Absorbance of 0.39), inoculate a soft agar tube with 100 uL of the target strain and immediately vortex.

7. Immediately pipet 1.5 ml of this target layer, using 2 x 750 ul, into a well that should have a target layer and swirl the contents around immediately to cover the entire well.

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

9. Incubate the plate at 37°C 5% CO2 overnight and check for zones of inhibition tomorrow.