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: Preparation

1. Set the small water bath to 60 degrees C.

Growing Producer and Target Strains

2. Grab producer and target strain plates from the 37°C 5% CO2 incubator. 3. 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) 4. Using a plastic inoculating loop, grab several colonies from the producer plate and inoculate the test tube labeled “<producer name>” 5. Vortex the tube using a vortex in the BSL-2 cabinet. 6. Repeat Steps 4 and 5 for the target strain. 7. Place the tubes in a tube rack and incubate them in the 37°C 5% CO2 incubator.

Making First Layer: Hard Agar

8. 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. 9. 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.