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

Growing Producer and Target Strains