King B Agar Lab Report

Agar and Media Preparation— Agar plates containing King’s B Agar were often used throughout the experiment to support growth of Pseduomonas fluorescens. A recipe was used that included a mixture of 10g Proteose Peptone #3, 1.5g Potassium Phosphate Dibasic (K2HPO4), 30ml 50% Glycerol, ~965ml water and 20g agar. The mixture, post- autoclave, was left to cool and 5ml 1M Magnesium Sulfate (MgSO4) was added and created about 40 plates. King’s B Medium was made using the same procedure as the King’s B Agar with the exception of adding 20g of agar. These recipes were referred to later in the protocol to create additional plates and liquid cultures when growing multiple strains. King’s B Agar and Medium served as enrichment media that enhance growth
Single colonies from the SM and WS+ lab stock were inoculated into 500μl of sterile King’s B medium and vortexed. 250μl of each inoculated suspension were added to a sterile screw top tube and 250μl of sterile glycerol was added. This was stored in the -80oC freezer and allowed for long term use and storage of an original strain or mutated strain. The stock cultures were only removed for plate streaking and static cultures to ensure the use of consistent strains throughout the entire experiment (Capuccino and Sherman, 1983). More stock cultures were created for any mutant strains that arose throughout the
To construct liquid cultures of bacteria, 6ml of King’s B medium was added to a 25mm test tube. In a microcentrifuge tube, a single colony of the corresponding strain was inoculated into 200μl of medium. 10μl of the cell suspension was taken and inoculated into the 6ml of medium and vortexed, which served as the final microbial liquid culture. The static cultures were incubated at 30oC, apart from the conditions in Rainey and Travisano at 28o C, and were observed after 3 and 5 days of growth, often yielding the formation of a biofilm at the liquid interface of each culture (Rainey and Travisano, 1998). The production of biofilms in liquid microbial cultures and colony growth on agar plates allowed for the contrast and comparison of SM cells, WS+ and mutations that