Working in the field is very dependent on the weather, and the upcoming forecast was rain! We needed to collect our samples before the rain hit and washed all of our bacteria away. So, with our samples tucked neatly away in sterile baggies, we were ready to begin taking our cultures. The most important aspect of this protocol was to keep everything sterile! Our goal of the day was to separate all living bacteria from the plants and grow them up in our own cultures. It was very important to avoid contamination in this first step so that we don’t end up mixing our plant bacteria with bacteria from our hands, or even from the air in lab. The procedures used were ones that we are familiar with, so the protocol did not take very long. The biggest and most important step of the day was to shake then sonicate our leaf samples to separate the bacterial cells and suspend them in water with Tween (detergent).
Before we plated our samples, we diluted them to 1 in 10, 1 in 100, and 1 in 1000 dilution samples. The purpose of the dilutions was to ensure that there will be an appropriate amount of bacteria on each plate. We need to be able to see small individual colonies of bacteria, so we would need about 30-300 cells initially. If we had too little amount, the cells of bacteria would be too small to see and culture, and it we had too many cells, then the cells would grow in a lawn across the plates and the individual cells would again be impossible to see. After dilutions, we spread our samples aseptically onto agar plates to induce bacterial growth. These plates contain cyclohexamine to kill any fungal cells so we had to wear gloves and a lab coat at all times since this drug is also toxic to humans.
Each different strain of microbe will grow differently in different environments. Since these cultures were taken from plants that live outside, we are incubating them at room temperature, underneath a window for about a week. We do not expect to see colonies until maybe Tuesday, and it will be exciting to see what ends up growing!