Hello again! As we approach the halfway point in the quarter, which I believe is officially Wednesday (but as of now we’re halfway to fall break!), we’re getting to the point where we’re beginning to identify the bacteria in our bacterial phyllospheres, which is really exciting—we can finally learn what lives on bamboo! Unfortunately, since we’re almost halfway through, if some part of our experiment isn’t working by the end of the week, we’ll have to abandon it and focus on other aspects of the project. Luckily for my partner and I, everything is looking great so far! On Friday, we focused on two separate tasks, simultaneously working on the culture-dependent and culture-independent components of the project. For the culture-dependent portion, we looked at the slides we made and stained on Wednesday with bacteria taken from the colonies we’ve been growing on agar the past few weeks. Under the microscope, we looked for various identifying characteristics of our bacteria: what shape it was, how individual bacteria grouped together, and what color it stained—which indicated whether it was gram positive or negative. Although we can’t identify the species of individual bacteria based on these stains (since there are probably thousands of species of bacteria that are gram-negative cocci), but it helps in preliminarily identifying what the bacteria isn’t. Later this week we’ll begin preparing the bacteria to be sequenced so we can get a better idea of their identities.
Along with looking at our stained slides, we also worked on the culture-independent portion of our experiment. On Wednesday, we performed ligation on our bamboo DNA samples, which involved inserting the various sequences that we cloned using PCR into plasmids, so each plasmid should have a different sequence, representing the entire phyllosphere of our leaf. On Friday we transformed these plasmids into competent bacteria and plated it on new agar plates, 18 hours later I went in and moved the plates from the incubator to the cold room, and didn’t notice any bacterial growth, but hopefully by class on Monday we’ll see something! For now, I’ll post pictures that my lab partner took of some of our bacteria viewed through the microscope. We have so many different kinds of bacteria that after class on Friday we hadn’t finished looking at all of them under the microscope, so we ended up coming back in until 11:30pm. Taking pictures was probably the hardest part, but at least we have something to show for our work!