After giving our cultures long incubation times, we finally were able to see colonies growing! It is amazing how diverse and abundant bacteria thrive on these small pieces of leaves. We re-streaked all of the visibly distinct and individual colonies onto agar plates of their own and grew those up. Two weeks later, Harry and I are proud to announce that we have 23 beautiful and different organisms. We almost ran out of letters labeling them from A-Z! Having this many colonies also means that we will be doing many of the protocols from now on x23!
We have now come to the point in lab where every group starts to work at their own pace. The biggest deciding factor of what step a group is on is the result of the PCR (polymerase chain reaction). The purpose of this step is to amplify pieces of pure DNA so they can be cloned and analyzed in the future. So far, we have run two PCRs and have come up with negative results. When we ran our reactions on electrophoresis gels to visualize the DNA, we ended up getting bands (DNA indicating bands) in our negative controls both times. Troubleshooting is now our plan of action! We are currently in the process of running our third PCR reaction with plans to use nested primers if the need arises.
Last night, Harry and I spent time in lab working on a different protocol, Gram staining. We stain hand-made microscope slides to pigment the bacteria so we can visualize the cells under the microscope. We use a variety of dyes to account for both gram-positive and gram-negative bacteria. Gram-positive bacteria are bacteria cells that have one membrane and are stained with crystal violet. Gram-negative bacteria have two membranes and are stained with safranin counterstain. In order to account for both bacteria types, the protocol had us using first the crystal violent on all slides, then washing it off with water and staining with safranin. After quiet some time of tedious but deliberate work mixed with a good playlist and some visitors, we ended up with 26 beautiful slides (3 extra slides were made from the controls).
Our next step is to run our PCR on a gel and hope to see bands so we can continue on with the cloning process.