Blog #4: Bacterial Production Assay and Epifluorescence Microscopy

Hi guys!

Here’s some additional information on how my project has been going. First, though, I would like to do a recap of what I am working on. There are three major components involved in acquiring data for my project: I used a tritiated leucine assay to determine bacterial production; I used epifluorescence microscopy to enumerate bacterial and viral abundance; and I am currently doing DNA extractions to prepare for T-RFLP assays, which I will use to determine bacterial community composition (this shows how diverse the bacteria in my microcosms from Lake Matoaka are after being exposed to different treatments). I will talk more about T-RFLP later, but here is a description of epifluorescence microscopy and the results from the tritiated leucine assay.

During week #5, I continued to make epifluorescence microscopy slides and photographed them for later enumeration, which I undertook last week (week #6). I make the slides using a Millipore manifold, a disc-shaped box with filter slots that operates via vacuum suction. Slide-making usually takes an hour to an hour-and-a-half. I usually make six at a time to make efficient use of glassware and Eppendorf tubes, which are used for making dilutions of my samples before loading them onto Anodisc filters. Once I load the filters, I use the vacuum pump connected to the manifold to draw out most of the liquid, so that the microorganisms are distributed onto the filter. Then I add a small quantity of SYBR Gold, which binds to the nucleic acids in the organisms. After waiting 15 minutes for the filters to stain (SYBR Gold is sensitive to light, so most of the staining occurs under the protection of a piece of foil), I dry out the filters and mount them onto glass slides, adding some Antifade to prolong the utility of the slides. I have to be careful when using tweezers to handle the anodics and glass coverslips, since both are brittle and susceptible to being cracked.

It is best to take photographs with the epifluorescence microscope one to two days after the slides are made. The stain begins to fade after, so one week or at most two is the maximum amount of time the slides will last. When looking at the slides, the blue light from the epifluorescence microscope excites the stained nucleic acids in viruses and unicellular organisms, which then fluoresce at a lower energy level, appearing green. Once I focus in on the fluorescing dots, I can use a camera attached to the scope to take pictures, although it takes some effort and adjusting to get a good, clear image. I take 10 photographs per slide (so 30 per treatment), after which I can go back and use a tool to highlight the dots and enumerate them. Thus with this equipment, I can estimate the number of viruses and bacteria on the anodisc and then make some calculations based on the volume of sub-sample to determine the average number of cells per volume of water.

In addition to working with epifluorescence microscopy over the last few weeks, I logged all the data from the bacterial production assays on an Excel spreadsheet, then transferred the data to a software system called PRISM for graphing and statistical analysis. From the graphs there is a visible difference among the four treatments. The control group remains fairly consistent for all three time points (t = 24, 48, and 72). Both the grazer-enhanced and grazer-free treatments show a dip in productivity over time, although the latter shows more productivity overall. The dip for the grazer-free treatment is interesting because it would make sense for bacterial production to increase over time as microbial biomass grows in the absence of grazers, yet this dip is the opposite result. In contrast, the virus-reduced treatment shows a drastic increase from t = 24 to t = 72, but then goes back to original levels at t = 72. This is an important observation because it not only suggests that viruses may have a greater effect on bacteria than do grazers, but it also shows a pattern in which viruses may affect bacteria. It would be interesting to take measurements for a longer period of time to see whether or not bacterial production oscillates on a long-term basis. Biologically this would make sense: in a virus-reduced environment the microbial biomass would increase, providing more hosts for viral infection, which would then diminish the number of bateria and thus the number of hosts, and the cycle would then start over again. Despite these visual differences, a statistical test showed that there are no significant differences between any of the treatments. After analyzing my epifluorescence microscopy data and T-RFLP assay I will hopefully have a clearer picture of how viruses and protists affect bacterial abundance and diversity.

This week (week #6) I have been doing DNA extractions using the nitrocellulose filters, which contain bacterial DNA. Next week I will work on PCR to amplify the genes I will need to conduct a T-RFLP assay. I will go into details of this third component of my experiment later.

Have a nice weekend!