Blog #1 Literature Review and Protocol Preparation

Hello!

It has now been two weeks since I began my summer research in the Bacteriophage Ecology Lab at William & Mary. I have spent this time reviewing articles and books for ideas as I’ve prepared the details of my experimental protocol. I have also shadowed some lab techniques that I will be using for my experiment once I have collected samples and set up microcosms, which will be incubated in the lab. On Monday, June 6, I learned to use tangential flow filtration to obtain filtrates and concentrates with which to create treatments. Other lab members have shown me how to prepare slides for enumeration via epifluorescence microscopy. I will later explain these lab techniques in further detail.

As I’ve spent time in the lab planning out my project, I have become acquainted with my fellow lab mates and learned a little bit about their projects. There are several. One student is studying general microorganism community patterns in Lake Matoaka. There is work being done on whether a type of tea can kill bacteriophages, thus removing top-down control on bacteria and increasing microbial biomass. A few lab members are working with specific primers they have designed. There is an experiment underway on whether it is possible to use smaller samples to study viral community composition and get similar results (so that heavy 20-L samples do not have to be collected in the future). Lastly, one student is studying a variety of diatoms after exposing them to viruses to see whether or not the viruses can break apart their silica frustules, or hard outer “shells,” during cell lysis. This has very important implications for research on biofuels. Essentially, scientists are looking for an approach where the benefits of obtaining lipids from diatoms outweigh the costs of breaking apart their outer protection. As you can see, the opportunities for research in this lab are very diverse!

My project will involve utilizing several techniques to determine the effects of viral infection versus protist grazing on bacteria. I have been perusing Web of Science and compiling a RefWorks list of articles related to my research, and in the process I have learned about different ways in which to analyze protist grazing and viral production. For the former, I plan to create a “grazer-enhanced” microcosm by filtering out predators of grazers using a 5-μm filter (Simek et al., 2001). This seems like a good way to approach the problem that arises when filtration allows one to remove organisms by their size, so that it is difficult to isolate grazers because smaller and more numerous bacterial cells will invariably remain in a grazer concentrate. Other methods have been described in which fluorescently labeled bacteria (FLB) or other fluorescent dyes are used to track protist grazing (Epstein and Jeffrey, 1995; Sherr et al., 1987). However, because of the extra materials and techniques involved, I have decided to consider trying one or more of these methods out in a separate project in the future. For now, tangential flow filtration should be sufficient to determine whether or not there is a difference between microorganism community dynamics in raw lake water, grazer-enriched water, and virus-enriched water.

For analysis of viral production, there are also a variety of possible methods. Papers often mention the use of FVIC, or frequency of visibly infected cells, as a means of assessing viral infection of bacteria. This is analyzed through a TEM (transmission electron microscope). It is a very laborious and time-consuming process, and given the fact that I will already be spending a lot of time on enumeration via epifluorescence microscopy and analysis of bacterial community composition (BCC) via t-RFLP. I have thus discussed an alternative method with Professor Williamson. It involves the use of potassium cyanide (KCN) to kill viruses and measure their decay rate (Fischer and Velimirov, 2002; Heldal and Bratbak, 1991). The assumption is that viruses decay at a particular rate, and that they must grow at the same rate to maintain their abundance levels. Thus, if I obtain a line with a negative slope corresponding to a decrease in viral abundance, the positive slope can represent the viral growth rate.

I have also found some books in Swem Library that have been very helpful in providing background information on microorganisms and their biotic and abiotic interactions. These include Aquatic Microbiology 4th Edition by G. Rheinheimer, which has a very comprehensive description of this field. It covers both freshwater and marine microbiology, and has information on bacteria, fungi, and viruses, among other microorganisms. There are sections on environmental factors that influence microorganisms, interactions among organisms, and the role of microorganisms in the production and cycling of nutrients. These are all topics that are significant to my research, and they provide the big picture for why such studies are useful. I am happy I was able to find these resources, because it is important for me to understand how my project fits into the context of other experiments and knowledge in microbiology.
I have met with Professor Williamson several times to discuss my protocol, timeline and materials needed for my project. In short, I will be setting up triplicates of five treatments, which means that I will need 15 2-L polycarbonate containers to serve as my microcosms. The treatments will be:

(1) Raw freshwater (control) with grazers + bacteria + viruses
(2) Grazer-enhanced
(3) Grazer-free
(4) Virus-free (bacteria in virus-reduced water)
(5) Virus-enhanced

I will be using cryo vials, Eppendorf tubes, and Falcon tubes to collect and store subsamples at t = 0, 24, 48, and 72. In my next blog, I can discuss my experimental protocol and subsampling plans in more detail.
This week we are waiting for the KCN to arrive, after which I hope to do a trial run with a few microcosms to make sure the procedure works out. Once I have all the materials, I should be able to proceed with the sampling form Lake Matoaka, filtration steps, and set-up of microcosms starting this Monday, June 20. The set-up of microcosms will probably take up all of Monday and some of Tuesday, and then I will be taking subsamples through Friday. After next week I can begin using the lab techniques I have discussed to look at bacterial and viral abundance and production in my subsamples. Although I will not directly measure production by grazers, I can look for differences in bacterial abundance and production between the treatments and draw conclusions about the effects of grazers versus viruses on BCC.

I am very excited about having developed a detailed protocol, and I also have realized through this process that my next weeks will very busy as I continue to work on this project.

 

References:

Epstein, Slava S., & Jeffrey Rossel. 1995. Methodology of in situ grazing experiments: evaluation of a new vital dye for preparation of fluorescently labeled bacteria. Marine Ecology-Progress Series. 128: 143-150.

Fischer, U. R., & B. Velimirov. 2002.. High control of bacterial production by viruses in a eutrophic oxbow lake. Aquatic Microbial Ecology. 27(1): 1-12.

Heldal, M., & G. Bratbak. 1991. Production and decay of viruses in aquatic environments. Marine Ecology-Progress Series. 72(3): 205-212.

Sherr, Barry F., Evelyn B. Sherr, and Robert D. Fallon. 1987. Use of Monodispersed, Fluorescently Labeled Bacteria to Estimate In Situ Protozoan Bacterivory. Applied and Environmental Microbiology. 53(5): 958-965.

Simek, Karel, Jakob Pernthaler, Markus G. Weinbauer, Karel Hornak, John R. Dolan, Jiri Nedoma, Michal Masin, and Rudolf Amann. 2001. Changes in Bacterial Community Composition and Dynamics and Viral Mortality Rates Associated with Enhanced Flagellate Grazing in a Mesoeutrophic Reservoir. Applied and Environmental Microbiology. 67(6): 2723-2733.