Blog #2: Preparations – Timeline, Equipment, and Materials

This is a blog I had been writing last week, but I got so caught up in my trial run and project preparations that I never got around to posting it. Last week during the KCN trial run and yesterday during the set-up of my microcosms, I ran into some obstacles that required some modifications of my project, which I will explain later. Overall, though, filtration and set-up ran very efficiently, and my project is now underway!

06/14/2011:

The KCN has arrived! This means I can do a trial run with a few treatments over the rest of this week to test out the KCN/virus decay assessment and also practice making slides for epifluorescence microscopy. I have finished rinsing out 19 2-L carboys with a 2% HCl solution, so they should be ready for use soon (after they have dried). I will need 15 carboys for my microcosms and 2 or 3 for the transfer of water from the larger carboys during filtration.

Before I sample from Lake Matoaka and set up my experiment, I have thought about how long this process will take, which materials I will need, and what measurements I should collect during the four-day incubation of my microcosms. Since I will have 15 2-L microcosms, I need at least 30 L of water plus a few more since my end goal is to obtain a virus-free filtrate, with which I can then reconstitute the bacterial concentrate (BC) and viral concentrate (VC) I obtain through tangential flow filtration. Below is my plan for the sampling methods and filtration process I will use.

Filtration

On Monday morning, I will collect lake water from Keck Pier in two 20-L carboys (large plastic containers) for a total of 40 L. I can store one carboy in a cold room while I obtain three of my treatments from the other carboy. First I will remove 3 2-L samples of unfiltered freshwater for my control treatments. Then I will set up a filter stand with 5-μm filter and attach a10-μm filter preceding it, with tubes running from the water to be filtered through a peristaltic pump and filters, and then into another container. I need to remember to run bleach and di-H2O through the filters to make sure they are sterilized before I move on with the filtrations. I will filter the remaining 14 L through a 10-μm filter and a 5-μm filter simultaneously. The 5-μm filter will remove larger plankton that may prey on grazers; thus, top-down control on grazers will be reduced, which should lead to an increase in their abundance over time. At this point I’ll remove 3 x 2L for grazer-enhanced microcosms and set those up. During this filtration, I can also set up and start the filtration for the second carboy. I also need to filter the remaining 7-8 L of filtrate through a 0.8-μm filter to obtain grazer-free filtrate. After that I will remove another 3 x 2L of filtrate for grazer-free microcosms, which contain only bacteria and viruses (as well as some particulate and dissolved organic carbon, or POC and DOC, respectively).

For the second carboy, I will pump the water through a 10-μm filter to remove debris that may block even smaller filter pores. I will then pump the remaining ~19 L through the 0.22-μm filter (using tangential flow filtration) in order to obtain ~1 L BC and ~18 L bacteria-free filtrate. At some point I need to clean the 0.22-μm filter with 0.1 M NaOH for 30 min and run sodium azide through after for storage. There are a lot of steps to keep track of for both filtering and cleaning the filters! Lastly I will pump the bacteria-free filtrate through a 30 kDa tangential flow filter to obtain ~17 L virus-free water and ~ 300 mL VC (30 kDa is such as small pore size that this filter traps viruses!). Eventually I will add a portion of BC to VFW (with heat-inactivated VC) as one treatment, and the other will consist of BC with an enhanced viral abundance through addition of VC. After this last filtration, I need to clean the 30kDA filter with 0.1 M NaOH, di-H2O, and sodium azide (maybe later in afternoon once microcosms have been set up), and then store it in the cold room.

Since filtration will take a long time (last time it took about 7 hours!), I will probably be done with it by Monday evening. If I am not able to set up all microcosms by Monday, I will set up the remaining ones by Tuesday morning (I’d rather have the microcosms set up on different days but around the same time of day so that my 24-hour subsamples can be done about the same time). That way I will either be taking subsamples Monday, Tuesday, Wednesday and Thursday, or Tuesday, Wednesday, Thursday, and Friday. I need to keep in mind that at t = 0 (time that microcosm has been set up), and I must take enough sub-samples to analyze the raw sample for BA and VA, BC for BA, and VC for VA. Once all the microcosms are set up and intitial t = 0 sub-samples have been collected, I can begin subsequent sub-sampling and data collection for my project!

Microcosms and Sub-sampling

For each microcosm I set up (except the virus-reduced one), I will remove a 50-mL sub-sample (t = 0, 24, and 48) and add KCN in order to analyze viral decay – and thus viral production (VP) – simultaneously with the VP in the (-)KCN microcosm. I will thus remove sub-samples for epifluorescence microscopy, t-RFLP and bacterial production. For each of these lab techniques, the number of sub-samples and amount per sample with vary. For example, for epifluorescence microscopy I will collect samples once I set up the microcosms (t = 0) and every 24-hour period after for three more days (t = 24, 48, and 72). For t-RFLP, I will only collect samples at t = 0 and t = 72 to analyze bacterial community composition (BCC) change over the 3-day incubation period. Fir bacterial production, I will begin sub-sampling at t = 24 and collect additional samples at t = 48 and 72. These samples will be analyzed immediately for bacterial uptake tritiated leucine. Since this technique involves a radioactive substance, Professor Williamson will undertake most of it while I shadow.

There is a lot to keep track of, but I feel prepared now that I have a detailed protocol that can serve as a road map for my project. I’ll post more once I have some results!