How to Capture Changes

Photo Jun 07, 2 12 00 PM

Sampling during a storm!

Viruses must be studied differently than bacteria. Not only are they orders of magnitude smaller than bacteria on average, they are much more varied in their genetic material. Due to their small size, we have to collect larger numbers of them to analyze, which can be difficult, because they’re invisible to the naked human eye. In fact, most viruses are smaller than 0.22 micrometers in size, while bacteria are larger. So, how do we study viruses? What tools do we have at our disposal, and what kind of data can we get?

The most basic and most well-known method of analysis is microscopy. In order to actually distinguish virus like particles (VLPs) from normal sediments? When using a light microscope, we have to stain the particles for their genetic material. I will be doing this with a stain called SYBR Gold, which flouresces. Another, more direct way of looking at viruses is with the Transmission Electron Microscope (TEM) which shows a high resolution image that can detail even a single bacteria and the surrounding particles very well. While this can be useful, especially when monitoring something like an infection and needing snapshots, but does not help count how many are present in any given.

The next method of studying viruses is in one of the expanding fields of research, genomics. Two main methods exist, PCR and Genome Sequencing. PCR helps to find out what is in a sample. It works by essentially “melting” the strands of DNA then randomly adding nucleotides until a double stranded section is made. Then that is melted and more are made, adding exponentially more genetic material per cycle. This is used to identify what sequences or sizes of sequence are present within a sample. This can monitor whether or not these sequences are present over several time points. Genome sequencing on the other hand provides the actual code present within a sample. With recent advances in sequencing technology, entire megabases are sequenced simultaneously. Both methods provide information about the viruses in question, but the scope of the data desired dictates which will be used in the study.

The purpose of my experiment, the VEE Storm Water Project, is to study how a community changes due to rainwater, and whether viruses from runoff affect the community they end up in. To do this, sampling will be done from the soil samples near a storm water retention pond. The idea of a storm water retention pond is to capture and filter runoff from nearby communities, so sampling from soil near a pond, and from the water in a pond both before and after a rain event will tell whether or not viruses are transferred between environments. If they are, late time points will identify how late these persist in the system. The primary approach for this method is to do PCR on a community sample to obtain a large quantity of DNA or RNA, and then use that to identify what sequences are present over a period of week both before and after a rain event.

A simplified experimental design:

  • Gather soil and water samples during time before a storm
  • Gather water samples at the beginning of a rain event
  • Gather water samples late in the rain event
  • Gather water samples once the system has had time to stabilize
  • Put samples through analysis to determine changes in community composition. If so, are the changes present in the soil/sediment before the rain event?

While the sampling will be fairly straightforward, the analysis will take some time. In addition, each of these steps is fairly resource intensive, so while one set of samples is being processed, I will have other opportunities to work on other projects going on in the lab. More details on those coming soon!

Comments

  1. scgilliand says:

    Your project sounds really fascinating! I had assumed that researchers who work with viruses would be in the lab all day. It sounds like you have a great mix of field work and lab work. If the viruses are indeed transferred in storm water, it would be interesting to know what organisms would be affected by them. I also wonder if there are lower amounts of viruses transferred between communities in dry years versus rainy years.