Transmission Electron Microscopy of phages

SEA-PHAGES (Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science) is a two semester program. The courses give students hands-on training in bacteriophage research. The Salish Sea Research Center Associate Director recently completed the third year of SEA-PHAGES at Northwest Indian College. A phage genome was sequenced by students and named Lahqtemish, "People of the Sea."

Dr. Arnold was awarded a second round of SEA-PHAGES grant funds. During the pandemic we do not have students in the lab. In the meantime, I'm working on imaging a phage isolated from the Lummi Nation. Rosa Hunter, the Salish Sea Research Center lab research manager, has provided me with her notebook from the SEA-PHAGES program that details how to prepare Lahqtemish for imaging. We just received our order of electron microscopy forceps and mesh carbon formvar coated copper grids (see below). The grids are about 1mm in diameter. The formvar is 10nm thick and the carbon is 1nm thick and the mesh is 74um-sqaured.

We have freezer stock of Lahqtemish. The Lahqtemish will be prepared for imaging at the University of Washington using Transmission Electron Microscopy.

The image above shows the process of applying the suspended phage sample to a TEM grid and blotting the excess liquid from the sample. Then, staining the phage with Uranyl Acetate and blotting the excess away.

Transmission Electron Microscope (TEM) allows us to image with a resolution of 1.5 Angstrom. The diameter of an atom is on the order of 1 angstrom, so the unit is particularly handy when referring to the atomic and ionic radius or size of molecules and spacing between planes of atoms in crystals. Individual phage particles are too small to be seen with light microscopes, or Scanning Electron Microscopy (resolution to 1 micron to a few nanometers). Please refer to the image below for relative size differences, ranging from an atom to a frog egg. To observe Lahqtemish using TEM, the phage needs to be concentrated and cleared of protein and bacterial membrane debris. This is accomplished by pelleting your phage in a microcentrifuge and resuspending the phage pellet in clean phage buffer.

History of the Nooksack River

Figure 1 from Grah O., Beaulieu J. (2013)

This post is all about the Nooksack River and Hooligans. The headwaters of the Nooksack originate from the glaciers on Mount Baker, Mount Shuksan, and other nearby peaks of the North Cascades. There are at least 8 source glaciers within the Noosack River watershed on Mount Baker that include the Deming, Thunder, Coleman, Roosevelt, Mazama, Sholes, Heliotrope and Hadley glaciers (Figure 1; Grah and Beaulieu, 2013). The river is made up of three forks (North, Middle, and South) that converge near Deming, Washington.

There is an online series by Dr. Kleinknecht that emphasizes the importance of the Nooksack River for Salmon and Orca.

Part 1:http://whatcomwatch.org/index.php/article/middle-fork-diversion-dam/

Part 2: http://whatcomwatch.org/index.php/article/the-nooksack-river-the-history-and-current-state-of-the-river/

Part 3:http://whatcomwatch.org/index.php/article/the-nooksack-river-a-treasure-to-preserve-2/

Part 4: http://whatcomwatch.org/index.php/article-categories/salmon-streams-and-tributaries/

At the Salish Sea Research Center we pay close attention to the Longfin Smelt in the Nooksack. I am working on qPCR of eDNA of Longfin Smelt (Spirinchus thaleichthys) or in Indigenous language, the Tiokowe. They are also known as Hooligans, or Hoolies. These species are also endangered in the Nooksack River. Anadromous (a fish such that migrates up rivers from the sea to spawn) fish like the Tiokowe need places to spawn.

The Whatcom Watch articles discuss how the Nooksack River has been modified for the use of local prairies for farmland. Since European arrival the numbers of fish that return to spawn in the Nooksack has greatly diminished due to loss of habitat due to human-caused alteration o the watershed.

Since the river was their main mode of transportation, [the settlers] first cleared and built along [the Nooksack] banks. Because they wanted boats larger than dugout canoes to bring equipment and supplies up the river, they cleared out an enormous natural log jam just south of Ferndale that ran nearly a mile. Removal of this log jam allowed them to get a steamboat upriver to Ferndale and to Lynden. This modification of the river might have been the first significant blow to the Nooksack’s salmon spawning territory.

A USGS report by Anderson and others (2019) describe the physical location and the history of flooding and sediment load into the Nooksack River over the past 15 years. They reference Nugent's Corner, found close to convergence of the north fork to the south fork (see map above).

There is a quite a lot of sediment that washes into the Nooksack River. Based on estimates by Anderson et al., (2019), about 100, 000 cubic yards per year is distributed into the Nooksack upstream of Nugent’s Corner near Glacier Creek and likely represents a significant source of coarse sediment to the lower mainstem river. To gain some perspective, if you covered a football field with this sediment to the height of the goal post cross bar (10 feet) this would give you a volume of 21,333 cubic yards.

Anderson, S.W., Konrad, C.P., Grossman, E.E., and Curran, C.A., 2019, Sediment storage and transport in the Nooksack River basin, northwestern Washington, 2006–15: U.S. Geological Survey Scientific Investigations Report 2019-5008, 43 p., https://doi.org/10.3133/sir20195008.

Grah O., Beaulieu J. (2013) The effect of climate change on glacier ablation and baseflow support in the Nooksack River basin and implications on Pacific salmonid species protection and recovery. In: Maldonado J.K., Colombi B., Pandya R. (eds) Climate Change and Indigenous Peoples in the United States. Springer, Cham. https://doi.org/10.1007/978-3-319-05266-3_12

This post is pulling together four post on the Whatcom Watch Online community forum by Ron Kleinknecht, professor emeritus of psychology and dean emeritus of Western Washington University’s College of Humanities and Social Sciences.

qPCR: how to do it right, the first time

The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time Taylor et al., 2019

As I am new to qPCR, I found this review article extremely helpful.

qPCR is a technique that provides precise and quantitative data on nucleic acids. The minimum information for publication of quantitative real-time PCR experiments (MIQE) provides guidelines for validation and data analysis procedures. Major sources of error are outlined in Taylor et al., 2019.

To validate a qPCR experiment and to minimize error and maximize data quality, you must first validate primers. To validate primers, an equalized pool of samples form each biological group is diluted 1:20 and initially tested suing a thermal gradient to determine the optimal annealing temperature, average level expression and unique product for each target from melt curve and gel analysis. The quantitative cycle (Cq) value from the optimal annealing temperature range can be used as a guide to establish the standard curve dilution factor for each target (i.e., if the Cq value for optimized temperature range is between 10 and 16, use a 1:8 serial dilution series of the pooled cDNA sample in water). An eight-point standard curve is tested for each primer pair using the same pooled sample and the appropriate dilution factor as determined from the thermal gradient data.