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.