Lab Safety-General No eating or drinking Only registered students allowed in the class Long hair must be tied back Familiarize yourself with the emergency stations Do not mark on the models Inform me of any medical conditions that will interfere with participation in the lab
Lab Safety-Cleaning Clean up your workstation! Return all materials to the storage sites Clean glassware and wipe down countertops Follow directions for disposing used slides, solutions, or other biohazard waste. Wash your hands
Microscope (and The Cell) Lab Exercise #1
Lab Safety-The Microscope Carry the microscope with 2 hands Clean oculars and objectives with lens paper before and after use Do no drag/slide the microscope, fully lift it to move it We will not be using immersion oil
Objectives Identify and know the functions of the parts of the dissecting and compound microscopes Learn to use the microscope Prepare a simple stained slide and view it under the microscope Identify the basic structures of the cell and learn their functions
Cells The microscope is an essential tool used to view cells and tissues. You must treat the microscope with CARE! Some of the cells and tissues we use in the labs are stained with dyes so that the cells and some of their parts are visible.
Resolution versus Magnification Resolution is the ability to distinguish between two points The ability to see details Magnification refers to the enlargement of the image of the object being examined Typically, the better the magnification, the better the resolution.
Resolution Resolution is a measurement of how well the smallest details of an image can be discerned. The wavelength of the light source used in a microscope limits the resolution, and thus the magnification that can be achieved. The white light limits magnification to 1000X, but other microscopes like the UV and electron microscopes can achieve high resolution and magnification Does A or B have better resolution? A B
Magnification Magnification is simply the number of times an image's size is enlarged. In practice this simply means that if we view a 1 cm line at 2x it appears to us as a 2 cm line.
Dissecting versus Compound Dissecting miscroscope is used to view opaque specimens 10-20X Compound microscopes have higher resolution and magnification 40-1000X
FIVR Class 2012 SEM image of a retina Electron Microscopes Electron Cryomicroscopy (TEM) Structure of TRPV1 Used to investigate subcellular structures. Use an electron beam instead of light and provide high resolution and magnification (1,000,000X). There are 2 types: transmission electron microscope (TEM) and the scanning electron micrscope (SEM). TEM uses ultra thin tissue sections stained with heavy metal ions, and the electron beam passes through the specimen and the image is projected onto a fluorescent screen. In SEM, the electron beam bounces off the surface of the specimen to reveal detail.
Components of the compound microscope Oculars (eye pieces)-pair of lenses used to look into the microscope and magnify the image 10X The distance between the oculars should be adjusted to match the distance between your eyes so that the 2 images overlap and you only see 1 image. Body tube-allows the light to pass from the objective to the oculars and holds the ocular and revolving nose piece. Revolving nose piece-holds the 4 objective lenses each of which can be moved into position above the opening in the stage. Feel it click into place. Objective- lenses attached to the revolving nose piece. Scanning 4X (find the specimen) Low power 10X (initially focus the slide) High-dry 40X (to see greater detail) To calculate total magnification, the power of the objective lens is multiplied by the power of the ocular.
Components of the compound microscope Stage- platform on which the slide is placed Use the metal clips to hold the slide in place 2 knobs that move the slide up/down or back/forth (image moves in opposite direction because the image is inverted) Course and Fine adjustment knobs- move either the stage or body tube. The image can be focused by increases or decreasing the distance between the objective and the slide. Use course for initial focusing and fine for precise focusing In-base illuminator-at the base and adjusted by the rheostat dial. Condenser lens-located under the stage used to focus the light from the in-base illuminator onto the slide. Can affect the intensity and clarity of the image. Position can be adjusted by pinion knob. Iris diaphragm and lever- increases or decreases the amount of light coming from the in-base illuminator. Base and arm- flat bottom part of the microscope. ALWAYS use two hands when carrying the microscope. One hand to support the base One hand to hold the arm
Preparation of the Wet Mount Obtain clean glass slides, coverslips, lens paper, bottle of methylene blue, paper towel and toothpick. Place the slide on a papertowel and put a drop of methylene blue Scrape the inner lining of your cheek with the blunt end of the toothpick. Stir the blunt end into the stain and dispose of the toothpick Add the coverslip to the slide. Place one edge of the coverslip at a 45 degree angle and slowly lower to avoid getting bubbles. What does methylene blue stain? Why do we use it? Cells are transparent. Methylene blue stains negatively charged molecules in the cell, including DNA and RNA. This dye is toxic when ingested and it causes irritation when in contact with the skin and eyes. The cells seen are squamous epithelial cells from the outer epithelial layer of the mouth. The small blue dots are bacteria from our teeth and mouth. Label the nucleus, cytoplasm, and plasma membrane in the 400X drawing
Focusing the compound microscope Clean lenses with lens paper only! Do not use other types of tissue or paper. Turn on illuminator and adjust light to high Click lower power lens into position over the stage Fit your slides inside the calipers Use stage knobs to move the slide into the middle of the stage --make sure the specimen is in the center of the stage so that light passes through it. Adjust oculars to correct distance Turn course adjustment until image comes into view and becomes sharp Use the fine adjustment for complete focusing Adjust the light If a specific part needs to be further magnified, bring it to the center of the field of view using the stage knobs. At higher magnifications the field of view is smaller. Use the revolving nosepiece to switch to a higher power objective (do not use course adjustment) The high power objective will come close to the slide! Do not use the course adjustment or you will snap your slide in half! Use only fine adjustments. Skip the oil immersion step Draw what you see with a total magnification of 40X, 100X, and 400X Discard the slide and coverslip in the biohazard box. Look at prepared slides: skeletal muscle, nervous tissue, and blood
Returning the microscope I must approve your microscope condition before it is placed in the cabinet. Before you unplug the microscope, turn off the light source switch. Clean all the lenses with lens paper Loosely wrap the electric cord around the base of the microscope Use 2 hands when transporting the miscoscope Use textbook to review the structure of the cell Complete all lab report questions before leaving