BIO 131 LAB Microscopy: Types and Principles PDF

BIO 131 LAB MICROSCOPY: TYPES AND PRINCIPLES Mai Sater MD, PhD Department of Medical Biochemistry Objectives ■ Recognize the principles and uses of different types of microscopes ■ Identify the parts of compound light microscope and understand their function ■ Find out the total magnification (ob...

BIO 131 LAB MICROSCOPY: TYPES AND PRINCIPLES Mai Sater MD, PhD Department of Medical Biochemistry Objectives ■ Recognize the principles and uses of different types of microscopes ■ Identify the parts of compound light microscope and understand their function ■ Find out the total magnification (objective and ocular lenses) ■ Understand and follow the rules of microscope use. ■ Focus the compound light microscope at low power and high power magnifications ■ Be familiar with terms like: (1) inversion (2) parfocal (3) Field of view (at low and high power) ■ Prepare a wet mount for: (1) human epithelial cells (2) onion epidermal cells ■ Focus stained slides (films/smears) of: (1) parasite (2) bacteria (3) human blood Microscopy ■ Biological objects can be very small to see with the naked eye. Accordingly scientists developed microscopes to view them. Microscopes come in many different types and forms and ranges from a simple light microscope to a complex electron microscope Longitudinal section vs cross section Microscopes ■ Electron microscope – Scanning electron microscope (SEM) – Transmission electron microscope (TEM) ■ Light microscope – Binocular dissecting microscope (stereomicroscope) – Compound light microscope Electron microscope ■ Uses a beam of electrons that is magnified and focused on the object by means of electron magnetics. ■ Can view much smaller objects compared to light microscopes, with far greater detail. ■ Very expensive. Very sophisticated. ■ Two main types: – Scanning electron microscope (SEM) à analogous to dissecting light microscope – Transmission electron microscope (TEM) à analogous to the compound light microscope Scanning Electron Microscope (SEM) Transmission Electron Microscope (TEM) Binocular Dissecting microscope (stereomicroscope) Compound Light Microscope Lenses and their magnifications Scanning objective Lower power objective High power objective Oil immersion Total magnification OBJECTIVE LENS POWER OF OCULAR LENS POWER OF OBJECTIVE LENS TOTAL MAGNIFICATION SCANING POWER 10X 4X 40 LOW POWER 10X 10X 100 HIGH POWER 10X 40X 400 OIL IMMERSION 10X 100X 1000 Total magnification = ocular x objective Rules for microscope use ■ Keep both eyes open while using the microscope and do not touch the eyepiece with your lashes ■ Lowest power objective (scanning) should be in position both at the beginning and end of microscope use ■ Use only lens paper for cleaning the lens ■ Do not tilt the microscope when viewing ■ Do not remove parts of the microscope ■ To locate small objects in slide, first find them under low power, then place them in the center of the field before rotating to high power Focusing the microscope- low power 1. Always begin focusing with the scanning power objective 4x 2. With coarse adjustment knob lower the stage until it stops 3. Place a slide on the stage and stabilize it with a clip 4. While looking through the eye piece with both eyes, slowly raise the stage using the coarse adjustment knob until the object comes into view 5. Use the fine adjustment knob to sharpen the focus if necessary Focusing the microscope- high power Compound light microscopes are parfocal, meaning once an object is In focus with the low power field, it should be almost in focus with the higher power 1. Always find the object under the low power field before viewing it with the high power field 2. Make sure the object is centered in the middle field 3. Move the objective lens to the high power objective. You should hear a “click” sound. (note: Parfocal microscope objectives will not hit normal slides when changing the objective if the lowest objective was initially used to focus) 4. If any adjustment is needed, use the fine adjustment knob only. Inversion ■ Inversion refers to the fact that a microscopic image is upside down and reversed Field of view ■ The area of the slide that you see when you look through a microscope's eyepiece. It is shown as a circle ■ Field of view of high power is ¼ of low power HP Field of view (40 x) LP Field of view (10x) Low-power field diameter (LPD) ■ Use the 10X (low power) objective ■ Use a clear ruler to measure the field diameter in mm ■ Convert from mm to micrometer ■ This will be your LPD (always expressed in micrometer) ■ You can calculate the size of a single cell which is equal to: Example: LPF = 2 mm (measure by ruler) = 2000 micrometer 1 cell = 2000/ 5 = 400 micrometers e.g. 2 mm diameter LP Field of view (10x) How many cells would you expect to see if you switch to high-power objective? High-power field diameter (HPD) ■ Since the high power magnification is four times the low power magnification, the field of view of the high power is one fourth of that of the low power. So to obtain the HPD you simply have to divide the LPD by four. If LPD is 2000 micrometers The HPD will be 2000/4 = 500 micrometer HP Field of view If you see 5 cells along the LPD, you (40 x) expect to see approximately 1.25 cell along the diameter once you switch to high power objective LP Field of view (10x) Microscopic observations ■ Wet mount preparation Human Epithelial cells ■ Obtain a toothpick and gently scrape the inside of your cheek ■ Place the scrapings on a clean dry slide ■ Place one drop of methylene blue stain and cover it with cover slip (wet mount) ■ Methylene blue is a basic dye that penetrates the cell and stains the acidic parts of the cell ■ Observe under microscope ■ Identify: – Cell nucleus, cell membrane, and cytoplasm Onion epidermal cells ■ Strip a small, thin, transparent layer of cells from the inside of a fresh onion ■ Place it gently on a clean, dry slide ■ Add a drop of iodine solution and cover it with a coverslip ■ Iodine solution stains starch which is present in plants ■ Locate the cell wall, the nucleus, and the cytoplasm Gram staining ■ is a method of staining used to distinguish and classify bacteria species into two large groups: gram-positive bacteria and gram- negative bacteria. ■ Gram staining differentiates bacteria by the chemical and physical properties of their cell wall by detecting peptidoglycan, which is present in the cell wall of grampositive bacteria. Gram-negative cells also contain peptidoglycan, but at very small layer. Classification of Bacteria Giemsa staining ■ is a classic stain for peripheral blood smear and bone marrow specimens. ■ Erythrocyte stain pink, Platelet show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta. ■ It is also used to visualize parasites, fungus and bacteria ■ Mixture of: Methylene blue (blue), eosin (pink), and azure B (dark green) Hematoxylin & eosin (H&E) staining ■ is one of the principal staining in histology. It is the most widely used stain, in medical diagnosis, and is often the gold standard test, for example when a pathologist looks at a biopsy to identify cancer. ■ Mixture of: hematoxylin (blue) and eosin (pink)