BIO 204 — Biological Techniques: Lecture Note

About this lecture note

This is the complete lecture note for BIO 204 Biological Techniques. You can read it online below, open it in the full viewer, or download the PDF for offline study. It pairs with the course's video lectures, so you can read and watch together.

Preview: opening of the note

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS Course code: BIO 204 Course title: Biological Techniques Course credit load: 2 Units Series 1: Microscopy and Slide Preparation • Part 1.1: The Light Microscope o Sub Part 1.1.1: Parts of the light microscope and their functions o Sub Part 1.1.2: Handling and care of the microscope o Sub Part 1.1.3: Magnification and resolution • Part 1.2: Slide Preparation (Microtomy) o Sub Part 1.2.1: Types of microscope slides and mounting media o Sub Part 1.2.2: Preparation of temporary wet mounts o Sub Part 1.2.3: Staining techniques • Part 1.3: Biological Drawings and Diagrams o Sub Part 1.3.1: Rules and conventions for biological drawings o Sub Part 1.3.2: Use of the hand lens o Sub Part 1.3.3: Scale, labelling, and annotation • Part 1.4: The Electron Microscope o Sub Part 1.4.1: Types and principles of electron microscopy o Sub Part 1.4.2: Comparison of light and electron microscopes o Sub Part 1.4.3: Applications and limitations of electron microscopy Introduction Microscopy is the foundation of biological laboratory work. The ability to observe cells, tissues, and microorganisms depends on understanding how microscopes work and how to prepare

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS specimens for examination. This Series covers the light microscope and its use, slide preparation techniques, biological drawing conventions, and an introduction to electron microscopy. Mastery of these practical skills is essential for all subsequent laboratory work in Biological Techniques. By the end of this Series, you will be able to handle and operate a light microscope correctly, prepare and stain slides, produce accurate biological drawings, and distinguish between light and electron microscopy. Series Learning Outcomes Upon completing this Series, you should be able to: • SLO 1.1 identify the parts of a light microscope and state their functions (Linked to Part 1.1), • SLO 1.2 describe the correct procedures for handling and focusing a light microscope (Linked to Part 1.1), • SLO 1.3 explain the stages in preparing a temporary wet mount and applying stains (Linked to Part 1.2), • SLO 1.4 produce accurate biological drawings following accepted conventions (Linked to Part 1.3), • SLO 1.5 use a hand lens correctly and apply scale and labelling conventions (Linked to Part 1.3), • SLO 1.6 distinguish between light and electron microscopes and state their applications (Linked to Part 1.4), and • SLO 1.7 describe the limitations of electron microscopy and the conditions required for specimen preparation (Linked to Part 1.4). 1.1 The Light Microscope 1.1.1 Parts of the light microscope and their functions The light microscope uses visible light and a system of glass lenses to magnify small objects. Its main structural components are: eyepiece (ocular lens, usually x10; the lens through which the observer looks); body tube (connects eyepiece to the objective lenses); revolving nosepiece

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS (holds and rotates objective lenses); objective lenses (low power x4 or x10, high power x40, oil immersion x100); stage (flat platform holding the slide); stage clips or a mechanical stage (hold the slide in place); and the base (supports the instrument). Focusing components: coarse adjustment knob (large knob; moves the stage or body tube for initial focusing); fine adjustment knob (small knob; gives precise focusing); condenser (directs light upward through the specimen); iris diaphragm (controls the amount of light reaching the specimen); and light source (built-in lamp or mirror). Total magnification = eyepiece magnification x objective magnification. Fill in the blank: Total magnification of a microscope is calculated by multiplying the ________ magnification by the objective lens magnification; for a x10 eyepiece and x40 objective, the total magnification is x400. Figure 1.1: Labelled diagram of a compound light microscope

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS 1.1.2 Handling and care of the microscope Correct handling protects the microscope and ensures reliable results. Always carry the microscope with one hand under the base and the other gripping the arm. Place it on a flat, stable bench away from the bench edge. Begin with the lowest-power objective in position. To focus: place the slide on the stage, lower the objective close to the slide using the coarse knob while looking from the side (not through the eyepiece), then slowly raise the objective while looking through the eyepiece until the image is in focus. Care and maintenance: clean lenses only with lens tissue (never paper or cloth); never force the coarse adjustment knob; always store the microscope covered with its dust cover; rotate back to the lowest objective before removing a slide; and never tilt the microscope when using water-immersion or oil-immersion objectives. Report damaged parts to the laboratory instructor immediately. Fill in the blank: When focusing a microscope, always begin with the ________ power objective and use the coarse adjustment knob to bring the objective close to the slide before looking through the eyepiece. 1.1.3 Magnification and resolution Magnification is the ratio of the image size to the actual size of the object. It tells us how many times larger the image appears compared to the specimen. Resolution (or resolving power) is the ability of the microscope to distinguish two closely spaced points as separate. A microscope may magnify an image many times but still produce a blurry result if its resolution is poor. The limit of resolution of a light microscope is approximately 0.2 micrometres (200 nm). Resolution depends on the wavelength of light used and the numerical aperture of the objective lens. Shorter wavelengths give better resolution. The formula for actual size is: Actual size = Image size / Magnification. For example, if a cell appears 40 mm wide under x400 magnification, its actual width is 40 mm / 400 = 0.1 mm = 100 micrometres. Understanding this relationship is essential for making accurate measurements from microscope observations.

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS Fill in the blank: The limit of resolution of a light microscope is approximately ________ micrometres; resolution is the ability to distinguish two closely spaced points as separate structures rather than as one blurred image. Pilot questions 1.1 1. Name six parts of a light microscope and state the function of each. 2. Describe the correct procedure for carrying and setting up a light microscope. 3. Distinguish between magnification and resolution. 4. A cell measures 20 mm in a micrograph taken at x200. Calculate its actual size in micrometres. 5. State three rules for the proper care of a light microscope. 1.2 Slide Preparation (Microtomy) 1.2.1 Types of microscope slides and mounting media Microscope slides are flat glass rectangles (approximately 75 x 25 mm) on which specimens are placed for examination. A cover slip (cover glass) is a thin square of glass placed over the specimen to protect the objective lens and flatten the specimen. Temporary slides are prepared fresh and used immediately; permanent slides are preserved with mounting media such as Canada balsam or DPX and can be stored for years. Mounting media serve to: hold the specimen flat; improve the refractive index match between the specimen and glass (reducing light scatter); preserve the specimen; and seal the cover slip. Aqueous mounting media (e.g. water, glycerol) are used for temporary preparations of living or unfixed material. Resinous media (e.g. DPX, Canada balsam) are used for permanent preparations after dehydration and clearing of the specimen. Fill in the blank: A ________ slide is prepared fresh and used immediately, while a permanent slide is sealed with resinous mounting media such as DPX and can be stored for long periods.

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS 1.2.2 Preparation of temporary wet mounts Steps for preparing a temporary wet mount: (1) clean the slide and cover slip with a lint-free cloth; (2) place a small drop of water (or appropriate mounting fluid) at the centre of the slide; (3) place the specimen (e.g. a thin section of onion epidermis or a smear of cheek cells) into the drop; (4) hold the cover slip at 45 degrees and gently lower it onto the drop to exclude air bubbles; (5) remove excess water with filter paper at the edge of the cover slip. Sectioning: thin sections improve light transmission and image clarity. For soft plant tissue, hand sections are cut with a sharp razor blade; for harder tissue, a microtome (a precision sectioning instrument) is used. Sections should be as thin and uniform as possible. Squash preparations crush soft tissue (e.g. root tip meristems) to spread cells in a single layer; smear preparations spread liquid material (e.g. blood, cheek cells) directly onto the slide. Fill in the blank: When lowering a cover slip onto a wet mount, hold it at ________ degrees to the slide and lower it slowly to avoid trapping air bubbles beneath the cover slip.

BIO la-NET |

This is the opening of the note. Read the full note or download the PDF to continue.