LAB 2: INTRODUCTION TO MICROSCOPY, CELL STRUCTURE AND TISSUES

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1 2-1 LAB 2: INTRODUCTION TO MICROSCOPY, CELL STRUCTURE AND TISSUES The purpose of this laboratory is to learn the correct care and use of the microscope. This lab also provides an introduction to epithelial tissues and reviews the proper procedure for making a temporary wet mount of a specimen. Prelab Review microscope use (p 7) Complete introduction to cell structure self review (p 8) Complete table of organelle functions (p 9) Label animal cell diagram (p 10) Complete p 12 Fill in the representative locations for the tissues (p 13-15) Complete p I. INTRODUCTION TO MICROSCOPY Introduction The microscope provides a means of viewing specimens that are not visible with the naked eye and allows small details of visible specimens to be examined. This instrument is expensive and proper care is critical to ensure good results and to prevent damage. In order to understand the instructions for use, you must first become familiar with the components of the microscope and their functions. Once you are familiar with the components, you may proceed to set up critical (Kohler) illumination if time allows. Following the steps for critical Illumination (p 6) will ensure that the proper amount and intensity of light is focused on the specimen to allow you to see minute details. Failure to follow the instructions can cause distortion of the images and interfere with specimen identification. Objectives 1. To identify the component parts of the microscope 2. To learn the functions of the component parts of the microscope 3. To describe and demonstrate the proper techniques for care of the microscope 4. To demonstrate the proper technique of illuminating and focusing the microscope using Kohler illumination 5. To define terms relevant to microscopy

2 2-2 PART A. IDENTIFICATION OF COMPONENT PARTS OF THE MICROSCOPE AND THEIR FUNCTIONS With the assistance of your instructor, locate the microscope parts identified below on your microscope, and label them on the diagram provided. Oculars, Head, Head clamp screw, Diopter rings, Revolving nosepiece, Objectives, Stage, Condenser, Iris diaphragm ring (or lever), Mechanical stage with slide holder, Stage travel knobs, Condenser adjustment knob (not shown in diagram), Coarse focus knob, Fine focus knob, Power switch, Rheostat NOTE: there are several different models of microscope in the lab; some of the labeled parts may not be in exactly the same location.

3 Become familiar with the functions of the component parts 1. Oculars (eyepieces) Function: To magnify the specimen. Magnification is usually 10X and is engraved on the top of the objectives. The oculars are actually simple magnifying glasses and are useful if you wish to examine the lenses for dirt or oil. 2. Diopter rings Function: To correct for differences between the viewer's eyes. Your microscope may have one or two diopter rings, depending on the model. 3. Revolving nosepiece Function: To house the objectives and allow the various objectives to be moved into or away from the viewing position 4. Objectives Function: To increase magnification and resolution. The objectives are engraved with several numbers. These numbers give information about the quality, requirements and capabilities of the objectives. Magnification of the four objectives on the Kyowa Medilux-12 are 4X (scanning), 10X (low), 40X (high dry) and 100X (oil immersion). The 100X objective is only used when immersion oil is placed between the slide and objective. This objective will not be used this term unless instructor demonstrates and/or adds the oil. 5. Stage Function: Where slide is placed (moved up and down by focus knobs) 6. Mechanical stage with slide holder Function: To hold the specimens in place for viewing and allow movement of the slides in two directions to enable the viewer to examine the entire slide. The mechanical stage has readings which permit relocation of specific sites. 7. Condenser clamp screw Function: To hold condenser in place. 8. Condenser Function: To focus the light from the illuminator on the object being viewed. N.B. Incorrect adjustment of the condenser is the most common error in microscopy. The condenser must be adjusted as described in the following section. The condenser is always at or near the stage. Generally the condenser is not moved to reduce light. Moving the condenser out of its correct position causes distortion of the image. 9. Iris diaphragm ring (or lever) Function: Controls the amount of light from the light source.

4 Head clamp screw Function: To hold head in place and to allow rotation of the head. Care should be taken to ensure that the head clamp screw is secure. 11. Stage travel knobs Function: To move the slide over the stage surface 12. Head Function: To house the two oculars 13. Condenser adjustment knob Function: To move the condenser up and down for critical illumination. 14. Coarse focus knob Function: To focus specimen with scanning objective. 15. Fine focus knob Function: To fine focus on the specimen. 16. Power switch for light Function: To provide light for viewing specimens. 17. Interpupillary distance scale Function: To give setting for distance between the viewer's eyes.

5 2-5 PART B. CARE AND USE OF THE MICROSCOPE 1. Students are responsible for the care and maintenance of the microscopes for the semester. 2. The microscopes should always be stored in the microscope cabinet with the 4X objective in place and the cord neatly and securely wrapped around the microscope. 3. Take the microscope assigned to your station from the cupboard. Carry with one hand on the arm or limb and the other underneath the base. Ensure that the microscope is held in its normal position with the base horizontal to the floor. If microscope is tilted, there is a danger of the oculars or condenser falling out. 4. Check your microscope at the beginning of each laboratory period and report any problem or malfunction to your instructor so that you will not be held responsible. 5. Check that all objectives are screwed in firmly and that the head clamp screw is tight. 6. Lower stage as far as possible, using the coarse adjustment to separate the objective and stage as widely as possible. 7. Wipe all lenses of the oculars, objectives, condenser, and the field diaphragm with kimwipes before use. 8. Obtain a stained, prepared slide, turn the light on, center the specimen over the light and using the 4X (scanning) objective, raise the stage as high as it goes. Now lower the stage to focus with the course focus knob. N.B. The coarse focus knob is only used with the scanning (lowest power lens) 9. Using the handling strips, adjust the interpupillary distance so that it is comfortable for you and you see only a single circle of light (viewing field). Note the reading on the scale. My reading on the interpupillary distance scale is 10. Rotate the diopter ring on the right eyepiece tube to the same reading as noted on the interpupillary scale. Switch to the 10X objective. Keep your left eye closed, look through the right ocular and adjust with the fine adjustment until the best image is obtained. Then close the right eye, look through the left ocular and adjust if necessary. Use ONLY the left diopter. Do NOT use either focusing knob during this adjustment. 11. To focus with the 10X objective, rotate the low power objective into place, and focus with the fine focus only. These microscopes are parfocal and remain basically in focus when you change from one objective lens to another. 12. Repeat the last step with the 40X lens to focus at high power. NOTE: the working distance is very small (the lens almost touches the slide); NEVER turn the coarse focus knob with any lens except the scanning objective...you can crush slides and damage lenses. 13. When you are finished using the microscope, rotate the scanning lens back into place, then lower the stage fully to remove your slide. Return the microscope to the cupboard.

6 2-6 CRITICAL ILLUMINATION (optional) 1. With the specimen in focus, move the condenser up toward the top of its range. 2. Open the substage condenser diaphragm. 3. Close the iris diaphragm. 4. Move the condenser up or down until the iris diaphragm is in sharp focus, i.e., the edges are clear and are surrounded by a bluish light. N.B. If the cone of light is not centered, ask the instructor for assistance. 5. Open the iris diaphragm until it clears the periphery of the field. Turn down the light if it is too bright. N.B. Once you have set the condenser, do NOT move it out of this position. The condenser should not be lowered to improve illumination, instead adjust the condenser diaphragm 6. Adjust the condenser diaphragm until the image is optimal. 7. Switch to high dry (40X) power. 8. Adjust the condenser diaphragm until the light is optimal for you. 9. Turn off the light when you have completed the observations. It is not necessary to turn the light off between slides but if the microscope is not being used for part of the lab, the light should be turned off. 10. Clean all lenses, rotate the 4X objective into place, wrap cord firmly and neatly around the microscope and return to the correct location in the microscope cupboard.

7 2-7 RELATED TERMINOLOGY: 1. Magnification - the enlargement of a specimen 2. Primary magnification - magnification of one of the components of the microscope, e.g., the primary magnification of oculars is 10X, objectives are 4X, 10X, 40X or 100X 3. Total magnification - the combined magnification of the oculars and the objective being used, e.g., the total magnification when using the 10X objective is 100X 4. Resolution - the ability to separate fine lines or structures so that they can be seen clearly as separate entities; the ability to see fine differences in detail. With poor resolution, two lines or points that are very close together could or would appear as one line or point. 5. Depth of focus (field) - the vertical range of the specimen that appears to be in sharp focus. As the magnification increases, the depth of field decreases. 6. Working distance - the space between the lowest part of the objective and the specimen when the specimen is in focus. 7. Viewing field the actual area that you see when looking through the microscope. The field of view decreases as the objective magnification increases. 9. Parfocal - the characteristic of a microscope that allows you to maintain focus as you change from one objective to another once the specimen has been focused with one of the objectives. Only small adjustments with the fine focus should be required. Complete the table below for your microscope: Objective lens Objective magnification Ocular magnification Total magnification

8 2-8 II. INTRODUCTION TO CELL STRUCTURE (self review) A. THE EUKARYOTIC - CELL 1. Briefly - distinguish between Eukaryotic Cell Prokaryotic Cell 2. Complete the following flow chart showing the levels of organization in living organisms: (eg. ) (eg. ) cells (eg. ) (eg. ) (eg. ) (eg. )

9 Using your text as a reference, complete the table below by summarizing the function of each of the organelles. Locate the following structures and organelles on the animal cell model and in the electron photomicrographs, then label the diagram of the cell which follows. Table 1. Organelles and their functions ORGANELLE plasma membrane FUNCTION nucleus with its chromatin nucleolus nuclear envelope nuclear pores cytosol ribosomes smooth endoplasmic reticulum rough endoplasmic reticulum golgi complex lysosomes mitochondria centrioles vesicles or vacuoles cilia or flagella cytoskeleton

10 2-10 THE ANIMAL CELL

11 Examine a living eukaryotic cell: Using a toothpick, gently scrape the inside of your cheek and spread the material on a slide. Add a drop of methylene blue stain to the smear, and cover the preparation with a coverslip. Examine the preparation with your compound microscope and locate the flat, squamous epithelial cells that line your oral cavity. Bring the cells into focus with the high power objective (400X) and sketch a few cells below (to scale, as you see them in the viewing field) What organelles are visible? Why are most of the "organelles" you just studied not visible? Fig 1. Sketch of squamous epithelial cells from lining of mouth Magnification power =

12 2-12 PART III: INTRODUCTION TO TISSUES In this lab you will be examining various epithelial tissues. You should be able to identify the cells and tissues studied, know the functions of the tissues, and know where in the body each tissue type is found. As the tissues are reviewed in the lab, you should identify each tissue image (at the back of the lab), paste the image in the appropriate space and label any structures / cells indicated. TISSUES 1. Define a "tissue" 2. Name the 4 major tissue types a) b) c) d) EPITHELIAL TISSUES AND GLANDS Epithelial tissues cover body surfaces and line body cavities, organs and vessel lumens. are characterized by a basement membrane, on which the cell layer(s) sit. form both exocrine and endocrine glands You will examine slides of various epithelial tissue types. You should learn to locate and recognize the various types and become familiar with their functions and locations in the body. Describe 4 functions of epithelial tissue:

13 2-13 Note the flow diagram below indicating the major types of epithelial tissues in terms of cell shape, (squamous, cuboidal, columnar); surface specialization, (plain, ciliated, microvillous): and the number of layers, (simple, pseudostratified, stratified, transitional). Differentiation Simple squamous Apical specializations Layering Nonkeratinized stratified squamous Keratinized stratified squamous Newly mitosed cell Simple cuboidal Microvilli Transitional Cilia Stratified cuboidal Microvilli Simple columnar Stratified columnar Pseudostratified ciliated columnar Cilia SIMPLE EPITHELIAL TISSUES A. Simple squamous FUNCTION: Rapid diffusion of materials through the tissue DESIRED FEATURES: Shortest possible diffusion distance, i.e. thinnest possible tissue REPRESENTATIVE LOCATIONS IN THE BODY: SLIDES: Mammalian lung, section (label squamous cell, alveolar sac)

14 2-14 B. Simple cuboidal FUNCTION: Form walls of small-diameter tubules, some secretion and absorption; general metabolism DESIRED FEATURES: Thicker cells than squamous, but not too thick for small tubules REPRESENTATIVE LOCATIONS IN THE BODY: SLIDES: Mammalian kidney, section. (Note the presence of microvilli in some, but not all of the tubules near the periphery. Those that have them are responsible for about 80% of the kidney's impressive reabsorption capacity) Label cuboidal cell, basement membrane. C. Simple columnar FUNCTION: Absorption of nutrients DESIRED FEATURES: Relatively large cells to house the necessary metabolic machinery and many microvilli REPRESENTATIVE LOCATIONS IN THE BODY: SLIDE: Duodenum, section (label columnar cell nucleus, basement membrane, connective tissue)

15 2-15 STRATIFIED AND PSEUDOSTRATIFIED EPITHELIAL TISSUES A. Stratified squamous FUNCTION: Protection against abrasion and tearing; can be stretched DESIRED FEATURES: Layers of cells on the surface that are expendable and that can be replaced continually from below REPRESENTATIVE LOCATIONS IN THE BODY: SLIDES: Esophagus, section (label squamous cells, basement membrane, connective tissue) B. Pseudostratified ciliated columnar FUNCTION: Trapping of airborne particles that could otherwise become lodged in the lungs DESIRED FEATURES: Production of mucus in which to trap particles and presence of cilia to move the mucus and trapped particles to where they can be disposed of REPRESENTATIVE LOCATIONS IN THE BODY: SLIDE: Trachea, section (label basement membrane, connective tissue, cilia, goblet cell). NOTE: locate a goblet cell; goblet cells are unicellular exocrine glands which secrete mucus NOTE: Glands are typically formed from epithelial tissue.

16 2-16 GLANDULAR EPITHELIUM Epithelial tissue has also become specialized for secretion. Modified epithelial cells form the functional basis of all the glands of the body including both exocrine and endocrine glands. Examine the text and answer the following: A. DISTINGUISH BETWEEN: a) Endocrine gland b) Exocrine gland B. STRUCTURALLY: exocrine glands may be... a) Unicellular: Locate the unicellular exocrine gland, the goblet cell, in the pseudostratified, ciliated, columnar epithelium. Goblet cells secrete a polysaccharide called mucin. When mixed with water, what does this secretion become? What are the functions of this substance? Where else in the body would you expect to find goblet cells? b) Multicellular: If multicellular, duct structure may be i) Simple ii) Compound Both simple and compound multicellular glands may have secretory units described as i) Tubular ii) Alveolar (acinar)

17 2-17 C. FUNCTIONAL CLASSIFICATION OF EXOCRINE GLANDS on the basis of how they work. Define each of the following terms - with examples. a) Merocrine b) Holocrine c) Apocrine EXOCRINE GLANDS Unicellular Multicellular Simple Compound Tubular Acinar Tubular Tubuloacinar Acinar D. ENDOCRINE GLANDS (Structure and Function will be studied in association with the endocrine system).

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19 EPITHELIAL TISSUES

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