BMED 2000 Laboratory 1 - Histology & Basic Microscopy PDF

BMED 2000-Laboratory Techniques 2 Humber College UNIT 1: Foundational Laboratory and Microscopy Skills LABORATORY 1 – HISTOLOGY & BASIC MICROSCOPY Rationale The purpose of this laboratory exercise is to observe a variety of different cell types – from microbes to mammalian cells – under micr...

BMED 2000-Laboratory Techniques 2 Humber College UNIT 1: Foundational Laboratory and Microscopy Skills LABORATORY 1 – HISTOLOGY & BASIC MICROSCOPY Rationale The purpose of this laboratory exercise is to observe a variety of different cell types – from microbes to mammalian cells – under microscope. Students observe the varied characteristics of these organismal samples while also developing familiarity with the compound microscope and some of the various staining/visualizing methodologies at our disposal as biomedical scientists. As such, students will practice wet mount slide preparation, and comparison of the various structures of bacterial, yeast, mammalian, and plant cells. Objectives 1. Practice the application of oil immersion microscopy to view a variety of microrganisms. 2. Compare the relative size and shape of various bacterial and mammalian cells on prepared slides. 3. Prepare and analyze wet mounts of plant cells. 4. Understand the process behind adding color to translucent layers of tissues and cells for proper visualization of general histology. 5. Explain the mechanism behind H&E staining. 6. Perform H&E stain on cultured cells. INTRODUCTION Compound Microscope A light microscope is an instrument that uses a combination of magnifying optical lenses and light to aid in the enlargement and visualization of extremely small objects, invisible to the naked eye. The invention of the microscope over three centuries ago has revolutionized the field of biological science by allowing scientists to visually observe and investigate the most fundamental and basic unit of life: the cell. While compound microscopes of differing size and shape are available, all function to serve a similar purpose: to magnify and permit visualization of extremely small objects invisible to the naked eye. For more information regarding the Parts of the Compound Microscope, Magnification Power, Calibration and Size Determination, and Slide Preparation, see Appendix – Lab 1. Bacteria Bacteria are unicellular prokaryotic microorganisms. They can be characterized based on shape, structure, metabolism, and by differences in genetic makeup. Most bacteria are approximately BMED 2000 Unit 1: Laboratory 1 1 0.5-1.0 micrometer (μm) in diameter, and 1-8μm in length. There are three common shapes of bacteria: the coccus, the bacillus, and the spiral. In these shapes, bacteria can exist as single cells, pairs, chains, or clusters. Bacteria commonly reproduce by binary fission. Bacteria can also possess cytological features such as spores, flagella, and capsules. Coccus A coccus-shaped bacterium is usually spherical or oval, 0.5-1.0μm in diameter, and may be observed in one of the following arrangements: Diplococcus arrangement: cocci in pairs Streptococcus arrangement: cocci in chains Tetrad arrangement: cocci forming a square of four Sarcina arrangement: cocci forming a cube of eight (2x2x2), or Staphylococcus arrangement: cocci in irregular, often grape-like clusters Bacillus A bacillus, or rod, is a “hot-dog” shaped bacterium typically on the order of 0.5 to 1.0μm wide and 1 to 4μm long. Bacillus bacterium can exist in one of the following arrangements: Bacillus: a single bacillus Diplobacillus: bacilli in pairs Streptobacillus: bacilli in chains, or Coccobacillus: oval and similar to a coccus Spiral Spiral-shaped bacteria occur in one of three forms, as outlined below: Vibrio: an incomplete spiral or comma-shaped Spirillum: a thick, rigid spiral, or Spirochete: a thin, flexible spiral that can be >100μm in length. Plant Cells Plant cells also belong to the Eukarya Domain, and fall within the Plantae kingdom. As such, plant cells have overlapping features with mammalian cells, such as prominent nuceli, as well as a variety of distinguishing features including cell walls, chloroplasts, and other intracellular vacuoles, which can likewise be observed under microscopic magnification. Additionally, as the Plantae kingdom gives rise to >350,000 species, the size of plant cells can range from anywhere between 10-100 μm long. Mammalian Cells Like yeasts, mammalian cells are eukaryotic, thus they contain nuclei, in which their DNA is found, as well as other important cellular organelles such as mitochondria, lysosomes, and endoplasmic reticulum, among numerous others. Additionally, since mammals consist of billions of cells arranged in specific arrangements across different organs and tissues. These specific patterns and combinations give rise to specialization of mammalian cells which are designed to carry out specific organismal functions, and can be evaluated under the microscope. Histology is the practice of applying specific stains to cells in order to view certain characteristics of the cell or tissue more clearly at higher magnification. Given the diversity and specialization of mammalian BMED 2000 Unit 1: Laboratory 1 2 cells, a wide variety of morphologies such as size and arrangement can be observed under the microscope, based on the tissue from which the cells are derived and the specific functions that they carry out. Histology Histology can be broadly defined as the study of the microanatomy of cells, tissues, and organs under magnification, with the overarching goal of identifying correlations between structure and function. To effectively observe this microscopic world, special techniques are used to prepare specimens for observation, and highlight areas of interest. Samples can include cultured cells or isolated tissue. Before any tissue is observed (and often cells as well), it is chemically fixed (to prevent decay). While cells are typically grown in a thin monolayer, tissues are embedded within a resin matrix (such as paraffin) for sectioning into thin cross-sectional slices, typically on the order of 2-7μm in thickness, To prepare these sections, the hardened resin is sliced with an instrument referred to as a microtome, or cryostat. Depending upon the thickness of the cross- sections prepared, tissues (almost always) appear colorless and transparent, making general visualization, structural observation, and identification difficult due to the apparent lack of contrast; staining methods are thus employed to enhance the contrast and permit visualization. Several different methods exist in histology to enhance the contrast of tissues for observation and study. Each histological method is unique, and exploits the fundamental chemical composition of the tissue being observed to achieve effective staining for visualization. Figure 1.1 An example of Hematoxylin and Eosin staining of mammalian tissue. H&E staining can be quickly applied to obtain well-identified differentiation within tissue (Humber College, 2017). Hematoxylin and Eosin (H&E) is a commonly used stain for general tissue identification and pathology. It can be used to rapidly stain tissue samples, producing a clearly observable differentiation between cellular structures (Figure 1.1), making this stain favorable if immediate observational feedback is required. This stain is so commonly and widely used that it is often referred to as routine staining in histopathology laboratories. Hematoxylin behaves as a basic dye (is positively charged) and appears purplish-blue. It acts to stain acidic or basophilic structures (those of opposing charge), such as the DNA and RNA. Following hematoxylin treatment, a bluing step (the addition of a bluing reagent) converts the insoluble red color of hematoxylin in the nuclei to an insoluble blue color. Eosin Y is an acidic dye (negatively charged) that acts to stain basic or acidophilic structures, producing a reddish-pink color for cytoplasm among other structures. The following biological structures appear the indicated color when treated with H&E stain. BMED 2000 Unit 1: Laboratory 1 3 Cytoplasm ® light pink Erythrocytes ® pink/red Collagen ® pink Nuclei ® blue Muscle ® pink/rose H&E staining provides the ability to rapidly observe the general organization of the cell being examined; as a result, immediate information regarding any morphological abnormalities present can be obtained. H&E staining is often used in conjunction with special or advanced stains, to provide supplemental information that could help, for example, facilitate the process of making a definitive diagnosis in a medical/pathological setting. Masson’s trichrome stain is typically used in conjunction with H&E staining in pathology laboratories to identify the accumulation of fibroids in tissues and can be used to aid in the diagnosis of muscular dystrophy. Hematoxylin and Eosin (H&E) staining is one of the most widely used stains in histology (anatomy of cells and tissues), pathology (diagnosis of disease), and cytology (structure and function of cells) applications within the field of medicine and research. This laboratory exercise will practice histological staining of specific tissues and cells to obtain a general understanding of the staining approach. It is important to stringently monitor the timing of each procedural step as it becomes critical in producing the standardized histological outcomes. Oil Immersion Microscopy Oil immersion is a microscopy technique used to increase the resolving power of the microscope by immersing the objective lens and the specimen in a transparent oil. The refractive index of the empty space (air) between the slide and the objective lens is less that of the slide, allowing light to be bent or scattered as it is directed through the air in between the slide and the objective lens, ultimately resulting in a distorted image of the object being viewed. This refractive distortion, however, is ameliorated when a mineral oil, which has a refractive index similar to that of the microscope slide (glass), is placed in between the slide and lens. The oil allows for the light passing through the slide to be directed into the objective lens with (little to) no distortion, resulting in a highly resolved, clearer, sharper image. Focusing with Oil Immersion 1. Focus in on the specimen to be observed under medium power objective lens (refer to the procedure outlined in the Appendix – Lab 1). 2. When the specimen is focused and the 100x objective lens is ready to be positioned into place, place a rounded drop of immersion oil directly on the cover slip over the area to be observed. 3. Gently slide to 100x objective lens into position, ensuring there is enough clearance between the lens and the slide. 4. Bring the image into sharp focus using only the FINE adjustment control. 5. Using the iris diaphragm lever, adjust the light to obtain optimum contrast and view the specimen. BMED 2000 Unit 1: Laboratory 1 4  6. When observations are completed, gently rotate the turret to position the 4x scanning objective lens into the line of sight. Using lens paper, wipe the oil off the oil immersion objective lens (100x). 7. Lower the stage using coarse adjustment to its lowest position, and then follow the appropriate steps to turn off and put the microscope away in the microscope cabinet for later use. References Abnova (2010, May 17). H&E Staining. [Video file]. Retrieved from https://www.youtube.com/watch?v=2D0rj0m6dVs Anderson, J (n.d.). An introduction to routine and special staining. Retrieved from http://www.leicabiosystems.com/pathologyleaders/an-introduction-to-routine-and-special- staining/ Everyday English with Byron (2014, Dec 7). Microscope calibration: a short tutorial [Video File]. Retrieved from https://www.youtube.com/watch?v=HXTqaUTGrKg Kaiser GE (2016). The Grapes of Staph: BIOL 230 Microbiology Lab Manual, Community College of Baltimore County, Catsonville Campus. Retrieved July 2015 from http://faculty.ccbcmd.edu/~gkaiser/goshp.html Lee CS, Yi J-S, Jung, S-Y, Kim B-W, Lee N-R, Choo H-J … & Ko Y-G. TRIM72 negatively regulates myogenesis via targeting insulin receptor substrate-1. Cell Death and Differentiation (2010) 17, 1254–1265; doi:10.1038/cdd.2010.1 Lewis IM (1941). The Cytology of Bacteria. Bacteriol Rev, 5(3), 181-230. See LA (2013, Sep 14). Microscope measurements [Video File]. Retrieved from https://www.youtube.com/watch?v=An5Aq3GqRmM R&D Systems. (n.d.). Protocol for the Preparation and Fluorescent ICC Staining of Cells on Coverslips. Retrieved from https://www.rndsystems.com/resources/protocols/protocol- preparation-and-fluorescent-icc-staining-cells-coverslips Sorenson RL and Brelje TC (2014). Atlas of Human Histology – A guide to microscopic structures of cells, tissues and organs (3rd ed). ISBN 2810000007289b, USA: University of Minnesota Bookstores. Retrieved from http://histologyguide.org/about-us/atlas-of-human-histology.html Vector Laboratories Inc. (n.d.). Hematoxylin and Eosin Stain Kit. Retrieved from http://docs.vectorlabs.com/protocols/H-3502.pdf BMED 2000 Unit 1: Laboratory 1 5 EXPERIMENTAL PROCEDURE Materials & Reagents C2C12 cells grown in a 60 mm dish (x4) HEK293 (or equivalent) cells grown in a 60 mm dish (x2) Ethanol (anhydrous, 20mL) Formaldehyde (10% in PBS, 2mL) Triton X-100 (0.5% in PBS, 2mL) Hematoxylin and Eosin Stain Kit (Cat # H-3502, Vector Labs) PBS (cold, 20 mL) Permount Mounting Medium (Cat # SP15-100, Fisher Scientific) Prepared bacterial structures slide set (x1; Caroline Cat # 292702) o Bacterial types – one slide with a smear of 3 bacteria: § Micrococcus luteum (coccus) § Bacillus megaterium (bacillus) § Rhodospirillum rubrum (spirillum) Onion (x1) Tissue culture incubator (x1) Light microscope (x1) Moticam microscope digital camera (x1) Micropipettes (P100 and P1000) Pipette tips (200 μL and 1000 μL) Tube holder (x1) Forceps (x1) Microscope slides and coverslips (x2) Wax pencil (x1) Prepared slides (x1) Lens paper (x1) Bibulous paper (x1) Immersion oil (x1) Timer (x1) Tissue culture waste beaker (250 mL) Organic waste beaker (250 mL) Glass waste container (x1) Ruler (x1) Chemical & Consumable Waste Disposal Safely dispose of all materials in the appropriate waste container: Leave at station Cold PBS 100% Ethanol Garbage Gloves Pipette tips Liquid cell culture waste All filtrate/solutions that came in touch with cells Wash with 10% bleach then wash glassware BMED 2000 Unit 1: Laboratory 1 6  Glassware Wash with soap and water, leave at station to dry Organic waste bottle Formaldehyde Biohazard bag 60 mm C2C12 and HEK293 (or equivalent) plates Procedure Part A: Visualization of Microorganisms NOTE: some procedures used in slide preparation may cause some arrangements to break apart or clump together (typically for cocci). The correct form, however, should predominate. Make sure to look around the slide to assuage what the majority of the bacteria look like. 1. Use the 4x objective lens to determine the low power field of view. Calculate the field of view at 1000X magnification power using the method described in the Appendix – Lab 1. Show all of the work required to complete this calculation. 2. The bacterial slide labelled, “Bacterial Types” includes three different types of bacteria. Pick one of the three bacterial samples prepared for you on the slide and obtain a microscope photograph at 1000X MP. Include along with your photograph: a. Name, shape and arrangement/form, b. True size of the specimen, and c. Magnification of the drawings. 3. Organize your result using the template provided in Table 1.1 Part B: Tissue observation 1. Obtain a prepared H&E-stained slide from your instructor. 2. Observe the cells using a light microscope using the 4x, 10x, 40x, and 100x objectives. 3. Take a photo of the stained cells using the microscope digital camera as viewed under magnification of your choice and begin to complete Table 1.2 Part C: Cell culture staining Note: The protocol will be performed by each member of your lab group. Follow the protocol in the proper order and pay close attention to the incubation times. 1. Obtain a 60 mm tissue culture dish containing C2C12 myoblasts, and discard media into the waste beaker. BMED 2000 Unit 1: Laboratory 1 7  2. Using a P1000 micropipette, wash the cells with 2mL cold phosphate buffered saline (PBS), discarding the PBS into a 250 mL waste beaker. Repeat this wash step twice (x3 rounds in total), discarding the PBS into the waste beaker after each wash. 3. Using a wax pencil, make a nickel-sized circle, in the centre of the plate. The cells contained within this circle are the ones that will be fixed and stained. 4. Use a P1000 micropipette to transfer 200 μL of a 10% formaldehyde solution within the circle on the plate to fix the cells. Allow the cells to incubate at room temperature for 10 minutes, and then discard the formaldehyde solution into an organic waste beaker. 5. Use a P1000 micropipette to transfer 200 μL of a 0.5% Triton-X-100 solution within the circle on the plate to permeabilize the cells. Allow the cells to incubate at room temperature for 5 minutes, and then discard the solution into the waste beaker. 6. Add enough hematoxylin within the circle on the plate to completely cover the fixed and permeabilized cells. Allow the cells to incubate at room temperature for 5 minutes. 7. Gently rinse the cell culture dish (continuously) for 15 seconds using distilled water from a squirt bottle, collecting the rinse waste into the waste beaker. Repeat the rinsing procedure two more times (15 seconds each round, x3 rounds in total). 8. Add enough bluing reagent within the circle on the plate to completely cover the cells. Allow the cells to incubate for 15 seconds at room temperature. 9. Rinse off the excess bluing reagent following the same washing procedure outlined in Step 7 above. 10. Treat the cells within the circle on the plate with sufficient anhydrous ethanol, allow to incubate for 10 seconds, and then blot the excess off using a Kimwipe. 11. Add enough Eosin Y solution within the circle on the plate to completely cover the cells. Allow the cells to incubate for 3 minutes at room temperature. 12. Rinse within the circle on the plate with anhydrous ethanol for 10 seconds, collecting the runoff into the waste beaker. 13. Add sufficient anhydrous ethanol within the circle on the plate to cover the cells and allow to incubate for 2 minutes. Repeat this dehydration procedure two more times, discarding the used ethanol into waste beaker each time. 14. Apply permount to the area around the circle of stained cells. Use forceps to gently cover the fixed and stained cells with a glass coverslip, ensuring no air bubbles form. Press down on the cover slip gently but firmly to create a seal. 15. Observe the cells using a light microscope using the 4x, 10x, 40x, and 100x objectives. Have the instructor view the stained cells for evaluation. 16. Take a photo of the stained cells using the digital camera mounted microscope at the designated bench viewed under your choice of magnification and begin to complete Table 1.3 in the Post-laboratory Assignment. BMED 2000 Unit 1: Laboratory 1 8  17. Obtain a 60 mm tissue culture dish containing HEK293 cells, and discard media into the waste beaker. 18. Repeat steps 2 to 15. 19. Take a photo of the stained cells as viewed under your choice of magnification and begin to complete Table 1.3 in the Post-laboratory Assignment. Part D: Slide Preparation – Wet Mount of Onion Epidermis 1. Cut a small 10mm x 10mm segment from an onion bulb 2. Using your forceps, carefully remove the epidermis (skin) from the onion. a. The epidermis is a single layer of cells from the onion, which makes it perfect for visualizing under the microscope 3. Obtain a clean glass microscope slide and apply a small drop of water to the center of the slide 4. Place the Onion Epidermis that you dissected in step 2 on top of the water 5. Add 1 drop of iodine to the onion epidermis on the slide and cover with a cover slip 6. Dap the excess water and iodine with a bibulous paper 7. Using proper technique, focus the microscope starting with the scanning objective lens. 8. Observe using oil immersion microscopy at 1000X MP (refer to Focusing with Oil Immersion). 9. Obtain a microscopic photograph of your sample at 400X MP. a. Include the shape, b. True size (diameter) of a single onion cell, and c. Magnification of the drawing. 10. Organize your drawing and result using the template provided in Table 1.4 Dispose of waste beaker material in biological waste container. Wash and rinse all glassware with soap and water. Hang all washed glassware on the glassware racks to dry. All glass microscope slides should be placed in designated container located at the instructor bench. They will be autoclaved and then disposed of in the glass waste container. All used tissue culture plates should be disposed in a biohazard bag. All other solid materials should be disposed of in the solid waste. BMED 2000 Unit 1: Laboratory 1 9 DATA COLLECTION AND POST-LABORATORY ASSIGNMENT Laboratory data, observations, experimental notes, and results will be submitted via BlackBoard, and must be submitted before the next scheduled laboratory session. Please consult Blackboard for submission details. The instructor will indicate compliance or non- compliance with the following criteria: Preparedness, Adherence to Safety Guidelines, and Cleanliness. ALL PARTS Proper adherence to the SOP, operation of equipment, and preparation of experiments as outlined in the procedures. Upload the following in a single document to blackboard: Part A: Visualization of Microorganisms Table 1.1 – Observation of bacterial samples Field of View Photo Photograph Specimen Identity Shape & Arrangement Objective Lens MP FOV size Real size Magnification BMED 2000 Unit 1: Laboratory 1 10  Part B: Tissue observation Table 1.2 – Observation of mammalian tissue samples Field of View Photo Photograph Specimen Identity Stain type Objective Lens MP FOV size Real size Magnification Part C: Cell culture staining Table 1.3 – Observation of mammalian cells samples Field of View Photo Photograph Photograph Specimen Identity Stain type Objective Lens MP FOV size Real size Magnification BMED 2000 Unit 1: Laboratory 1 11 Part D: Slide Preparation – Wet Mount of Onion Epidermis Table 1.4 – Observation of onion epidermidis via wet mount preparation Field of View Photo Photograph Specimen Identity Stain type Objective Lens MP FOV size Real size Magnification Discussion Questions: 1. Based on the experimental data presented in the Table 1.1 and Table 1.2, comment on whether the staining procedure was successful for cells and tissue. Explain your reasoning. 2. If you are tasked with locating and identifying a subcellular structure, you may choose to use a different staining procedure than H&E. Choose and name a subcellular structure and the appropriate staining procedure. Accurately explain why you chose this staining procedure and what information it will provide you. 3. 4. Provide sample calculations for each of the following using any of the values from Tables 1.1-1.4. Include all steps and show all work. a. Using the field of view information obtained in Part A under 4x magnification, determine the field of view diameter under 10x magnification. b. Calculate the high power field of view diameter (under the 100x objective lens). c. Using the scientific drawing obtained in Part B, show all of the steps required to determine the actual size of a single yeast cell. d. Calculate the magnification of the spherical bacterial drawing obtained in Part A if the cells are visualized under 1000X MP. BMED 2000 Unit 1: Laboratory 1 12 APPENDIX – LAB 1 Parts of the Compound Microscope 1. The components of a compound microscope are shown in Figure 1.2 and described below: 2. Light Source: Illuminates the specimen using a concentrated source of light. 3. Light Switch: Turns the light source on and off. The light switch also adjusts the intensity of light supplied by the light source to optimize the contrast of the viewed image. 4. Condenser: Directs the light that is projected from the light source onto the specimen. 5. Condenser Diaphragm: Regulates the intensity of light entering the condenser. 6. Stage: The platform on which slides and samples are placed to be viewed. 7. Stage Clips: Used to hold a slide securely in place on the stage. 8. Objective Lens: The magnifying lens used to enlarge the image of the specimen being observed. Typically, a compound light microscope has three or four different objective lenses mounted on the rotatable ring (turret). 9. Turret: A rotating ring holding all objectives in place. It aids in changing objectives from one to the other. 10. Coarse and Fine Adjustments: Used to move the microscope stage up and down to adjust the distance between the slide on the stage and the objective lens. Coarse adjustment (9b) moves the stage at a fast rate while fine adjustment (9a) moves the stage at a slow rate. 11. Ocular Lens/Eyepiece: Eyepiece through which a scientist looks to observe the magnified image. Additionally, the ocular lens further magnifies the image and is usually equipped with a 10X lens. Compound microscopes can be equipped with one ocular lens (monocular) or two ocular lenses (binocular). 12. Tube: Directs the light through the eyepiece and to the viewer’s eyes. 13. Neck/Arm: Body of microscope and handle to be used to carry the microscope safely from one place to another. 14. Base: The base of the microscope. BMED 2000 Unit 1: Laboratory 1 13  10.$Ocular$Lens$/$ Eye$Piece$ 11.$Tube$ 7a.$Lowest$ Objec:ve$Lens$ 12.$Neck/Arm$ 7b.$Medium$ Objec:ve$Lens$ 8.$Turret$ 7c.$Highest$ Objec:ve$Lens$ 5.$Stage$ 6.$Stage$Clips$ 9b.$Course$ Adjustment$ 9a.$Fine$ 4.$Condenser$ Adjustment$ Diaphragm$ 1.$Light$Source$ 3.$Condenser$ 2.$Light$Switch$ 13.$Base$ Figure 1.2 Illustration of a monocular compound microscope and its key components. Note: A binocular compound microscope will have similar parts except for two ocular lenses instead of the one shown in this figure (Humber College, 2016). For more information regarding Handling & Care, Microscope Set-up, and Focusing, see Appendix – Lab 1. Magnification Power (MP). Total MP is how much a microscope can enlarge an image being observed. It is calculated by multiplying the magnification power of the ocular lens (which is usually 10x) by the magnification power of the objective lens (could be 4x, 10x, 40x, or 100x) in use. See Table 2.1 to view the total MP for all of the different objective lenses found on a compound microscope. 𝑀𝑃 = (𝑝𝑜𝑤𝑒𝑟 𝑜𝑓 𝑜𝑐𝑢𝑙𝑎𝑟 𝑙𝑒𝑛𝑠) × (𝑝𝑜𝑤𝑒𝑟 𝑜𝑓 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑙𝑒𝑛𝑠𝑒) Table 1.2 A list of the different objective lens, the associated magnification, and total MP for each (adapted from (2)). Lens Magnification Ocular Lens Total MP Scanning 4x 10x 40X Low power 10x 10x 100X High power 40x 10x 400X High power 100x 10x 1000X It is important to note the difference in convention used in the table above. Magnification is described using the convention nx, where n is the numerical degree of magnification of the specific component and the lower-case x indicates that it is component specific. Magnification power (MP) values are described using the nX convention, where n is the numerical degree of magnification, and the upper-case X signifies that this value is representative of MP, which is a compounded magnification of multSCD components (this is why the light microscope is often also referred to as a Compound Microscope!). BMED 2000 Unit 1: Laboratory 1 14 Handling & Care 1. Always carry the microscope using two hands. Hold the microscope arm with one hand and support the weight of the microscope with the other hand, palm facing upwards directly under the base 2. Clean the lenses with lens paper ONLY at the start and at the end of each laboratory session. NEVER clean the lenses with paper towel to avoid damage. 3. Avoid any spills on the microscope stage or any other parts. If a spill occurs, immediately consult with the laboratory instructor for proper clean-up protocol. 4. When specimen examination is complete, carry out the following steps to safely put away the microscope: a. Rotate the turret to ensure the lowest magnification objective lens (4x) in is the viewing position; b. Use the coarse adjustment knob to lower the stage to its lowest position away from the objective lenses; c. Remove any and all slide(s) from the microscope stage; d. Clean all lenses using lens paper; e. Turn the light switch off; f. Carefully wrap the cord (tightly) around the base of the arm to avoid damage during storage; g. Cover the microscope with its protective plastic cover; h. Carry with both hands to its storage location (microscope cabinet); Microscope Set Up The following steps should be carried out sequentially before attempting to focus any image of a specimen on a slide: 1. Remove the microscope’s protective plastic cover, fold neatly, and set aside 2. Carefully unravel the cord from the microscope base, and plug it in to the nearest outlet. 3. Turn on the light source using the light switch, and adjust intensity to the highest setting; 4. Lower the microscope stage to its lowest position (nearest to the light source) using the coarse adjustment knob (DO NOT force any knobs beyond their stopping point); 5. Position the slide to be observed on the stage, and secure it in place using the stage clips; 6. Looking at the slide move the stage horizontally or vertically until the specimen on the slide is positioned directly in the light path going through the stage hole; 7. Proceed with focusing as outlined below. Focusing To effectively observe the details of a specimen under magnification, the image must be brought into focus. To adequately focus a specimen for observation, the steps outlined below must be followed: 1. Begin with the lowest objective lens (scanning objective lens, 4x magnification). Gently rotate the turret to position this lens directly over the sample to be observed; 2. Look through the ocular lens(es) and observe the field of view. If a binocular compound microscope is being used, adjust the spacing between the two oculars such to achieve BMED 2000 Unit 1: Laboratory 1 15  only a single viewing area (NOT two). Do this by merging the two individual viewing areas through adjustment of the eyepiece spacing; 3. Adjust the light intensity going through the condenser using the condenser diaphragm (NOT light switch knob) until the correct contrast has been achieved. If adjustment of the condenser diaphragm does not adjust the light intensity sufficiently, the light switch knob can be used to further reduce the light intensity; 4. Use the coarse adjustment knob (moving the stage fast) to bring the specimen into focus. NOTE: Coarse adjustment of the microscope stage is permitted ONLY when the scanning/4x objective lens is in place. The use of coarse adjustment with higher magnification objective lenses may result in slide breakage and irreversible damage to the objective lens. 5. Bring the image into sharp focus using the fine adjustment knob; 6. Increase magnification (if desired) by carefully rotating the turret to bring the next higher magnification (medium objective lens, 10x or 40x magnification) objective lens into position. Observe the distance between the objective lens and the slide during turret rotation to ensure sufficient clearance between the two objects; 7. Use fine adjustment ONLY to bring the specimen into sharp focus, ensure the specimen is centred in the field of view before turning to higher magnification; 8. Increase magnification (if desired) by carefully rotating the turret to bring the highest magnification objective lens into position. Observe the distance between the objective lens and the slide during turret rotation to ensure sufficient clearance between the two objects; 9. Use fine adjustment ONLY to bring the specimen into sharp focus; ensure the specimen is centred in the field of view before turning to higher magnification; 10. If the compound microscope in use is equipped with 100x objective lens (oil immersion objective), special oil is required to be placed on the slide before attempting to use the 100x objective. Calibration and Size Determination Although all compound microscopes are designed similarly, there may be small variations in the magnitude of magnification from one instrument to the next. To determine the actual size of a specimen, each microscope needs to be calibrated using two types of rulers: Stage Micrometer: A slide that has a ruler printed on its surface. The distance between each line on stage micrometer is 10μm (Figure 2.3A). NOTE: If a stage micrometer is not available, then a regular ruler can be used instead. Ocular Micrometer: Located on the ocular lens of the microscope with no specific measurements assigned to it (Figure 2.3B). The distance between each line needs to be determined using a stage micrometer. Calibration of the ocular micrometer is the exercise of determining the exact distance between the lines of the ocular micrometer by superimposing the lines of the stage micrometer and the ocular micrometer. The ocular micrometer is used to calculate the real size of the viewed BMED 2000 Unit 1: Laboratory 1 16 specimen. The following steps are carried out to calibrate the ocular micrometer of the microscope, and determine the real size of an object under observation: Place a stage micrometer or a ruler on the stage of the microscope and focus as outlined above; Carefully, superimpose the zero lines of the stage micrometer with the lines found on the ocular micrometer (Figure 2.3C); Using the lines of the stage micrometer calculate and record the exact distance between the lines of the ocular micrometer. This is outlined in the worked example presented below; Recorded measurements will be used to determine the actual size of a specimen (Figure 2.3D). Figure 1.3 Calibrating using an ocular and stage micrometer. A: Stage micrometer is 1 mm long with 100 divisions. B: Ocular micrometer with unknown distance between each line, divided into 100 units. C: Superimposed image of stage and ocular micrometers, with the point of overlap, clearly marked at the 35 μm mark on the ocular micrometer. D: The size of the specimen is determined. (Humber College, 2016). BMED 2000 Unit 1: Laboratory 1 17 Example: Figure 1.3A above shows a stage micrometer that is 1mm long and contains 100 equivalent divisions. Therefore, each division of the stage micrometer is (1mm/100 = 0.01mm) 10μm apart. The ocular micrometer with an unknown distance between each line, divided into 100 equivalent units, is shown in Figure 1.3B. The stage and ocular micrometers are superimposed, with the point of overlap clearly identified at the ‘35’ mark on the ocular micrometer (Figure 1.3C, top), which corresponds to 30 divisions on the stage micrometer (Figure 1.2C, bottom). Since each division on the stage micrometer is exactly 10μm apart, 30 divisions on the ocular micrometer spans an area of (10μm x 30) exactly 300μm. Therefore, 35 divisions on the ocular micrometer corresponds to an exact length of 300μm; each division on the ocular micrometer is then (300 μm/35) exactly 8.6μm in length. Only after calibration of the ocular micrometer can the true (or actual) size of a specimen be determined. This is done by measuring the widest part of the specimen using the ocular micrometer (Figure 1.3D). In this example, the widest part of the cell is ~20 unit divisions on the ocular micrometer; thus, the diameter of the cell is (20 x 8.6μm) exactly 171.4 μm. Magnification Magnification is a value describing the degree of image enlargement. It specifies how many times larger a drawing or image of an object is relative to the true (or actual) size of the object. It is calculated by dividing the size of the drawing/image by the actual size of the specimen. The resulting value (n) is then expressed as nX, where n represents the magnitude of enlargement and X is used as a designation for magnification. 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑑𝑟𝑎𝑤𝑖𝑛𝑔(𝜇𝑚) 𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 = 𝑟𝑒𝑎𝑙 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑠𝑝𝑒𝑐𝑖𝑚𝑒𝑛 (𝜇𝑚) NOTE: Magnification is not to be confused with total Magnification Power (MP), which is described above. Measuring Using a Ruler If the microscope does not come equipped with an ocular micrometer, a standard ruler can be used to measure the field of view, and the subsequent measure of the size of the microorganism. 1. Using a clear ruler with a cm/mm scale, measure the diameter of the field of view using the 4x scanning objective lens (or 40X magnification, Figure 2.4); 2. Convert the measured distance to micrometres (1 mm = 1000 μm). In Figure 2.4, the field of view measures approximately 5mm across. 1000𝜇𝑚 5𝑚𝑚 × = 5000𝜇𝑚 1𝑚𝑚 BMED 2000 Unit 1: Laboratory 1 18  Figure 1.1 Illustration of a ruler spanning the field of view at 40X magnification (Humber College, 2021). 3. Repeat the process at 100X MP using the 10x objective lens (low power). The field of view for the same ruler viewed at 100X MP measured to be 2 mm (image not shown) 1000𝜇𝑚 2 𝑚𝑚 × = 2000𝜇𝑚 1𝑚𝑚 Note: You can only use the 4x and 10x objective lenses to make rough estimates of the size. Measuring using a ruler at a high power is more complicated as the individual ruler marks may not be visible at 1000X MP (using the 100x objective lens). 4. A formula is used to estimate the size of the field of view at 1000X MP: ℎ𝑖𝑔ℎ 𝑝𝑜𝑤𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑜𝑓 𝑣𝑖𝑒𝑤 (𝜇𝑚) 𝑙𝑜𝑤 𝑝𝑜𝑤𝑒𝑟 𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 (𝑀𝑃) = 𝑙𝑜𝑤 𝑝𝑜𝑤𝑒𝑟 𝑓𝑖𝑒𝑙𝑑 𝑜𝑓 𝑣𝑖𝑒𝑤 (𝜇𝑚) ℎ𝑖𝑔ℎ 𝑝𝑜𝑤𝑒𝑟 𝑚𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 (𝑀𝑃) Solving for High Power Field of View (x), where Low Power Field of View = 2000μm Low Power Magnification = 100X High Power Magnification = 1000X 𝑥 100 = 2000 𝜇𝑚 1000 100 × 2000 𝜇𝑚 𝑥= 1000 𝑥 = 200 𝜇𝑚 Therefore, the High Power Field of View diameter is 200μm. 5. Now using 200μm as the approximate field of view at high power, the size of the specimen can be estimated. For instance, if a cell takes up approximately 1/10th of the FOV, then you can calculate the specimen size as: 1 × 𝑟𝑒𝑎𝑙 𝑠𝑖𝑧𝑒 FOV 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 10 1 × 200𝜇m 10 BMED 2000 Unit 1: Laboratory 1 19  = 20μm Scientific Drawings and Recording Observations Scientific drawings are used to illustrate the key features of a viewed specimen. Below are the standard rules to be followed for a scientific drawing (Figure 1.5): 1. Scientific drawings must be produced on white paper (NOT lined or coloured paper). 2. They are to be drawn using only black pencils (NOT pen or coloured pencils). 3. Do not shade or colour scientific drawings. Instead, use lines and stippling (dots). 4. An appropriate and descriptive title must be included at the top of each drawing. 5. All relevant details must be identified as follows: a. Labels must be placed on either the left or the right of each diagram (NOT on the top or the bottom of the diagram). b. All labels must line up parallel to one another. c. Use lines to connect the specific location on the diagram with the label (NOT arrows or dashed lines). d. Do not cross lines when labelling. 6. Include magnification on the bottom of the illustration. NOTE: For assessment purposes, an accurate scientific drawing is defined as a drawing that provides an accurate representation of the specimen AND meets all criteria/standard rules for a scientific drawing. Figure 1.2 Scientific drawings. A: The correct, and B and C: incorrect methods of labelling a scientific drawing. (Humber College, 2016). BMED 2000 Unit 1: Laboratory 1 20 Slide Preparation Slides can be prepared in several different ways, however, the two types of preparation that are most commonly used are wet mount and dry mount. Wet Mount A wet mount slide preparation requires the suspension of the sample in a small amount (one drop) of a liquid medium. Samples are either already in suspension before placing a drop on the slide, or a drop of suspension liquid is added onto the slide containing the specimen. Dry Mount As the name implies, a dry mount slide preparation does not use water or any type of liquid solution to suspend the sample for observation. Similar steps are followed as outlined above (wet mount) to prepare a dry mount slide with the obvious exception of the addition of any liquid medium. Wet Mount Slide Prep Procedure To properly prepare a wet mount slide for observation under a microscope, the following steps must be followed: 1. Place sample specimen in the middle of a clean slide. 2. Place one single drop of the appropriate suspension solution on top of the sample specimen. NOTE: The excessive use of suspension solution will cause the specimen suspended solution to run off the edges of the slide. 3. Place the edge of the coverslip at a 45-degree angle on one side of the sample drop. 4. Slowly lower the coverslip such to sandwich the drop between it and the microscope slide. Gently release and drop the coverslip on top of the sample. 5. Using one finger, gently tap the microscope slide to eliminate any bubbles that may have been formed between the slide and the coverslip. Figure 1.3 The preparation of a wet mount slide (Humber College, 2016). BMED 2000 Unit 1: Laboratory 1 21