LAB 1 INTRODUCTION TO THE MICROSCOPE AND SCIENTIFIC DRAWINGS LEARNING OBJECTIVES AFTER COMPLETING THIS LABORATORY, YOU SHOULD BE ABLE TO PERFORM THE FOLLOWING: 1.
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Mount and stain a specimen on a slide and prepare it for microscope viewing. Identify parts of a compound microscope and operate it effectively. Make proper biological drawings of both and an animal and plant cell. Describe the cell cycle. Identify stages of mitosis from prepared slides. By examining the mitotic region of an onion root tip (Allium cepa), calculate the percentage of time a cell spends in the various stages of the cell cycle. This work is licensed under the Creative Commons License. ACTIVITY 1: INTRODUCTION TO THE COMPOUND MICROSCOPE The following diagram illustrates the parts of a compound stereo microscope. There are many types of microscopes used for a variety of applications. Choosing a type of microscope will be dependent on the size of sample you are viewing and its translucency. An object that will allow light to pass through it, such as a jellyfish, can be viewed with the light source coming from below, but an opaque object, like a lady bug, will have to be illuminated from above. This microscope has the light source coming from below the stage, and is good for viewing relatively small, translucent objects such as individual cells. The two eyepieces of the stereo microscope allow you to view an image using both eyes, therefore you can see the image in three dimensions, as opposed to the flat, 2‐D image you would see with a monocular scope. This microscope has a 10X magnifying ocular lens (or eyepiece) as well as 4X, 10X, 40X and 100X objectives. It is called a compound microscope because the total magnification is compounded by multiplying of the magnification of the ocular lens and the objective lens. This results in a choice of 40X, 100X, 400X, and 1000X magnification. Refer to “Use of a Compound Microscope” in Appendix A. Connect to the following link at the University of Delaware: http://www.udel.edu/biology/ketcham/microscope/scope.html Click the “start tour” icon. While working through the tour, label the parts of the microscope on the diagram below, and then practice your skills at manipulating a slide on the stage. (5 marks) After you have practiced with the virtual scope, you will be ready to try the RWSL remote compound microscope. You will need to run through the RWSL Tutorial before viewing the prepared slides to complete the following exercises. Image from: http://www.enasco.com/pdfs/sci_microscopesSB28945.pdf This work is licensed under the Creative Commons License. MATERIALS Unlined paper Pencil Computer (access to remote microscope) PROCEDURE 1.
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Log into the remote microscope. Set the magnification to the lowest setting (X40) Use the micrometer (the ruler that appears in you field of vision) to measure the width of the letter ‘e’ at each of the three magnifications. Use the micrometer to calculate the width of the field of view (edge to edge of the viewing area) at each of the three magnifications. a.
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With the lowest power objective (4X) in position, place the millimeter ruler on the center of the stage so that the scale is visible through the microscope. Line up the 0 mm. vertical line with the left side of the circular field of view. Count the number of millimeters included from one side of the field to the opposite side. If the right side of the field of view does not coincide with one of the lines, you will have to estimate to a fraction of a millimeter. Ensure that the edge of the ruler is across the middle of the field of view, so you are measuring the full diameter. Turn the 10X objective into place and fine adjust. Estimate this new field of view of 100X. Now turn the 40X objective into place. You will observe the field of view is less than 1 mm. Instead of measuring the field directly it is more accurate to calculate the diameter by the following equation: (Field of View 1) X (Magnification 1) = (F of View 2) X (Mag. 2) TABLE 1: FIELD OF VIEW MEASUREMENTS mm µm 40X field of view 100X field of view 400X field of view This work is licensed under the Creative Commons License. There are two slides for you to view under the microscope, one with the typeface letter e, and one with three coloured threads overlapped. Use these slides the answer the following questions: QUESTIONS 1.
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The “e” slide has been placed right‐side up on the stage. Which orientation do you view it in? (1 mark) Slide the stage left. Which way does the “e” move? Now slide the stage right, up, and down and record the movement you see through the lens. (2 marks) The three threads have been placed on the slide, overlapping in a particular order. Use the fine focus to determine which thread is on top, in the middle and on the bottom. Practice seeing different depths of the slide using the focus (i.e. focal length of the lens). (2 marks) ACTIVITY 2: BIOLOGICAL DRAWINGS One of the most important skills a biologist can have is keen observation. A second is communicating what they have seen. Biological drawings are an excellent way to practice both of these skills, and will be something that we revisit often in your labs. There have been specific standards set as to what constitutes an acceptable biological drawing. Please read through Appendix A to review these standards before doing the following activity. PROCEDURE: PREPARING BIOLOGICAL DRAWINGS (10 MARKS‐ 2 DRAWINGS, 5 MARKS EACH) 1.
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Prepare proper biological drawings of both the animal cell and plant cell on the slides provided to you for the remote microscope. Be sure to label your diagrams in the manner explained in Appendix A, including the actual magnification as calculated (you can use either the micrometer or the measurements of your field of view calculated in Activity 1). These drawings will be submitted to your instructor via mail. ACTIVITY 3: PREPARATION OF A WET MOUNT AND FIXED SLIDE WET MOUNTING Though some samples can be placed directly under the microscope, many samples look better when placed in a drop of water on the microscope slide. The water helps support the sample and helps light pass more evenly through the slide, the sample, and the cover slip. A slide prepared in this way is known as a "wet mount". To make a wet mount place your sample on the slide. Using a pipette, put a single drop of water on the sample. Place one edge of the cover slip on the slide at a 45° angle and slowly approach the drop of water. Once the cover slip edge touches the sample, lower it carefully all the way down. The purpose of this is to avoid air bubbles between the cover slip and the specimen. Air and water refract light differently and it will be hard to make out what you are looking at if air is trapped under your cover slip. Do not pat or press the cover slip onto the sample, as it will damage it. Follow this link for a video demonstration of this procedure. http://www.youtube.com/watch?v=HCQNyjl‐iFQ 4 | P a g e This work is licensed under the Creative Commons License. The water should just fill the space between the cover slip and the slide. If there is too much water and the cover slip is floating around, remove some water by holding the edge of a paper towel next to the edge of the cover slip. If there is too little water and some of the space under the cover slip is still dry, add more water by placing a drop right next to the cover slip. A little practice will help you learn how much water to add. MATERIALS Slide and slide cover Cotton swab Distilled water Paper towel PROCEDURE: CHEEK CELL WET MOUNT 1.
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Using a clean slide, place a drop of distilled water in the center. Using a cotton swab (Q‐tip) or toothpick, rub the inside of your cheek. This should not hurt! Lightly scrape the inside of your mouth to removes sloughed cells. Take the end of your toothpick or cotton swab and dab it in the distilled water on your slid. Place a cover slip over the sample. Be careful to use the technique described above to ensure no air is trapped. Look at you slide under your hand microscope. Focus first, at the lowest power, and work your way up to 100X’s magnification. Can you readily see the cheek cells? STAINING AND FIXING SAMPLES When viewing specimens, sometimes it is difficult to distinguish them from the medium they are in. For example, a cheek cell appears clear, very much like the water it sits on the wet mount lide. One way to assist in visualizing such a sample is to add a dye or stain to the slide. Staining makes certain specimens more viewable, allowing their morphology (e.g. size and shape) to be seen more easily. In some cases, specific stains can be used to visualize particular cell structures (flagellae, capsules, endospores, etc). Several staining methods are used routinely for slides, especially with bacteria. These methods may be classified as 1) simple, or nonspecific, and 2) differential, or specific. Simple stains will react with all microbes in an identical fashion. They are useful solely for increasing contrast so that morphology, size, and arrangement of organisms can be determined. On the other hand, differential stains give varying results depending on the organism being treated. These results are often helpful in identifying the specific type of bacterium. Stains are generally salts in which one of the ions is coloured. Remember a salt is a compound composed of a positively charged ion and a negatively charged ion. For example, the dye methylene blue is actually the salt methylene blue chloride. This salt compound dissociates in water into a positively charged methylene blue ion (blue in colour) and a negatively charged chloride ion (colourless). The cytoplasm of all bacterial cells has a slight negative charge (if in a neutral environment) and will therefore attract and bind with basic dyes. Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue. 5 | P a g e This work is licensed under the Creative Commons License. When staining a sample, the stain is usually introduced to the wet mount matrix. Unfortunately, there is generally too much background (unbound dye) to allow for visualization of the cells. Therefore, you need to remove the unbound dye. Simply washing off the dye would result in removal of the cells along with the excess dye. The cells need to be “fixed” to the slide, in order to allow the removal of the background dye. A simple method is that of air drying followed by heat fixing. The organisms are heat fixed by passing an air‐dried smear of the organisms through the flame of a gas burner. The heat coagulates the organisms' proteins causing the bacteria to stick to the slide, much like cooking an egg in a frying pan. The organisms must not be overheated when fixing them to a slide, or the sample will distort. Keep in mind the fried egg: when you put a raw egg onto a cold frying pan, it has a certain shape. Start heating it and the proteins (albumin) on the lower surface of the egg precipitate and fix the egg to the pan. At this point the initial egg shape is maintained, and is stuck to the pan. If you keep heating the pan the egg eventually changes shape as the proteins really denature, until eventually is nothing but charred remains. The goal is to maintain the samples shape, but just fix it enough that it sticks to the slide. You will be heat fixing a slide of natural yogurt, which contains live bacterial culture. You may be familiar with terms such as “probiotic” that food companies are using to promote live culture yogurts. Many bacteria are either harmless, or in fact beneficial to our health, and the cultures found in yogurt are a good example of such bacteria. MATERIALS Cheek cell slide (prepared in previous procedure) Methylene Blue Paper towel or blotting paper Plain, live culture yoghurt Tea light candle Plastic wrap Distilled water PROCEDURE: STAINING A WET MOUNT 1.
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Using the wet mount slide you prepared of your cheek cells, place a small drop of methylene blue to the left edge of the cover slip. Place the very edge of a piece of paper towel to the right edge of your cover slip. You should see the paper towel absorbing the water from underneath the cover slip and the methylene blue stain begin to be drawn underneath the cover slip and into your sample. Once the stain has been drawn into the sample, take a second look at it under the microscope. The methylene blue stain should have stained the nuclei of your cheek cells. The cells should appear to be to something like a fried egg with a blue yolk. The “yolk” is the cell’s nucleus. PROCEDURE: HEAT FIXING AND STAINING (10 marks) 2.
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Place a thin smear of the plain yogurt on a clean slide with a clean dissecting blade. Do not use water. Spread the suspension over the entire slide to form a thin film. Allow this thin suspension to completely air dry. 6 | P a g e This work is licensed under the Creative Commons License. 5.
Pass the slide (film‐side up) quickly through the flame of the tea light candle 3 or 4 times to heat‐fix. You should notice a slight dulling of the surface of the smear (like paint drying). If the smear begins to turn brown at the edges you have heated it too much and need to start over with a clean slide. 6. Cover the smear with methylene blue 7. Allow the dye to remain on the smear for approximately 1 minute. (Note staining time is not critical. Somewhere between 30 seconds and 2 minutes should give you an acceptable stain. The longer you leave the dye on, in general, the darker the stain.) 8. Wash the excess stain off the slide using the distilled water wash bottle, holding it over your largest beaker. Gently wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide. Wash off any stain that got on the bottom of the slide as well. 9. Blot off excess stain using absorbent paper. DO NOT rub the slide, rather place the slide between two sheets of absorbent paper and press down gently. Paper will absorb excess dye. Let the slide dry completely. 10. This slide will be submitted by mail to your instructor to be viewed at a later date on the RWSL microscope. Wrap it in plastic wrap and place it in the envelope provided. DISCUSSION QUESTIONS 1.
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Why are dyes used in microscopy? (2 marks) Describe the appearance of the cheek cells both with and without the use of methylene blue. (2 marks) In your own words, describe the chemistry of how the methylene blue dye has been taken up in the cell tissues. Do you think the shape/appearance of the cells or organelles have changed because of the dye? Explain. (4 marks) Bibliography Hodgson, C. (2005). Laboratory manual for Bio 102, Principles of modern Biology, 6th Ed. Courtenay: North Island College. Johnson, R. a. (1992). Biology, Third Edition. St. Louis: Mosby‐Year Book Inc. University, T. P. (2001, 01 08). Simple Stains: Direct and Indirect Staining. Retrieved 09 30, 2009, from ENVE 301: Environmental Microbiology Laboratory: http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/Lab2_Simple_staining.html 7 | P a g e This work is licensed under the Creative Commons License.