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2: Lab First Microscopy Lab I. Purpose The purpose of this lab is to familiarize the students with the inner workings and proper use of light microscopes, as well as to introduce certain key terms such as magnification, resolution, contrast, phase contrast, and brightfield. II. Procedure After viewing a short film on microscopy and identifying the pieces and functions of the light microscope, students began to use their microscopes using a prepared slide. They were instructed to practice using objectives, focusing, and basic microscopy maneuvers, and then set the Kohler illumination. Once these steps had been completed, the next phase instructed the students to calculate the resolution of each objective lens and compare it to the labeled resolution. Steps were then undertaken to calibrate the eyepiece micrometer in the microscope by utilizing a micrometer on a given slide. The conversions were: 4X: 1 mm = 40 retical units 10X: 1 mm = 100 retical units 40X: 1 mm = 400 retical units The next part of this lab involved the examining of an onion root tip. 3 drops of 1N HCl was added and allowed to soak into a 2 mm piece of onion root tip, and then 3 drops of aceto-orcein solution was added to the tip and 2-3 more minutes were allotted for the root to sit. A wet mount of the root tip was then made, coverslip added, and the students began looking through the microscope hoping to find various stages of mitosis in action. Once at least a few good examples of the stages of mitosis were found, the slide was taken to a set-up involving an Olympus research microscope, a CCD camera, and a computer, and the students captured digital images of the better examples of mitosis found in the onion root tip sample. Once this was completed, the students returned to their lab stations and began the next part of the lab, in which they used a toothpick to scrape some epithelial cells from the inside of their cheek. These cells were placed on a clean slide, and a drop of methylene blue stain was added and allowed to sit for 1 minute. Students then observed and made notes on various details of their stained cheek cells, and after this they were instructed to repeat the process a second time, only this time no stain was added. Once they took note of the various differences in the non-stained cells from the stained cells, students examined the cells using phase contrast instead of brightfield, and the Olympus/camera set-up from earlier was utilized to take a snapshot of the cells. Once this was done, the students observed as the TA and Instructor showed Elodea slime molds, a Kalanchoe leaf, and a Paramecium sample. The final phase of this lab involved students working with Diatom specimens, and testing the resolving power of the light microscope. Students worked with the Olympus microscope and camera set-up, and photographed high resolution images of the Diatom samples. III. Results and Analysis 1. Label the parts of the microscope: 2. To focus a specimen on the stage, slowly move the coarse focus knob until the specimen is in view, then use the fine focus knob to bring it into focus. 3. Moving the field diaphragm controls the positions of the field plane that are illuminated. 4. Closing the aperture diaphragm creates greater contrast; it however comes at the cost of reducing resolution. 5. Changing the objectives from 4X to 10X and then to 40X zooms in on the specimen on the stage, allowing us to see closer, yet less of the specimen. 6. To find an object on a slide, begin by using the 4X. Once you have identified the general location of your specimen on the slide, you can choose to change objectives and zoom in as necessary. 7. To view an object seen at low magnification in a higher magnification, use the coarse focus knob to move the stage lower. Once it's sufficiently clear of the higher objective lens, rotate the lens (going through 4X if rotating more than once) and then use the focus knobs as needed to bring the object back into view. 8. To place immersion oil on a slide, rotate the nosepiece to a position between the 40X and 100X objective. Once the nosepiece is in the correct position, place a drop of immersion oil on the center of the slide. 9. To return to a low magnification, remove the oil by wiping the oil off of the objective using lens paper. 10. The image obtained with the 100X objective is much more detailed, and certain details are only visible in this higher objective. The area of the specimen we can see is greatly reduced, and sometimes a 40X lens should be used to increase the area while keeping a decent amount of detail visible. 11. To look for your specimen using the 100X objective would be difficult, because it's hard to tell exactly what you're looking at that close up, especially because it's most likely out of focus. 12. 13. 4X: 1 mm = 40 retical units 10X: 1 mm = 100 retical units 40X: 1 mm = 400 retical units 14. The diameter of one red blood cell is 1 retical unit under 10X objective. Since the conversion is 100 retical unit = 1 mm, 1 retical unit is equal .01 mm. 15. 16. 17. The nuclei are visible, and it's relatively easy to find cells undergoing mitosis. It's easier to see the specimen that is stained versus unstained, because staining creates contrast. 18. 19. The diameter of a cell: 7 retical units (@10X) = .07 mm The diameter of a nucleus: 1 retical unit (@10X) = .01 mm Size of objects on cells (if you see them): 1 retical unit (@40X) = .0025 mm 20. When the focus was moved slightly up and down, not much remained in focus. This leads me to believe that the cell isn't very thick at all. The shape is almost block-like, though more circular. 21. This shape of epithelial cells allows them to fit together nicely, form a nice wall, and also assists in the intercellular transport. 22. The objects around the cells are pieces of bacteria from inside my mouth. 23. stained nucleus, cytoplasm, bacteria, and a clear cell wall unstained nucleus, fuzzy cell wall 24. 25. The structures flowing inside the walls of the Elodea cells are green chloroplasts. 26. The velocity of the flowing structures I estimate to be about 20 microns every 7 seconds. 27. The objects moving in the Plasmodial slime mold are chloroplasts, which move in one direction, slow down, and then reverse directions. 28. 29. Inside the guard cells of the Kalanchoe leaf, the central openings, or stomata, are very noticeable. The function of these openings is to let water in and out of the plant, and they're usually closed at night.

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