Microscope-lab Manual PDF

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Microscope-lab Manual...

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Lab 1 MICROSCOPE AND ITS USES Compound Microscope Microscopes are very important tools in biology. The term microscope can be interpreted as “to view the tiny,” because microscopes are used to study things that are too small to be viewed by naked eyes. The type of microscope that we will be using in this lab is a compound light microscope. Light microscopes magnify the image of the specimen using visible light and lenses. The term compound means that this microscope uses more than one lens to magnify image.. Objectives 1. Learn the parts of a compound light microscope and their functions. 2. Learn how to calculate the magnification of a compound light microscope. 3. Learn how to make a wet mount slide. 4. Understand how the orientation and movement of the specimen’s image changes when viewed though a compound light microscope. 5. Learn the proper use of the low and high power objective lenses. 6. Learn the proper use of the coarse and fine adjustments for focusing.

Microscope Vocabulary Magnification – is to make something larger in size than it really is by use of lenses. In microscopes, this is usually indicated by the abbreviation “X”. So a 10X eyepiece will magnify the image on a slide by 10 times. To determine total magnification of a specimen by a microscope, simply multiply the eyepiece magnification times the objective magnification. (For example, if the eyepiece is 10X and the objective is 10X, then the total magnification would be 100X.) As magnification increases, generally more light is needed to maintain the same level of resolution (defined below). Resolution - is the ability to distinguish between two points on an image (i.e. the amount of

detail) and is determined by the amount and physical properties of the visible light that enters the microscope.

Field of view– is the area of observation on the slide when viewed through the microscope. As the magnification goes up, the field gets smaller in diameter, and requires more light for visualization of the slide. Parfocal – This is the ability of a microscope to stay relatively in focus as the user switches among the objectives. In a good, parfocal microscope, less than 1/8 of a turn of the fine adjust should bring the image back into focus.

Parts of microscope and their functions

Using the microscope with a prepared slide: Place your slide with a letter "e" slide, coverslip side up, on the stage. Use the low power objective (4x). Focus on the letter "e" using the coarse focusing knob. This phenomenon is known as inversion.

e

Inversion (40x total magnification)

400X total magnification Q 1. If you are looking at the “through the microscope and you push your slide to the left which way does the “e” in the microscope move? Q 2. If you push the slide away from you which way does the “e” in the microscope move?

Preparing and observing a wet mount Materials required

1. Clean microscope slide and coverslip, 2. Flat-tipped toothpick, 3. Dropper bottle of methylene blue stain,

Procedure 1. Place a drop of methylene blue stain in the center of the slide. 2. Using the flat end of the toothpick, gently scrape the inner lining of your cheek. 3. Agitate the end of the toothpick containing the cheek scrapings in the drop of methylene blue stain • These epithelial cells are nearly transparent and thus difficult to see without the stain, which colors the nuclei of the cells and makes them look much darker than the cytoplasm. 4. Immediately discard the used toothpick in the disposable autoclave bag provided. 5. Carefully lower the coverslip onto the preparation. Do not just drop the coverslip, or you will trap large air bubbles under it, which will obscure the cells. 6. Examine your preparation carefully. The coverslip should be closely opposed to the slide. If there is excess fluid around its edges, you will need to remove it. 7. Place the slide on the stage and locate the cells in low power. You will probably want to dim the light with the iris diaphragm to provide more contrast for viewing the lightly stained cells. Furthermore, a wet mount will dry out quickly in bright light, because a bright light source is hot. A coverslip must always be used with a wet mount to prevent soiling the lens if you should mis-focus.

Epithelial cells of cheek (400X

Waste Disposal 1. When you have completed your observations, dispose of your wet mount preparation in the beaker of bleach solution. 2. Before leaving the laboratory, make sure all other materials are properly discarded or returned to the appropriate laboratory station. Clean the microscope lenses and put the dust cover on the microscope before you return it to the storage cabinet.

Determination the approximate field diameter for each of the objective lenses by using ruler Procedure: To calculate the diameter of the field of view for low (40X) and medium (100 X) power. Calculate the total magnification of the low power objective lens by multiplying the magnification of the ocular lens by the magnification of the objective lens. The magnification of the lenses is etched on the sides of the actual lens holders. Record the Magnification of all power levels for your microscope. Example: Low power: Objective lens = 4X and Ocular lens = 10X Total magnification = magnification of eyepiece × magnification of objective lens Total magnification at Low Power = 10 × 4 = 40X 1. Take a clear plastic ruler and examine the millimeter scale under low power. 2. Place the center of one of the scale marks along the edge of the field as shown below.

The Diameter of the field of view of low power (40X) is 4.2 mm.

3. Count the whole number of millimeter spaces. If there is part of a spacing, estimate (in decimals) the size of the millimeter portion that shows. Record the field diameter in millimeters.

4. Convert the field diameter of low power into micrometers and record this number. (1 µm = 1/1000th mm) 5. Follow the same procedure for the medium power lens. 6. The field diameter for high power and oil emulsion cannot be measure directly using millimeter ruler. The diameter of the field of view under high power is calculated by using the information from the lower power objective.

To calculate the diameter of field of view at high power, use the following formula High power magnification = Low power field diameter Low power magnification High power field diameter For example let’s say, if the diameter of 40X field is 4.2 mm you can calculate the diameter of 100X field. Now put the values in above equation 100 = 4.2 mm 40 High power diameter (unknown)

168 = 100 X High power field diameter High power diameter = 1.68 mm or 1680 µm Once the field diameter in micrometers is known, it is possible to estimate the size of an object by comparing its size to the field diameter. How can we measure the size of objects under the microscope?

Let’s say Field Of view under high power is 1.68mm or 1680µm. How many same objects can fit in the field diameter. What is the size of one object 3 cells = 1680µm 1 cell = Diameter of field of view No. of cells that fit 1 cell = 1680µm = 560 µm 3

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