Microscope Lab - Using the Microscope and Slide Preparation

Name ________________

Date _______________

Examine the microscope and familiarize yourself with the parts of the microscope.

1. Magnification

The magnification written on the ocular lens (eyepiece) is _____________

The magnification on the Scanning objective ___________ Low Power Objective ___________ High Power Objective ___________

What is the total magnification for each lens (multiply ocular times objective)

Scanning _____________ Low Power ______________ High Power ________________

2. Diaphragm

Examine the diaphragm, what are the numbers written on it? ____________________

Which setting makes the specimen the lightest? ________________ The darkest? _______________

3. Lenses

Twist the ocular lens, does yours have a pointer? _____________ What is the purpose of the pointer? __________________________________________

Find out what happens to your viewing field if you do not have an objective fully clicked into place. ____________________________________________

4. Viewing a Slide

Obtain a prepared E slide. Focus the slide first with the scanning objective, then click to lower power and focus again. Finally, focus the slide under high power. Remember, at high power, you should ONLY use the fine adjustment knob.

Draw the E exactly as it appears in your viewing field for each magnification. The circles below represent your viewing field. The E should take up as much space in the drawing as it does in your viewing field while you're looking at it.

Scanning

Low Power

High Power


5. Depth Perception

Obtain a prepared thread slide. You will only need to view it under scanning at this point. Your task is to figure out which thread is on top, which is in the middle, and which is on bottom. You should notice that as you focus the thread, different thread will come into focus at different times. The one that comes into focus the first should be the top thread.

What is the color order of your threads?

 

6. Making a Wet Mount of a Slide

1. Gather a few strands of cotton from a cotton ball using forceps. If your specimen is too thick, then the coverslip will wobble on top of the sample like a see-saw, and you will not be able to view it under High Power.

2. Place ONE drop of water directly over the specimen. If you put too much water, then the coverslip will float on top of the water, making it hard to draw the specimen, because they might actually float away. (Plus too much water is messy)

3. Place the coverslip at a 45 degree angle (approximately) with one edge touching the water drop and then gently let go. Performed correctly the coverslip will perfectly fall over the specimen.

Draw the specimen as it appears in your viewing field under scanning, low and high power.

Scanning

Low Power

High Power

 

7. Staining a Specimen

1. Place one drop of stain (methylene blue) on the edge of the coverslip. Caution: Methylene Blue will stain clothes and skin!

2. Place the flat edge of a piece of paper towel on the opposite side of the coverlip. The paper towel will draw the water out from under the coverslip, and the cohesion of water will draw the stain under the slide.

3. As soon as the stain has covered the area containing the specimen, you are finished. The stain does not need to be under the entire coverslip. If the stain does not cover as needed, get a new piece of paper towel and add more stain until it does.

4. Be sure to wipe off the excess stain with a paper towel.

                                                                        

Draw your specimen as it appears under low power. Used color pencils to show how the stain appears. It may appear darker or lighter in spots. Use shading to show darker and lighter spots.

Scanning

Low Power

High Power

 

8. Investigation of Pond Water

1. Prepare a wet mount of pond water - a sample of pond water is provided in a jar. The best specimens usually come from the bottom and probably will contain chunks of algae or other debris that you can see with your naked eye. (Be careful that your slide isn't too thick)

2. Use the microscope to focus on the slide - try different objectives, some may be better than others for viewing the slide.

3. Use reference books (provided in class) to identify at least three different things (protists, algae, insects..etc) in your pond water. You may want to color your pictures.

4. Make three separate drawings below at different areas of the slide and at different magnifications. Label where appropriate.

Drawing Specimens

1. Use pencil - you can erase and shade areas
2. All drawings should include clear and proper labels (and be large enough to view details). Drawings should be labeled with the specimen name and magnification.
3. Labels should be written on the outside of the circle. The circle indicates the viewing field as seen through the eyepiece, specimens should be drawn to scale - ie..if your specimen takes up the whole viewing field, make sure your drawing reflects that.

 

E. Measurement of microscopic specimens

Very often you will want to know the size of microscopic specimens. To be able to estimate the size of these objects, you will want to know the width or diameter of your microscopic field.

1. Cut the metric ruler from the last page of this handout.

2. Place the ruler on a slide (otherwise you may not have enough range to get it into focus)

3. Place the ruler over the opening in the stage so that the scale may be seen.

4. Under the lowest power, line up one of the vertical lines so that it is just visible at the left side of the field of view.

5. Determine the number of millimeters distance from one side to the other side of the field of view. It may be necessary to estimate fractions of millimeters.

What is the diameter of your field of view using a magnification of 100X?

              (mm)               (µm)

A micrometer (µm) is one-thousandth of a mm (0.00l mm) or l000 µm = 1 mm. The µm is used often in microscopic measurements. (l micrometer is the same as l micron (µ), a slang term that is commonly used.)

To measure the diameter of the field of view under high power, since it is less than l mm, we will determine the field of view by knowing the relative magnification powers of the high power objective and the low power objective: First get the ratio of these by dividing the magnification of the low power objective by that of the high power objective. Then multiply this result by the diameter of the low power objective field.

(1) Therefore, what is the diameter of the high power field? mm µm

Does your answer make sense?                

Later, you will have an opportunity to measure the size of microscopic organisms using this information.

G. Observations of Cells under the Compound Light Microscope.

The Onion Cell

1. On the supply table is an onion that has been cut into quarters or eighths. Separate the thick modified leaves that make up the onion bulb. Each inner leaf is covered by a delicate, thin, layer of cells. Break a leaf and lift off a small piece of this thin cell layer from the inner leaf surface (you want a single layer of cells) and make a wet mount of it by placing it on a slide and then adding a drop or two of water. Carefully place a cover slip over it, taking care to force out as many air bubbles as possible. (But don't squash the cells!! We want to look at onions, not squash!)

2. Observe these cells under lowest power and adjust the light to low levels until you to see internal structures clearly.

3. Notice the dark, thick cell walls surrounding each cell. These are composed of cellulose and form much of the supportive framework of the plant. Much of the inner volume of the cell is the membrane  enclosed vacuole. The cell membrane would be present between the cytoplasm and the cell wall but it is too thin to see with out using electron microscopy. The nucleus can be seen as a slightly grayish blob pressed against the inner side of the cellulose cell wall. It is embedded in cytoplasm that is pushed against the wall by the large fluid-filled vacuole in the center of the cell. Some of the nuclei appear to be centrally located because they are pressed against the top or bottom part of the cell wall. Do you see any nucleoli in the nuclei?

 

                                

 

 

   

The smallest space on this scale slide is equal to 0.1 mm

Use the Following Steps to Calculate the Correct Size

  1. Determine the diameter of the field of view using the scale slide.
  2. Determine the number of cells needed to cross the field of view.
  3. Divide the distance of the diameter by the number of cells needed
  4. The result is the size of a cell

 

The Above Calculation Using the Slides At The Top Of The Page

  1. 10 spaces x 0.1mm per space = field of view = 1.0 mm
  2. 3 cells across the diameter
  3. 1.0 mm diameter/3 cells
  4. About 0.33 mm per cell

Magnification of the illustration = size of illustration/actual size

For example, a magnification of 1X means the illustration is the same size as the object. 100X means the illustration is 100 times the size of the object, etc. It is important that you first calculate the actual size of an object and from that calculate magnification of the illustration you have made of the object.