4 Lab Protocol: Introduction to Microscopes

Exercise 1 Parts of a Compound Light Microscope

Obtain a compound light microscope and identify the following parts:

  1. Stage and stage clip which are the platform on which the slide is placed and the clip which holds the slide in place.
  2. There are also two knobs that adjust the location of the slide over the condenser (light source). One moves the stage side to side, the other moves the stage front to back and vice versa.
  3. Ocular lenses which the person will look through to see the image. The ocular lenses have a magnification power of 10X. The distance between the ocular lenses can be adjusted according to the distance between the scientist’s eyes.
  4. The four objective lenses are located just above the stage. The scientist can choose which objective lens to use at a given time by rotating the nosepiece to a particular position. The magnification power of these lenses varies from microscope to microscope. We have microscopes with 4X, 10X, 40X, and 100X objective lenses as well as microscopes with 10X, 40X, 66X and 100X objective lenses.
  5. The course and fine focus knobs which move the stage up (closer to the objective lens) or down (further away from the objective lens). It is important that the course focus knob, which moves the stage quickly, should only be used with the lowest power objective lens.
  6. The light source can be adjusted using an iris diaphragm or dimmer knob. Adjustment is helpful according to the specimen and the magnification used.

 

Exercise 2 Using the compound light microscope by viewing a slide with the letter “e” 

  1. Make sure the objective lens being used is at the lowest power (4X or 10X, depending on the microscope).
  2. Place a prepared slide of the letter “e” on the microscope stage right side up and secure with the clip.
  3. Turn on the light to the microscope. Without using the ocular lenses, use the stage adjustment knobs to position the letter “e” directly above the light source.
  4. View the specimen through the microscope and use the stage adjustment knobs to center the “e” within the field of view and the course focus knob to bring it into focus. Using the iris diaphragm or dimmer switch, adjust the light intensity so that it is comfortable to your eyes.
  5. When initially focusing, it may be best to start with the objective lens as far from the slide as possible and then to move it closer and closer until you can see the “e” roughly in focus. Now use the fine focus knob to bring the “e” into sharp focus.

 

A. What is the orientation of the letter “e” in the field of view?

B. In which direction does the “e” move within the field of view when you move the stage to the left?

C. In which direction does the “e” move within the field of view when you move the stage closer to you?

 

6. Turn the nosepiece to change the objective lens to the next higher power. Without making any adjustments, view the “e” through the microscope. You should still be able to see the letter “e” or part of the letter “e” roughly in focus. Microscopes are built such that they are “par-centered” which means that the specimen will remain centered in the field of view after switching objective lenses. Microscopes are also built such that they are “par-focused” meaning that the specimen will remain roughly in focus after switching objective lenses. Thus, one will only need to make minor adjustments using the fine focus knob and stage adjustment knobs.

7.  Increase the magnification again by switching to the next objective lens. Re-center and re-focus the microscope. We will not be using the 100X objective lens today.

 

Excercise 3 Calculating the total magnification

When lenses are coupled in a microscope, the total magnification is calculated using the formula:   Total Magnification = Ocular Magnification X  Objective Magnification  

Calculate the 4 total magnifications that are possible with the microscope you are using to complete the table below.

Ocular Magnification

Objective Magnification

Total Magnification

 

 

 

 

 

 

 

 

 

 

 

 

 

Exercise 4 Determining the diameter (FOVd) and the area (FOVa) of the field of view  

  1. To use a clear ruler to determine the diameter of the field of view for the lowest power objective lens, place the ruler on the microscope stage the same way you would place a slide. In this way, you can use the stage adjustment knobs to position the ruler exactly the way you would like.
  2. Focus on the mm markings on the ruler placing one mark at the extreme left position in the field of view.
  3. Now count the number of intervals between gradations that you can see in the field of view. This number is the diameter of the field of view in mm.
  4. To determine the diameter of the field of view for higher power objective magnifications (Mag), we will use the mathematical formula:  FOVd1 X Mag1 = FOVd2 X Mag2.  FOVd1 and Mag1 would be based on the lowest power objective (or total magnification), Mag2 would be based on the higher power objective (or total magnification), and FOVd2 is what we are trying to calculate.
  5. Complete the table below to indicate the FOVd for each of the objective lenses on your microscope. Be sure to indicate the proper unit of measurement. Note that if the magnification is increased two-fold, the diameter is reduced two-fold.
  6. The area of a circle is calculated using the formula: area = πr2 .  Calculate the radius of each circle by dividing the corresponding diameter by 2 and include in the table on your data sheet. π is roughly equal to 3.14; therefore, calculate the area of the field of view (FOVa) by multiplying 3.14 x radius x radius and enter in the last column of the table. Be sure to include the proper unit of measurement. Note that if the magnification is increased two-fold, the FOVa is decreased 4-fold.
  7. Calculate the radius of each circle by dividing the corresponding diameter by 2 and include in the table above. π is roughly equal to 3.14; therefore, calculate the area of the field of view (FOVa) by multiplying 3.14 x radius x radius and enter in the last column of the table. Be sure to include the proper unit of measurement. Note that if the magnification is increased two-fold, the FOVa is decreased 4-fold.

Objective lens magnification

FOVd

radius

FOVa 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Exercise 5 Exploring the relationship between depth of field and magnification

Depth of Field refers to how much of the thickness of an object can be brought into focus at the same time. A low depth of field means only a small fraction of the specimen’s height will be in focus at a time.

  1. Place the cross thread slide onto your microscope and view at the lowest magnification.
  2. Determine how many different threads appear in sharp focus at the same time and record on the table below.
  3. Examine the slide at the two higher levels of magnification and do the same.
  4. Complete the following statement by choosing the correct option: The depth of field (decreases, remains the same, increases) as the magnification is increased.
  5. Continue viewing the slide of the cross threads at different magnification and pay attention to how the brightness changes.
  6. Complete the following statement by choosing the correct option: As the magnification increases, (less, the same, more) illumination is needed to maintain the same brightness.

     

 

Magnification

How many threads are in focus?

 

 

 

 

 

 

 

 

Exercise 6 Estimating relative size of an object 

In the previous exercises, you calculated the diameter and area of the field of view under various magnifications. You can use this information to make an estimate about the relative size of cells through the microscope. In this case, we will be looking at a sample of brine shrimp and brine shrimp eggs to estimate their relative size.

  1. Prepare a wet mount slide from the sample provided by your instructor. You should use the pipette to mix the solution of brine shrimp to suspend the organisms as they usually settle on the bottom of the container.
  2. Mount your prepared slide on the stage of the microscope and use proper technique to locate a place on the slide where you can observe several brine shrimp eggs.
  3. Take note of which magnification you are using and calculate the relative size of the shrimp eggs using the following equation: size= FOVd/ fit number

 

View through a light microscope showing a cluster of dark cicular structures which are brine shrimp eggs magnified at 100x. There are two clearly visible brine shrimp in the center of the image and a third one on the upper left that is blurry.
Brine shrimp and brine shimp eggs by Jovana Bustillos and Amelia Hall SLCC Spring 2024. Photo taken with 10X objective lens (100X total magnification).

 

Exercise 7 Practice using a dissecting scope by viewing a penny, fruit fly, and starfish

  1. Place the penny on the platform of the dissecting scope such that it is right side up. Turn on the light source.
  2. Now view the penny with the highest magnification possible. What is this magnification?
  3. Determine the field of view’s diameter at the lowest and highest magnification using the ruler.
  4. Using the highest magnification which still allows you to see an entire fruit fly, measure the length and width of a fruit fly.
  5. Draw a diagram of the underside of a starfish showing the tube feet of one of the arms. Include a size bar on your diagram.

 

 

 

 

 

 

 

 

 

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Biology 1615 - College Biology I Lab Copyright © by Dalia Salloum. All Rights Reserved.

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