INVESTIGATING THE COMPOUND MICROSCOPE
The microscope is an instrument designed to examine objects too small to be seen with the naked eye. Unaided, the human eye cannot distinguish objects much smaller than 0.1 mm. The microscope acts as an extension of the eye, allowing one to see smaller objects.
What is a compound microscope?
The most commonly used type of microscope is called a compound (multiple lenses) monocular (one-eyed) microscope because the image is seen through two lenses with one eye. Usually the light passes through the object being viewed, then through one of the objective lenses, into the microscope, through the eyepiece lens, out of the microscope and into the viewer's eye.
How does one focus a microscope?
Your eye has a lens that automatically adjusts to focus on an object being viewed. However, the lenses of a microscope must be brought into focus mechanically. A pair of knobs, called the course and fine adjustment knobs, move the lenses toward or away from the object being viewed. In some microscopes the stage holding the object is moved instead of the lenses.
The focal length is different for each objective lens.
The distance between the objective lens and the object viewed determines whether the image will be in focus.
• The high power lens needs to be much closer to an object to bring it into focus than the low power lens does.
• Because the lens is so close to the slide, there is greater risk of damaging a slide and the lens when using the high power objective than when using low power.
The total magnification is a product of two lenses.
When you look through the microscope it is important to know how much the object you are observing is magnified.
• To find the degree of magnification, multiply the number on the objective being used by the number on the eyepiece.
• If the eyepiece power is l0X (magnifies 10 times) and the objective power is 43X, for example, the total magnification is 10 multiplied by 43, or 430X. This means that you would be looking at something 430 times it’s normal size.
NOTE: The microscope is an expensive instrument that must be given proper care.
Always follow these general instructions when using a microscope.
1. Always carry the microscope with both hands, one hand under its base, the other on its arm.
2. When setting the microscope on a table, keep it away from the edge. When working with a microscope it is generally best to clear your lab table of everything that is not absolutely needed.
3. The lenses of the microscope cost almost as much as all the other parts put together. Never clean them with anything other than lens paper.
4. When getting ready to put the microscope away, always turn the nosepiece to return it to the low power setting. Wrap up cords.
5. Always remove all materials from the microscope stage and re-cover the scope with the plastic bag before returning it to the cart.
Materials
compound microscope scissors eye dropper
glass slide lens paper newspaper
pond water cover slip water
Procedure
You are going to set up some objects on microscope slides and examine them under increasing powers of magnification.
Setting up the compound microscope
1. Place the low-power objective in place if it is not already there. When you change from one objective to another you will hear a “click” when the objective lens is set in the proper position. If the objective lens is out of line with the arm, no light will get through the microscope.
2. Move the mirror so that light is reflected upward through the opening in the stage. You should be able to see the light through the hole in the stage. If your microscope has it’s own light source then you do not need to do this.
3. Most microscopes are equipped with a diaphragm for regulating light. Once you are looking at an object, turn the diaphragm until you get the right amount of light for the object being viewed. Some materials are best viewed in dim light, others in bright light.
4. Make sure that all the lenses are clean. Wipe the lenses with lens paper only.
Big, Bigger, Biggest!
1. Cut (or tear) out a small piece of newspaper containing the lower case letter "e" in small print. Place this on a clean slide and use a dropper to put one drop of water on the letter. Wait until the paper is soaked.
2. Hold a cover slip at about a 45° angle to the slide and then slowly lower it flat onto the slide. A gentle tapping (with a fingernail or the back of a pen) will usually remove any bubbles that may have been caught between the slide and the cover slip. You have now made a wet mount.
3. Now you will examine your slide with the compound microscope.
· Place the slide on the stage and clamp it down. Move the slide so that the letter is in the middle of the hole in the stage and the light is shining through the “e”.
· Make certain that the low power objective lens is in place. Watch the stage from the side while you use the course adjustment knob to lower the objective lens (or raise the stage depending on your scope). Lower it until it won't go any further down.
· Look through the eyepiece and use the fine adjustment knob to slowly raise the objective lens until the letter is in view. Use the fine adjustment to sharpen the focus.
Question: How does the "e" look different from what it looked like in the newspaper?
Draw exactly what you see. Note the magnification. Record any other observations you make.
4. Put one thumb on each end of the wet mount slide.
· Look through the eyepiece at the letter and move the slide slightly away from you.
· Move the slide to your right.
· Move the slide to your left.
Write: Make a general statement about the relative position and movement of objects as they are viewed through the compound microscope.
5. You are going to look at three different-colored hairs. (Use one of your own and "borrow" some from classmates.). Arrange them to look like the diagram below. Let’s do this only if we have time.
6. Add a drop of water to make a wet mount as you did with the “e”.
7. Now examine your slide with the compound microscope. Observe under low power. Position the slide so that the hairs cross in the very center of the field.
8. Turn the nosepiece so that the high-power objective is in viewing position.
CAUTION:
Do not change the focus while changing the objective.
Never focus the high power objective using the coarse adjustment.
Steps for using the high-power objective:
· Find the object with lower power.
· Focus and adjust the diaphragm for maximum light.
· Position the slide so that the object is in the center of the low power field.
· Turn the high-power objective into viewing position.
· Sharpen the focus using the fine adjustment only.
· Adjust the diaphragm to get the best lighting. If you are not successful in finding the object under high power the first time, return to low power and repeat the whole procedure carefully. Continue until you see the object under high power.
9. The crossed hairs should now be in view and need only slight focusing with the fine adjustment. Focus on the hairs at the point where they cross.
Question: Can you see all three hairs sharply at the same focus level? Explain why or why not.
Question: Can you tell, while looking through the microscope, which hairs are the top, middle, and bottom pieces? How can you tell?
10. Prepare a slide with one drop of pond water. Cover with a cover slip. Nematode drops may be available.
11. Now, examine your slide with the compound microscope, first under low power, and then under high power. In each case:
Draw your pond critters. Note magnification
Draw your nematodes (if available). Note magnification.
Microscopic measurements
NOTE: Be sure to use the same microscope for this entire section!
Many things that biologists study with a microscope are of definite sizes and shapes. In order to make quantitative observations of these features you must have some way of measuring them. The microscope can be used as a tool to measure things too small to be measured with an ordinary ruler.
You will use two techniques to measure the approximate diameter of the low-power field of your microscope.
1) Look at your microscope.
Ø Record the number of your microscope.
Ø Record the magnification printed on the ocular.
Ø Record the magnification printed on the low power objective.
Ø Record the magnification printed on the high power objective.
(If your microscope has more than two objectives or any missing data, see your teacher.)
2) Using an extra-sharp pencil:
Ø Carefully draw a row of small distinct circles on a piece of plain paper. Start with the smallest circle you can draw and make them progressively larger until you have about 10 circles, the largest about 4 mm in diameter.
Ø Cut a strip of paper containing your circles and make a wet mount. (You will probably need to use two cover slips.)
Low Power Field Size Estimation
3) Estimate your low power field size.
· Examine your slide under low power, starting with the smallest circle. This circle should be smaller than the field. Look at each circle in order from smaller to larger until you find the one that most nearly matches the diameter of the microscope field. If you don't have one that matches, try making other circles until you get one that does. Remove the slide from the microscope and use a ruler to measure the diameter of the matching circle to the nearest 0.5 mm.
Ø Record the approximate diameter of your microscope field.
Ø Calculate how much the circle is magnified when you look at it under low power?
· Lay the clear millimeter ruler on a slide and place it on the stage so that you can see the millimeter markings while you are looking through the low-power objective. Line up one millimeter line with one edge of the field. Note that the lines of the ruler look quite wide under magnification. Find the midline of each ruler marking in your field of view. Remember that ruler markings are 1mm apart.
Ø Determine the diameter of the low-power field to the nearest 0.5 mm.
Ø Explain how this measurement compares with the estimate of the field diameter that you made using the drawn circle procedure above.
NOTE: If the two measurements do not agree closely you should repeat both methods of determining the diameter of the field (steps A and B) to determine your sources of error.
4. Remember that the unit of measurement for objects too small to be seen except through the microscope is the micrometer (micron). One micrometer equals one one-thousandth (0.001) of a millimeter. One millimeter equals 1000 microns. The symbol for this unit is m (mu).
Ø Calculate and record the diameter in microns of the low-power field of your microscope. Show your work!
5. Now that you know the approximate diameter of the low power field of your microscope, you can use this information to estimate the size of objects under low power. Cut out a small section of a light-colored area of a picture from a magazine. Be sure the back side of your paper is light in color or you won't be able to get enough light through to see! Make a wet mount of the paper and examine it under low power.
Ø Describe in words and sketches what you see.
6. Choose one color dot and estimate the number of these dots that would fit across the diameter of the low-power field if the dots were touching each other.
Ø Record your estimate.
7. By comparing the size of the dots with the known diameter of the low-power field, estimate the diameter of one dot. (Divide the diameter of the field in microns by the number of dots that could fit across it.)
Ø Calculate the estimated size of one dot of the color you’ve chosen. Show your work!
8. Use the same procedure as in steps 6 and 7 to estimate the distance between two dots (space size).
Ø Calculate how many spaces would fit across the low-power diameter. Show your work!
Ø Count how many spaces you actually see across the diameter of the low-power field. Record the number.
Ø Divide the diameter of the field in microns by the number of spaces that would fit across it. Show your work!
Record your estimate for the size of each space.
9. You can check to see how good your "measurement" of dot and space size is.
Ø Add the size of all the dots that are in a diameter: (• multiply one dot size times the number of dots you actually counted) to the size of all the spaces that are in a diameter : (• multiply one space size times the number of spaces you actually counted). Show your work!
Ø Record the total diameter. This the diameter of the low power field using dot and space size estimates.
Ø Is this figure close to the estimated diameter of the field that you determined earlier in steps A and B above? Why or why not?
Ø If not, repeat steps 6 through 8 until you have a closer estimate of the dot size.
High Power Field Size Estimation
10. Working from your low power dots and spaces, you can estimate the diameter of the high power field of your microscope. Be sure you have a bright low power field. Carefully switch the objective to high power. Focus. Line up the edge of one dot of your chosen color along one edge of your field.
Ø Count and record how many dots are in the high-power diameter.
Ø Count and record how many spaces are in the high-power diameter.