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STUDENT HANDOUT:
STUDENT HANDOUT:
LABORATORY REPORT FORM:
LABORATORY REPORT FORM:
INTRODUCTION:
INTRODUCTION:
Most microbes are so small that they cannot be seen without the aid of the microscope. For this reason, the science of microbiology did not develop until the microscope was developed in the 1700's.
When you look at a microbe through the microscope, the image of the microbe is enlarged enough for you to see it. The magnification is accomplished by a series of glass lenses. Each lens by itself is similar to a hand-held magnifying glass, but the microscope is constructed so that we are actually looking through more than one lens at a time. The image magnified by one lens is further magnified by each succeeding lens so that we see a greatly enlarged image of the microbe. A microscope using more than one lens is called a compound microscope.
The lens at the top of the microscope, the one you look into, is the ocular lens (eyepiece). Its magnifying power is tenfold, or "10X." On the nosepiece of the microscope, above the stage on which the slide rests, there are three or four objective lenses. Only one of these is used at a time. Total magnification is the product of the ocular lens x the objective lens. It is best to start focusing the microscope on the specimen with the smallest lens, usually the scanning or low power, 4X-10X, objective lens and then move up gradually to the higher power objectives.
Once the subject is in focus, rotate the nosepiece to bring the high dry lens (with magnifying power between 40X and 50X) into position. After the image has been refocused using the fine adjustment knob, place a drop of immersion oil directly on the material being examined. Then rotate the longest of the lenses, the oil immersion lens, into place and using the fine adjustment knob, adjust the focus a final time. Do not place any other lenses in the oil.
Immersion oil is a special kind of oil through which light passes the same way it passes through glass. It must be used with the oil immersion lens to get a sharp image. Do not be concerned if the tip of the oil immersion objective lens gets into the oil; it is supposed to! You will need to remove all of the oil with a piece of lens paper at the end of your microscope session. A commercial glass cleaner such as Windex® is helpful in dissolving the oil. Make sure to dry the lens with lens paper when finished and remove any oil that is on the stage as well.
OPTICAL AND MECHANICAL FEATURES OF THE MICROSCOPE
OPTICAL AND MECHANICAL FEATURES OF THE MICROSCOPE
Figure 1. Parts of the monocular microscope.
USING THE MICROSCOPE
USING THE MICROSCOPE
You can watch a video of how to use the microscope below.
OBJECTIVES:
OBJECTIVES:
Identify the parts of the microscope.
Learn how to use the compound microscope.
Observe various kinds of microorganisms.
PRECAUTIONS:
PRECAUTIONS:
Use two hands to carry or move the microscope (Figure 3).
Figure 3. To properly carry the microscope, use one hand to hold the base of the microscope and the other to hold the arm.
MATERIALS (Work in pairs or individually, depending on how many microscopes you have):
MATERIALS (Work in pairs or individually, depending on how many microscopes you have):
Microscopes: Some microscopes are equipped with four objective lenses, some with two or three. Total magnification is the product of the ocular lens x the objective. You may need the oil immersion objective for this exercise if instructed.
Slides and coverslips: Slides and coverslips may be washed and used again. Plastic coverslips are acceptable.
Immersion oil: The refractive index of the immersion oil must match the refractive index of the glass in the lenses.
Prepared samples: the letter e, thread, a ruler, and other prepared slides will help you to practice focusing the microscope.
Water samples containing protozoa and algae: Pond water, aquarium water, or hay infusions may be used. Be sure to allow the samples to stand for a few days at room temperature.
Prepared slides of bacteria: Prepared slides to include the three basic shapes of bacteria: rods, cocci, and spirals.
(Alternatively, you can replace this activity with wet-mounts of live bacterial cultures).
PROCEDURE: OBSERVATION OF PREPARED SAMPLES
PROCEDURE: OBSERVATION OF PREPARED SAMPLES
Make sure the microscope is set to the lowest objective. Place the slide onto the stage of the microscope. Turn on the microscope. Bring the objective lens and stage as close as possible with the coarse focus adjustment knob.
Look through the ocular lens and, using the coarse and fine adjustment knobs, bring the specimen into focus. There is very little detail at this magnification, but you will be able to tell that you are focused on the specimen.
Rotate the nosepiece to the next higher power lens and refocus using the fine adjustment knob only. Most microscopes are parfocal which then allow for only the use of the fine adjustment knob at higher magnification if properly focused at lower magnifications.
Continue to increase the magnification of the nosepiece objective and refocus with the fine adjustment knob.
On Table 1 of the Laboratory Report Form, sketch any two images that you have viewed. Be sure to record the total magnification for each sketch by multiplying the ocular lens (10X) by the objective.
PROCEDURE: OBSERVATION OF LIVING PROTOZOA & ALGAE
PROCEDURE: OBSERVATION OF LIVING PROTOZOA & ALGAE
Place one drop of a water sample on a glass microscope slide.
Gently put a coverslip on the drop to prepare a wet mount (Figure 4).
Make sure the microscope is set to the lowest objective. Place the slide onto the stage of the microscope. Turn on the microscope. Bring the objective lens and stage as close as possible with the coarse focus adjustment knob.
Look through the ocular lens and, using the coarse and fine adjustment knobs, bring the specimen into focus. At this magnification you will not see the microbes yet, but you will be able to tell that you are at least focusing on the drop of water.
Rotate the nosepiece to the next higher power lens and refocus using the fine adjustment knob only. Most microscopes are parfocal which then allow for only the use of the fine adjustment knob at higher magnification if properly focused at lower magnifications.
Continue to increase the magnification of the nosepiece objective and refocus with the fine adjustment knob.
On Table 2 of the Laboratory Report Form, sketch the organisms you see in the water sample. Be sure to record the total magnification for each sketch by multiplying the ocular lens (10X) by the objective.
Hypothesize the microorganisms you have viewed and record your answer in Table 2. The rapidly swimming cells are protozoa; the green cells are algae. Here are 2 links to help you with your identifications:
(a) LINK #1
(b) LINK #2
Figure 4. Adding a coverslip. Hold the coverslip at an angle and drop it onto the slide. Doing this will reduce the amount of air bubbles trapped between the slide and coverslip. Image from Wikimedia Commons.
PROCEDURE: OBSERVATION OF PREPARED SLIDES OF BACTERIA
PROCEDURE: OBSERVATION OF PREPARED SLIDES OF BACTERIA
NOTE: The oil immersion is optional for this portion of the activity.
(Alternatively, you can replace this activity with wet-mounts of live bacterial cultures).
Make sure the microscope is set to the lowest objective. Place the slide onto the stage of the microscope. Turn on the microscope. Bring the objective lens and stage as close as possible with the coarse focus adjustment knob.
Look through the ocular lens and, using the coarse and fine adjustment knobs, bring the specimen into focus. There is very little detail at this magnification, but you will be able to tell that you are focused on the specimen.
Rotate the nosepiece to the next higher power lens and refocus using the fine adjustment knob only. Most microscopes are parfocal which then allow for only the use of the fine adjustment knob at higher magnification if properly focused at lower magnifications.
Continue to increase the magnification of the nosepiece objective and refocus with the fine adjustment knob.
On Table 3 of the Laboratory Report Form, sketch any two images that you have viewed. Be sure to record the total magnification for each sketch by multiplying the ocular lens (10X) by the objective.
QUESTIONS:
QUESTIONS:
What is the final magnifying power of each of the lenses on the nosepiece of your microscope?
Approximately how large is a protozoan cell?
How large is a bacterial cell? How does its size compare to the protozoan cell?
Apart from size, compare and contrast two of the microorganisms you have viewed today.
KEY TERMS:
KEY TERMS:
algae
coarse adjustment knob
compound microscope coverslip
fine adjustment knob
high dry lens
image
immersion oil
low power lens
objective lenses
ocular lens
oil immersion lens
protozoa
stage
wet mount
SUGGESTED READINGS:
SUGGESTED READINGS:
Click HERE to learn about the history of microscopes and the work of several famous individuals, including Zacharias Janssen, Antonie van Leeuwenhoek and Robert Hooke.
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