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Use of Microscope PDF
Use of Microscope PDF
The microscope is absolutely essential to the microbiology lab: most microorganisms cannot
be seen without the aid of a microscope, save some fungi. And, of course, there are some
microbes which cannot be seen even with a microscope, unless it is an electron microscope,
such as the viruses.
You will be using an assigned light microscope for a variety of lab exercises through the
semester, everything from viewing pond water to identification of your unknown bacterium.
Therefore, it is extremely important that you understand how to use the microscope
effectively and how to use different types of microscopy----brightfield, phase-contrast, and
darkfield. These Nikon microscopes have a revolving condenser, with specific settings for
the 3 kinds of microscopy: the settings are labeled with white letters that can be seen in the
front. Bright field is “0”, darkfield is “DF”, and phase-contrast is “PH.” There are 3 settings
of phase-contrast, one for each of the lenses—PH1 for 10X, PH3 for 40X, PH4 for 100X. If
you forget, look at the markings in red on the objective lenses.
Carry it with 2 hands---one on the arm and the other under the base.
Clean ALL objective lenses and the ocular with lens paper BEFORE you even place a
slide on the stage, and it is a good idea to wipe the condenser lens also. The last
person using the microscope may have left it dirty: it is imperative to begin with clean
lenses.
Use lens paper (ONLY) to remove any oil from the 100X lens.
Once oil has been added to the slide, do not move back to the 40X lens to focus: oil
should never get on this lens. If this happens, it will be very difficult to get all of the oil
off, and you will have to clean the lens thoroughly.
Place the microscope back into the correct spot in cabinet, with the arm toward you.
Loosely wind the electrical cord around the cord holder on the back of the arm. Do not
bend the cord at the microscope socket.
Determination of total magnification = ocular lens (10X) X objective lens (10X, 40X, 1 00X)
OBJECTIVES:
1
MATERIALS NEEDED:
microscope
lens paper
bibulous paper
oil dropper
prepared wet mounts or stained smears
THE PROCEDURES:
You will use a variety of specimens with your microscope----a wet mount, a bought
prepared smear, and a stained smear that you have made. Refer to the lab exercise
PREPARATION OF SPECIMENS.
2
10X magnification.
11. You will want to reduce the light coming through the condenser, so close your iris
diaphragm so that you get better contrast of the specimen.
TO VIEW A SPECIMEN:
1. Place the wet mount or prepared smear on the stage, and secure it inside of the stage
holder.
2. Try to guesstimate where the specimen is located on the slide, and place it in the
center of the hole allowing light through the stage.
3. While looking through the ocular eyepiece, lower the stage SLOWLY using the coarse
adjustment knob. Be sure that you are looking through the binocular head of the
microscope with BOTH eyes.
4. As soon as you see the specimen, STOP using the coarse adjustment, and switch
over to the fine adjustment knob. After focusing at the beginning with the coarse
adjustment knob, it is NOT TOUCHED AGAIN. All focusing will now be done with the
fine adjustment knob.
5. CHANGING OBJECTIVES:
1. Do NOT MOVE the focus knobs or the stage knobs. Swing the 40X objective (high
dry) out of the way. Place a single drop of immersion oil on the slide right over where
the light is coming through the stage, and rotate the 100X objective (oil immersion)
into place. The lens will actually GO INTO THE OIL DROP.
2. Now look through the oculars, increasing your light with your iris diaphragm lever.
Your object should still be in the field of vision, probably out of focus. Use the fine
adjustment knob to focus clearly.
3. Once you have gone into oil immersion, do NOT GO BACK TO THE 40X
OBJECTIVE. The objective will get oil on it, and you will have to really clean it to get
the oil off. The 1 0X can be returned to, since the lens should not touch the slide
anyway.
4. Once through with the microscope, use the lens paper to wipe the oil from the
100X objective lens.
3
USING DARKFIELD AND PHASE-CONTRAST:
Once you are looking at your object using brightfield microscopy, you can easily switch to
another type of microscopy: Just rotate the condenser knob. Darkfield and phase-contrast
microscopies have particular condenser lenses required for proper visualization.
HOWEVER:
Darkfield is used for wet mounts, using 10X and 40X (1 00X will not show well).
Be sure that your iris diaphragm is OPEN all the way.
TROUBLESHOOTING:
Focus fuzzy?
Probably oil on the lens. Clean with lens paper thoroughly. If that does not help, use lens
cleaner with lens paper. If that doesn’t work, ask instructor for some help. Xylene may be
used, but SPARINGLY since it can destroy the glue seating the lens.
Found the specimen on low power, but lose it when moving into a high power?
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PART OF MICROSCOPE FUNCTION
QUESTIONS:
2. How do the functions of the substage condenser and the iris diaphragm within the
condenser differ?
3. What term is used to describe the feature of the microscope that makes it possible to move
among the objective lenses with just MINOR focusing?
4. What condenser setting value do you want when you are using the 1 00X objective lens?
5. What would be the total magnification of a specimen using the 40X objective lens?
6. Why move the 10X objective lens into place when putting the microscope back into the
cabinet?
7. What is parfocal?
8. Why reduce your light when using the 10X objective lens?