A.

Identify and familiarize yourself with the following parts of the microscope and their functions:

1. Power switch - turns the lamp on and off.

2. Illumination control
i. controls the brightness of the lamp
ii. on most microscopes it is a continuously variable rheostat, controlled by a sliding switch
3. Illuminator
a. consists of a lamp bulb and one or more lenses to produce a cylindrical beam of light directed toward
the base of the condenser
b. may have a ground glass - a frosted sheet of glass that scatters the light from the lamp bulb to
provide more diffuse light
c. may have an iris diaphragm, called the field diaphragm
i. controls the diameter of the illuminating beam of light
ii. used as a guide to focus the light from the condenser onto the specimen
4. Condenser - contains a set of lenses that focus the light on the specimen. The condenser has the following
components:
a. Focusing knob - moves the condenser up and down to adjust the focus of the light on the specimen.
b. Front lens - the glass surface closest to the specimen. Be careful not to touch this surface as it is
easily scratched.
c. iris diaphragm
i. controls the aperture of the illuminating light.
ii. used to adjust contrast
d. filter holder
i. located at the base of the condenser
ii. allows insertion of a filter (usually blue) to adjust the color of the illuminating light
5. Revolving nosepiece - holds several objective lenses that can be rotated into position to change the lens.
6. Objective lenses - create a magnified image of the specimen
a. 4x lens - used to get an overview of the structures present in o a section and to find areas for more
detailed observation
 b. 10x lens - the most useful magnification to identify tissues
c. 40x lens - used to see the details of cell and tissue organization
d. 100x lens - because it requires the use of immersion oil, is used primarily to see subcellular details
Note: Most histology is done with the 10x and 40x lenses.
7. Eyepiece - forms an image that can be visualized by the eye or a camera.
a. ocular lens - magnifies (typically 10x) and rotates the primary image produced by the objective lens
b. eyepiece tube
i. Holds the ocular.
ii. In a binocular microscope, the distance between the two tubes can be adjusted to fit
the distance between the observers’ eyes.
8. Focusing controls - used to raise and lower the specimen stage to focus the image of the specimen
a. coarse focus - used to focus the specimen at 4x and 10x
b. fine focus - used to focus the specimen at 40x and 100x, but only after initially focusing at lower
magnification
c. focus stop
i. present on some microscopes to set the highest point to which the stage can be
raised
ii. prevents the slide from being pressed against the front of the objective lens
d. tension adjustment of coarse focus - present on some microscopes to adjust the tightness of the
coarse focusing knob
9. Specimen stage - holds the microscope slide
a. slide holder - spring-loaded device to hold the microscope slide in place on the stage
b. slide holder travel controls - allow the slide to be moved along two axes: longitudinal and lateral
c. stage verniers - present on some stages to measure the position of the center of the slide

B. Setting up and adjusting the microscope.

1. To adjust your microscope for optimal viewing of a slide, make sure you understand how to control the
following variables:
Focus - adjusted with the coarse and fine focus knobs
Brightness - adjusted with the illumination control and the condenser focusing knob
Magnification and resolution - adjusted by selecting the appropriate lens
Contrast - adjusted by opening or closing the condenser diaphragm.
Oil immersion - used only with specially designated lenses; involves placing a drop of immersion oil
between the slide and the front of the objective lens
2. Adjust your microscope by following the following steps:

Step 1: Adjusting focus of the specimen (4x objective lens)

- Place a slide on the specimen stage and at 4x magnification focus the microscopic image,
using the coarse focus controls. Adjust the brightness of the image as needed using the
rheostat control of the illuminator.

Step 2: Adjusting illumination (4x objective lens)

- If your microscope has a field diaphragm:

Reduce the diameter ("stop down" or "close") the field diaphragm until you can see
at least part of its edge. Focus the image of the diaphragm by raising or lowering the
condenser using the condenser focus knob until the edge is in sharp focus. The image should
also be in focus. If not, refocus the image using the coarse focus knob. Repeat until both the
image of the specimen and the image of the diaphragm are both in focus at the same time.
- If you microscope lacks a field diaphragm.
Raise the condenser as high as it will go using the condenser focus knob and then
lower it slightly until the illumination is uniform and maximum. If you see a granular
background it is probably the image of the ground glass present in the illuminator to diffuse
the light. Raise or lower the condenser until the image of the ground glass vanishes.

Step 3 (4x objective lens)

- Use the condenser centering screws to center the image of the field diaphragm, closing the
diaphragm down further, as necessary, to center it accurately.

Step 4 (4x objective lens)

- Open the field diaphragm until its edge are just within the boundaries of the optical field.
Adjust the focus of the image and of the diaphragm so that they are both in focus at the
same time.

Step 5 (10x objective lens)

- Open the field diaphragm until it just vanishes from the optical field. The height of the
condenser lens is now properly adjusted. You should not have to readjust it. Now it is time
to adjust contrast.

Step 6 (4x objective lens)

- Increase the magnification to 10x. Refocus the image and adjust the intensity of the
illumination using the rheostat control of the illuminator, as necessary.
 Step 7: Adjusting contrast (40x objective lens)

- Increase magnification to 40x. With the condenser diaphragm wide open, adjust the focus
(using the fine focus control) and brightness (using the illuminator control), as necessary.
Note that the image looks washed out. Now you will adjust the condenser diaphragm to
achieve optimal contrast.

Step 8 (40x objective lens)

- Close the diaphragm of the condenser until the image has enough contrast to see the
structures that you are interested in. Adjust the brightness of the image with the illuminator
control. The illumination is now properly adjusted for good microscopic work. As you
change magnification and slides, you will need to readjust the contrast - using the
condenser diaphragm - and the brightness- using the illuminator control - as needed to
achieve the optimal image. You should not need to readjust the field diaphragm.

C. Using the Oil Immersion Lens.

1. Some lenses, such as the 100x lens on standard microscopes, require that oil be placed between the lens
and the slide. Such "oil immersion" lenses are indicated by having the word "oil" engraved on the outside of
the lens.
2. Immersion oil is a specially formulated oil whose refractive index has been adjusted to allow the oil immersion
lens to achieve maximum resolution.
3. To use the 100x oil immersion lens, do the following:
 Adjust the illumination and focus the specimen at 40x.
 Rotate the nosepiece so that it is half way between the 40x and 100x lenses.
 Place one drop of immersion oil on the slide at the place that was under the lens.
 Rotate the nosepiece to bring the 100x lens into position, such that the end makes contact
with the drop of oil.
 Refocus if necessary using the FINE FOCUS ONLY. If you use the coarse focus you will run
the risk of forcing the 100x lens into the slide which may break the slide and/or damage the
lens.
 Adjust the contrast and illumination as necessary to produce a good image.
 When you are finished with the specimen rotate the nose piece so that it is half way between
the 100x and 4x lenses. Do not rotate the 40x lens through the oil. If you do it will not focus
properly and will need to be cleaned with lens paper moistened with ether.
 Remove the slide.
 Remove the excess oil from the slide with lens paper or another absorbent paper moistened
with ether.
 To continue, place a clean slide on the stage and rotate the 4x lens into position. You may
continue to use the non-oil lenses but be careful not to rotate the 100x lens over the slide as
it will transfer oil to the slide.
 To use the oil lens on a new slide repeat the preceding steps.
 When you are finished with the oil immersion lens, clean the tip of the oil immersion lens with
lens paper ONLY. Moisten the paper with ether and draw the wet paper over the surface of
the lens to float the oil off onto the paper. Repeat as necessary to remove all of the oil.
D. Cleaning the Microscope
It is important to keep your microscope clean:
 Lenses should be cleaned ONLY with lens paper that has been moistened with water (to remove
general dirt) or ethyl ether (to remove oil).
 Never rub a lens with lens paper. Use the paper to apply a film of fluid to the surface of the lens and
subsequently to remove the fluid from the surface of the lens.
 Use absorbent paper, such as Kimwipes, to remove dirt and oil from the stage and other mechanical
parts of the microscope.
 Stubborn dirt may require other solvents. Seek advice from a knowledgeable person before trying
this yourself.

E. Pay attention to the following do's and don'ts:

1. The Do's:
a. Wash your hands before handling slides to avoid transferring finger grease to the slides.
b. To remove oil from an objective, moisten a piece of lens paper with ethyl ether and slowly draw the wet
paper across the front surface of the lens.
c. To remove dirt from an eyepiece, breathe on the lens and GENTLY wipe off the condensed water with
lens paper.
d. Get help if these steps do not solve your problem.
2. The Don'ts:
a. Never touch a lens with anything but lens paper.
b. Never touch a lens with dry lens paper.
c. Never rub a lens.
d. Never put oil on a dry objective, and remove it at once if an accident occurs.
e. Never wear mascara when using a microscope.