PART III.

VIEWING YOUR SPECIMEN

1. Prepare a wet mount using a very small letter “e” cut from a magazine page.
a. Describe in detail the process of preparing the wet mount. Provide an illustration (picture or
drawing with correct labels).
Using a newspaper or a book, find a lowercase letter e, and cut it. Afterwards, place the
freshly cut paper in the middle of the clean glass slide.

● Use a pipette to put a drop of water that is 1 cm above the slide/paper so that the paper
won’t touch the pipette.

● After that step, cover the water drop with a clean cover slide. Hold the cover slip in a
45 degree angle.

b. How will the presence of air bubbles be prevented? What should be done in case air bubbles
are present? 
- As mentioned above, hold the cover slip in a 45 degree angle to the slide and gently
lower the cover slip to allow the water to evenly coat under the cover slide. It should be
dropped slowly and not rushly because by doing the latter, the air bubbles will be
trapped inside.
 c. What is the proper way of dealing with the following situations: there is too much water and
the cover slip is floating around and if there is too little water and some of the space under
the cover slip is still dry?
- A way to avoid these situations is to use a pipette and make sure that you only drop a
preferred amount of water into the slide. And since if too much water is added, the
cover slip will "float," creating an overly thick water layer, and if too little water is
added, the specimen may be crushed or dry out too quickly. So, control the way you use
the pipette to get the desired amount of water in the slide.
d. How are liquid substances added to a wet mount?
- The specimen is held in place between the slide and cover slip by a drop of water. On the
slide, place a sample. Put a drop of water on the sample with a pipette. Place the cover
slip's edge over the sample and slowly drop it into position.

2. Set up the microscope.

a. Enumerate the steps in setting up the microscope.  
- PREPARING THE HEAD. (1) Turn on the illumination and adjust the brightness before placing a
slide on the stage. (2) Make sure to rotate the 4X objective lens into position. (3) Also, rotate the
eyepiece diopter ring to ’0’. (4) Lastly, adjust the interpupillary distance so that the images on
the right and left merge into one.

- FOCUSING YOUR EYES. (1) Place the specimen on the stage. (2) Focus the 10X objective using
the coarse knob and adjust with fine focus on the smallest detail visible. (3) Position the 40X
objective and fine focus. (4) Change to a 4X objective and adjust the diopters of the eyepieces to
compensate. (5) Check the focus again at 40X.

b. What is the importance of adjusting the interpupillary distance of the lenses in a microscope
with two oculars?  How will you know that the correct width is achieved? Are we going to use
both eyes in viewing?
- When using a binocular microscope, adjusting the interpupillary distance is crucial. This is
because your interpupillary distance—which is determined by the distance between the centers
of your two pupils—must match the distance between the binocular microscope's two
eyepieces. Lastly, the interpupillary distance varies from person to person, so the microscope
must be adjusted for your particular distance.

c. While peering into the eyepiece, move the knob of the iris diaphragm towards and away from
you. Is there a difference in the brightness of the field? What causes the difference?
- When the power is low, the most light is delivered to your eye. Light decreases when you
increase the power, which also affects your ability to tell apart two objects in your field of view.
To compensate for this, use just enough light to provide adequate contrast and to illuminate the
object on the slide.
3. View the specimen with the scanner.
a. Turn the nosepiece until the 4X objective locks into place.

https://www.ncbionetwork.org/iet/microscope/

b. Place the slide with the mounted letter on the stage and secure it in place using the slide holder.
c. Center the slide over the light source using the stage control knobs.
d. Looking through the ocular lens, slowly turn the coarse adjustment knob until a clear image
appears.  LOOK OCCASIONALLY FROM THE SIDE TO MONITOR THE DISTANCE BETWEEN THE
SLIDE AND THE OBJECTIVE.
e. Slowly turn the fine adjustment knob until the object comes into sharp focus.
f. Adjust the light for maximum contrast using the iris diaphragm. Can you see the entire letter?
What is the position of the letter when seen through the eyepiece?
- Since the depth of focus is highest on the lowest power objective, it is clear that we can see the
entire letter since we are employing a low-power objective. The two lenses on a microscope give
the letter e the appearance of being turned upside down. When compared to the letter e seen
ordinarily, the letter e seen under a microscope seems inverted due to the magnification.

 g. Using the stage control knobs, move the slide until you find the letter “e” in your field of view. If
you have markedly different eyesight between your eyes, how will you make use of the
diopter ring to gain sharp focus?
- If the two eyes have differing vision, the diopter ring is utilized to help the eye have a sharp
focus on the specimen. By shifting each side according to how each eye sees until both are
sharply focused.
4. View the specimen with the low-power objective.  
a. Rotate the revolving nosepiece to the LPO, the 10× objective.

https://www.ncbionetwork.org/iet/microscope/

b. Once you have the specimen in coarse focus, use the fine adjustment knob to bring the specimen
into sharp focus. NOTE: Because of the parfocality of the microscope, you should only have to
adjust the fine focus knob ½ turn forward to bring the specimen into sharp focus.
c. Using the stage control knobs, move the slide to the right, left, up and down. Describe the
movement of the image. What is the implication of this observation?
- The slide moves to the left when you move it to the right on the stage, and the slide moves to
the right when you move it to the left on the stage. Same goes for the up and down movement.

https://www.ncbionetwork.org/iet/microscope/
 d. Draw the letter ”e” as seen under the LPO.

Ps. This drawing was recycled from my Grade 12 Biology Class

5. View the specimen with the high-power objective.  

a. Turn the nosepiece until the HPO, the 40x objective, locks into place.  At this point, the letter ”e”
should still be roughly in focus.

https://www.ncbionetwork.org/iet/microscope/

b. Turn the fine adjustment knob slowly to focus the image.  NEVER USE THE COARSE
ADJUSTMENT WHEN THE HIGH POWER OBJECTIVE IS IN PLACE!
 c. Open the iris diaphragm until optimum contrast is achieved. Can you still see the entire letter
or just a portion of the letter? Why is this so?
- No, since we are using a high-power magnification. With this, the depth of focus is at lowest
when it's in high-power objective making you see only a smaller part of the specimen. This is
recommended if you want to study the finer details of the specimen.
d. Draw the letter ”e” as seen under the HPO.

PS. This drawing was recycled from my Grade 12 Biology Class.

6. If the microscope has an oil immersion objective, then view the specimen using this lens.  
a. Turn the nosepiece so that no objective lens is directly over the slide.  Then place a small drop
of immersion oil on top of the cover slip. Provide an illustration (picture or drawing with correct
labels).
 b. Turn the nosepiece so that the oil immersion objective is locked into place over the specimen,
making sure that the lens is immersed into the oil.
c. Slowly turn the fine adjustment knob until the image comes into focus.
d. Adjust the iris diaphragm to achieve maximum contrast.  
e. When you are finished, remove the oil from the oil immersion lens using lens paper.  
f. Remove the oil from the slide by wiping it gently with a paper towel.

7. Turn off the microscope.  

a. Lower the stage and then remove the slide. Clean and dry the slide and cover slip.  
b. Clean and dry the stage and lenses USING THE RIGHT KIND OF PAPER!
c. Place the 4X objective over the stage, facing the front.
d. Center the mechanical stage and make sure it is fully lowered.
e. Unplug the microscope and wrap the cord correctly.
f. Cover the microscope with the dust jacket.
g. Return the microscope to the correct cabinet placing the oculars toward the BACK of the cabinet.
PART IV. PROPERTIES OF LIGHT MICROSCOPY
1. Total MAGNIFICATION is determined by multiplying the ocular lens power times the objective lens
power. Example: Ocular lens power 10X; Objective lens power 4X; Total magnification is 40X.
Calculate the total magnification when the following objectives are used: low-power lens,
high-power lens, and oil immersion lens. Show your solutions.
● LPO (10X) - 10 x 10 = 100X total magnification
● HPO (40X) - 10 x 40 = 400X total magnification
● OIL IMMERSION (100X) - 10 x 100 = 1000X total magnification

2. Shift the objectives from the lowest power to the highest power. Compare the distance between
the tip of each objective and the stage. This is called the WORKING DISTANCE. As the power of the
objective increases from scanning → LPO → HPO → oil immersion, does the working distance
increase or decrease? Show an illustration with proper labels.
- The working distance gradually decreases as the magnification gets bigger. In order to focus, the
high-power objective lens must be significantly nearer to the specimen than the low-power lens.
SCANNING

LPO
HPO

OIL IMMERSION

3. Move the scanning objective lens in place and put a clear plastic ruler over the light opening in the
stage, or use the micrometer in the ocular lens, or grid slide. Describe a micrometer and a grid
slide. Provide illustrations.
GRID SLIDE - Use with dry specimens or suspended specimens; all cells and specimens placed on top
of Slide-Grids can be counted and/or sized; inert and stable under most laboratory conditions.
 MICROMETER- Specimens are frequently measured or counted using microscope micrometers.
Eyepiece micrometers are tiny glass discs with markings on them, sometimes known as "reticles."
An image of the markings is superimposed over the image of the specimen thanks to the
micrometer, which is positioned in one of the two eyepieces. Stage micrometers are
microscope-viewable slides with measurements marked on them that can be used to calibrate
eyepiece micrometers.

4. Look through the ocular lens and move the ruler or grid slide so that a line touches the left edge of
the field and count the number of millimeter intervals that can be seen. Show an illustration.
Record your measurement and convert it into micrometers. Show your conversion.

Conversion: 0.25 mm = 0.25 x 1000 μm = 250 μm

5. Switch to the low-power objective lens and repeat this procedure to count the number of
millimeter intervals that can be seen. Record in millimeters.
- 2mm / 80 cells = 0.025 mm
6. Switch to the high-power objective lens. Can you still measure the field using a transparent ruler?
- No, I cannot measure the field since I am using a HPO lens.

7. Assume that you viewed the following object using the scanning objective. Estimate the length of
the object in μm. What is the importance of understanding the FIELD DIAMETER or the FIELD OF
VIEW of your microscope?

- Field diameter, which indicates how much of your entire field of view will be visible after you
look through the eyepiece lens, is more important in microscopy than magnification. The
amount of millimeters or micrometers you can see via the eyepiece lens is the field diameter. The
process is identical to counting the lines on a ruler while holding it up to a microscope.

8. Rotate the scanning objective into place, and place the slide with the three colored threads (red at
the top most followed underneath by blue and yellow) on the stage. Move the slide using the
mechanical stage controls until the threads come into the field of view. Position the slide so that
the point of intersection of the three threads is in the center of the field.

9. Rotate the low-power objective into place and then focus down until all three threads are out of
focus then slowly move the stage upward to focus and note which thread comes into focus first.
What happens when you are sharply focused on one colored thread?
- The top thread, followed by the middle thread, and eventually the bottom thread are
brought into focus when the lens is focusing downward. At low magnification, each level
was sharp.

10. Continue to focus upward, noting when the second and third threads come into focus. Predict what
happens to the DEPTH OF FIELD/DEPTH OF FOCUS as magnification increases.
- When using higher magnification, the depth of field will decrease since only a small part of the
specimen will be seen when using the said magnification.