LABORATORY ACTIVITY 1: BASIC DEVELOPMENTAL LABORATORY TOOLS

AND MICROSCOPY

Most of an embryologist’s tools are so fine that they have to be made by hand. The ones you will
be encountering are those used most routinely by embryologists. The only indispensable microdissection
tool that you will not be making is fine forceps. Tools to be used on living material must be kept separate
from those that have been in fixative. Your dissecting tools from other labs must never touch those that
you make for living material. Fixative is nearly impossible to remove completely from a tool, and the
smallest trace can severely damage living embryological material.

Microknives

Knives as fine as the iridectomy knives used by eye surgeons can be made from chips of razor
blades mounted on wooden dowels. The ideal knife is shaped like a half-spear with a long shank (see
Figure
2.1A). Microknives can be sterilized before use in 70% ethanol or in boiling water. Microtomes and knives
were created to work together in order to produce tissue sections to be observed under the microscope.

Microneedles

Microneedles (Figure 2.1B) are extremely valuable for fine manipulations and can be used
as microscissors by drawing one needle past another. Microneedles can be sterilized before use in 70%
ethanol or in boiling water.

Hairloops

Hairloops (Figure 2.1C) are one of those romantic additions to an embryologist’s arsenal. Dating
from the days of Hans Spemann, when experimental embryology was in its infancy, they have been used
to gently coax delicate tissues around a culture dish or to deliver grafts to a graft site. Blond baby’s hair
makes the finest quality loops, and for years’ embryologists have been known to hang around barber
shops waiting for the first haircut of a blond child. You will probably want to experiment with several
types of hair. Fine blond hair will make the smallest loops. Coarser hair will make larger loops. Basically,
you want to force the hair into the smallest loop possible to give you a firm instrument.
Pipettes

Pipettes are among of the most valuable instruments you can have. The appropriate bore size can
perform magic at the bench, and you won’t know the appropriate size until you’re sitting there needing it.
It therefore is very important to have made a variety of sizes in advance.

Embryo Spoons

An embryo spoon is one of those tools you seldom use and can’t do without. It is nothing more
than a perforated spoon that allows you to pick up embryos and eggs in the several-millimeter to several
centimeter range without having them float off the spoon. One way in creating your very own embryo
spoons is by the use of a really cheap, thin, relatively small plastic spoon is best. Heat a clean dissecting
needle in an alcohol lamp until it is glowing red, then perforate the bowl of the spoon, making 12–15
holes, evenly spaced, and enlarging each hole with the heated needle so that each is approximately 2
mm in diameter. You will have to reheat the needle periodically. Once the holes are made, you will notice
that their edges are very rough. Sand both the upper and lower surface of the spoon with sandpaper until
your fingers can no longer feel any rough edges. This sanding is extremely important, because a rough
edge can seriously damage an embryo. Test your spoon by putting water in it. The water should drain
through the holes. If the spoon holds water instead, the holes need to be enlarged.

Sterile Technique

Sterile technique is no more than a matter of common sense. If you simply imagine that
everything about you is filthy—your hands, your hair, your breath—and that germs are raining
down from the heavens, and let this principle guide your actions, then you will master
sterile technique quickly. All work areas, instruments, solutions, and cultureware must be
sterilized.
 Embryological Tools
Questions

Name: NEHEMIAH S. QUIANE_ Date: 08/21/2021

Kindly answer these questions once you have conducted your research or background checks on these
mentioned tools.

1. What use does an embryo spoon have? After making one, how do you test it to see if it will work
properly?

- Embryo spoons are used to pick up embryos and eggs in the several-millimeter to several
centimeter range without having them float off the spoon. To test the embryo spoon you made you
should try scooping some water and the spoon should drain it. If the spoon still stored some water u
should make the holes bigger or smoothen the edges of the holes.

2. What percentage alcohol do you use for sterilizing tools? Also, why do you think this percentage
of alcohol is considered the most germicidal or effective in sterilizing?

-70% alcohol is recommended for sterilizing tools. This percentage of alcohol is used
mostly in sterilizing since it can effectively denature the proteins and lipids of bacteria, viruses, and
fungi. Lower concentration of alcohol can only kill certain type of bacteria and can be inefficient for
sterilizing tools. It is also studied that higher concentration of alcohol (99%) will result in almost
immediate coagulation of surface or cell wall proteins and prevent passage of the alcohol into the
cell. The coagulation of the membrane can protect the virus or bacteria from the alcohol.

3. How can you use two microneedles to simulate microscissors?

- You can simulate microscissors using two microneedles by drawing one needle past
another.

4. What methods can you use for sterilizing your microknives and microneedles?

- Sterile or Aseptic techniques are the methods that can be used to sterilize microknives and
microneedles. Sterile technique is a set of specific practices and procedures performed to make
equipment and areas free from all microorganisms and to maintain that sterility (BC Centre for
Disease Control, 2010
5. What can a hairloop be used for?

-Hairloops are used to gently coax delicate tissues around a culture dish or to deliver grafts
to a graft site.

6. When making a hairloop, which will make a smaller loop, a fine or a coarse hair?

-A fine hair is used in making smaller loop.

 Using the Compound Microscope
OVERVIEW

Follow along in this online exercise, with guided instruction covering the basics of light
microscopy, comparable to what you would learn in a seated lab. You will identify components of the
microscope, understand the functions of those components, learn how to focus in on a specimen, and
review proper care and maintenance.

LEARNING OBJECTIVES

• Identify the parts of the light microscope and describe the function of each.

• List the steps in focusing a light microscope.

• Describe how to properly handle the light microscope, focus slides, and clean the microscope
when
finished.

BACKGROUND

When we walk out the door each day we see many examples of life on earth – trees, birds, other
humans. Unless we look closer we will miss the huge diversity of life that is too small to be seen with our
eyes alone. The majority of life on earth is microscopic and, until tools were available to visualize these
organisms, they were completely overlooked. One of the first tools that opened our eyes to microbial life
was the microscope. The compound microscope is an exquisitely refined, expensive piece of equipment.
Learning how to use it correctly not only will allow you to achieve the greatest resolution your
microscope can give, but it also will avoid costly damage. The light microscope uses lenses along with
light to magnify items up to 1000x. This allows us to see organisms like bacteria, archaea, yeasts,
protozoans, and algae that we would never notice because they are so small. In addition, we can also look
closer to see cells that are the building blocks of the macroscopic organisms (like ourselves, the trees, and
birds we noticed earlier.) In addition to magnifying organisms, most cells are colorless so many times
preparing slides also involves adding stains (dyes) to color the cells so they are more easily viewed. The
slides you will practice with today are either naturally pigmented or have been stained so we can find
them more easily. Look for these colors as you are focusing the virtual microscope.

PROCEDURE 1: Introduction to the Microscope and its Parts

1. Go to http://www.ncbionetwork.org/iet/microscope/.
2. Click on the Guide link (bottom of the home page).
3. Click through the six parts of the Guide, starting with the Introduction. You can use the
arrows at the bottom of the Guide box to guide you through the chapters.
4. When you have completed all six sections, click Close.
5. Next click on the Learn link (bottom of the page), which will take you to an image
of a microscope with question marks.
6. Starting at the top of the microscope, click on the question marks identifying the parts of the
microscope.
7. Read the description of the part of the microscope and take notes as needed.
8. Continue clicking on question marks until all turn to green check marks.
a) Do not forget to click on the question marks for items associated with the microscope.
b) You may click on any green check mark to review any part of the microscope.
 c) Use the Microscope Parts checklist on the next page to ensure all parts have
been identified.
9. Click on the Next button (bottom right).
10. Start on the left and click on the question mark. When the lens enlarges, click on each
question mark until each turns into a green check mark. Read the descriptions and take notes
as needed.
11. Click on the Next button (bottom right).
12. Click on the Dry Slide and Oiled Slide buttons to see the difference in why immersion oil is
used for the 100X objective lens.
13. Click on the Next button (bottom right).
14. Click on the Eyepiece Options and Lens Options to learn about calculating
total magnification. Try all combinations and see how the Letter E slide image changes.
15. Click on the Next button (bottom right) to return to the home page.

PROCEDURE 2: How to use a compound microscope to view slides

1) Click on the Explore link (bottom of the home page).
2) Click on the question mark on the slide box.
3) In the Slide Catalog, click on the Sample Slides.
4) Click on the Letter E slide. It will automatically be placed on the stage of the microscope.
5) When the Microscope View window opens, make sure that the 4X circle is highlighted in blue.
NOTE: Always begin examining slides with the lowest power objective.
6) Use the slider under Coarse Focus to find the E.
NOTE: The coarse adjustment knob should only be used when you are viewing a specimen with
the 4X objective lens.
7) Then use the slider under Fine Focus to make the image “crisp and clear.”
8) You can click on the E in the viewing window to move the image and visualize different parts.
Sketch your view of the letter E at 4X in the results area.
9) Next click on the 10X circle. The nosepiece on the microscope will rotate automatically.
10) Repeat steps 6 – 8 to see part of the E. Sketch your view of the letter E at 10X in the results area.
11) Click on the 40X circle and repeat steps 7 & 8. You may need to use the slider under
Light Adjustment for better visualization. Sketch your view of the letter E at 40X in the results
area. Click on the 100X circle. A notice to add immersion oil will open.
12) Click on the question mark on the immersion oil bottles to add oil to the microscope.
13) Repeat steps 7 & 8. You may need to use the slider under Light Adjustment again for
better visualization. Sketch your view of the letter E at 100X in the results area.
14) When you have visualized the Letter E slide using all 4 objective lenses, click on Remove Slide
(top right).
15) Read the notice about using lens paper to clean the immersion oil off the microscope and click on
the question mark over the lens paper. Choose wisely!
16) Click on the Main button (bottom left corner) to return to the home page.
17) You can now explore other slides that are available.
Care and Maintenance of Your Microscope: “Twelve Good Rules”

“‘The time has come,’ the Walrus said, ‘to talk of many things.’” And those things for us are a
most annoying, but most necessary, list of dos and don’ts that will make you a safe microscopist
rather than a liability.
1. Always carry the microscope with one hand grasping the arm and one hand supporting
the base, keeping the microscope level.
2. Never increase the light so that the meter shows in the red zone. If brief incursions into
the red zone are necessary (for photomicrography, for example), keep them short,
reducing the light as soon as possible.
3. Never leave the light on when the microscope is unattended.
4. Never force a knob or control. Get help from the instructor if something won’t move.
5. Always move from the lowest-power objective to successively higher-power objectives.
6. Never unscrew an objective unless instructed to do so. If you remove an ocular
for adjusting Koehler illumination, do not let go of it, and replace it immediately after the
adjustment is made. Dirt that gets down the ocular tube impedes microscopy and can be
removed only by a technician.
7. Always clean an oil-immersion lens immediately after use and clean any oil that might
have spilled on the stage. Never use alcohols when cleaning; these will dissolve the glue
that holds the lenses in place. A small amount of xylene or toluene may be used, but
always check with your instructor first.
8. Never leave a slide on the stage when storing the microscope.
9. Always put the lowest-power objective back into place for storing the microscope.
10. Always cover the microscope when storing it. If there is no microscope cover available,
get a plastic bag large enough to come down over the scope to a level lower than the stage
so that the condenser is adequately covered.
11. Always review these rules until they are reflex.
12. Always take pride in being a fine microscopist, and keep the naive show-offs away from
your precision instrument
Sources: ncbionetwork.org

Bradbury, S. 1980. Getting the best out of your micrscope. Royal Microscopical Society Proceedings 15: 270–279. A
concise and valuable set of tips and explanations.

Davidson, M. W. 1991. A photography primer. The Science Teacher 58(7): 12–18. This nontechnical, well-illustrated
paper gives practical information about adapting the microscope for polarized and reflected light as well as
photography.

Delly, J. G. 1988. Photography through the Microscope. Eastman Kodak Company, Rochester, NY. This is a
beautifully illustrated pamphlet that concentrates primarily on photomicrography but has an excellent discussion of the
principles of light microscopy, Koehler illumination, filters, dark-field, and polarizing methods.

Gage, S. H. 1920. The Microscope. Comstock Publishing Co., Ithaca, NY. Originally published in 1908, this book is a
testament to how sophisticated microscopes were even in the early 1900s. Packed with information about microscopy
and histological preparations, it is still extremely useful.
 Compound Microscope
Questions

Name: _NEHEMIAH S. QUIANE Date: 08/24/2021

Kindly answer these questions once you have performed the indicated procedures. You may also include
pictures or screenshots from the activity to further support your answers.
1. What is the typical magnification of an ocular lens? What other magnifications are possible?
-Ocular lenses usually have a magnification of 10x but also come in 5x, 15x, and 20x.

2. What are the magnification abilities of each of the objective lenses? What is the total
magnification with each objective?
a. Scanning (small lens), red ring = 4x (10x) (ocular lens) = 40x
b. Low-power (medium lens), yellow ring = 10x (10x) = 100x
c. High-power (large lens), blue ring = 40x (10x) = 400x
d. Oil immersion (largest lens), white ring = 100x (10x) = 1000x

3. If you are using a 40X objective and a 10X ocular, what is the final magnification that you are
viewing through the microscope?
- 40x (10x) = 400x is the final magnification of the microscope.

4. What is a diaphragm? What does it do?

- The diaphragm is used to adjust passing through the stage opening. You can adjust the
opening of the diaphragm depending on the specimen that you are using.

5. What does the condenser do?

-The condenser serves to gather wave fronts from the microscope light source and
concentrate them into a cone of light that illuminates the specimen with
uniform intensity over the entire view-field.

6. If the image looks grainy, what might be incorrectly adjusted on the microscope?

- The fine focus can be adjusted if the image looks grainy.

7. If the image looks washed out, what might be incorrectly adjusted on the microscope?

- If the image looks washed out, the coarse focus may be adjusted
8. Why do you use immersion oil with 100X objective lens?
- The 100x lens is immersed in a drop of oil placed on the slide in order to eliminate any air gaps
and loss of light due to refraction (bending of the light) as the light passes from glass (slide) → air
→glass (objective lens).

9. What is the total magnification of a sample with an ocular lens power of 15X and using a 40X
objective lens?

- 15x (40x) = 600x is the total magnification.

10. What is the proper way to carry a microscope?

- One hand holding the arm and another hand under the base is the proper way of carrying the
microscope.

11. RESULTS: Sketch the letter “e” at each of the resolutions

12. What did you notice about the letter E when you increased in magnification from the 4x to the
10x and then to the 40X:
a. Did the size (magnification) increase or decrease?
- the size or magnification increase.
b. Could you see more of the entire letter or less?
- I could see less of the e as I increase the magnification.