BIOS242 Lab 3
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Lab 3: Microscopy- Observation of Cells
Learning Objectives:
Use the compound light microscope for observation of cells.
Identify different types of bacteria based on their shapes.
Identify a variety of eukaryotic cells.
Introduction:
Microbiology is the study of organisms, which cannot be seen with naked eye. You will need help of a microscope to
observe these “tiny animalcules” as they were described by Antoni Von Leeuwenhoek. Microscopes can either be simple
(contains single lens) or compound (contains series of lens systems to achieve higher magnification).
Principle of microscopy: In a compound microscope, the light passes from the specimen into the objective lens which
magnifies the image to 4x, 10x, 40x, or 100x (depending upon the power of objective lens used) which then enters eye
piece and is further magnified 10x. Final magnification is hence the product of power of objective lens (4x, 10x, 40x, or
100x) multiplied by the power of the eyepiece (10x).
Total Magnification of a Microscope = Magnification of Objective Lens x Magnification of Ocular Lens
Exercise 1: Parts of a Microscope
Eyepiece Lens: The lens at the top of the microscope that you look through
Arm: Supports the tube and connects the head containing the eyepiece to the base of the microscope
Illuminator: A steady light source (110 volts) used in place of a mirror.
Stage: This is the flat platform where you place your slides. Stage clips hold the slides in place and can be moved around
by turning two knobs. One of the knobs moves the stage left and right (horizontal movement), the other knob moves the
stage up and down (vertical movement).
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They usually consist of 4X (scanning), 10X
(low power), 40X (high power) and 100X (oil immersion) lenses.
When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X, 400X
and 1000X.
Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen.
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and
is used to vary the intensity and size of the cone of light that is projected upward into the slide.
Coarse adjustment knob: A large, round knob on the side of the microscope used for focusing the specimen; it may
move either the stage or the upper part of the microscope
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Fine adjustment knob: small, round knob on the side of the microscope used to fine-tune the focus of your specimen
after using the coarse adjustment knob
Aperture: the hole in the stage that allows light through for better viewing of the specimen
Parfocal microscope objectives: The specimen being observed remains in focus when the magnification is changed, i.e.,
if the microscope is switched from a higher power objective (e.g., 40×) to a lower power objective (e.g., 10×), the
specimen remains in focus.
Field of View: Sometimes abbreviated "FOV", it is the diameter of the circle of light that you see when looking into a
microscope. As the power gets greater, the field of view gets smaller.
Complete the Table 1 in the Lab report for calculations of magnification and identifying parts of microscope.
Exercise 2: Observation of Different Types of Bacteria
Materials:
Compound microscope, immersion oil, lens wipes, prepared slides of various bacteria- Rods, cocci, and spiral
Notes to Students:
For this exercise, you may have a single slide containing three different types of bacteria or you may have three slides,
each containing only one type of bacteria. Check with your instructor about how many slides you will be observing.
Method:
1. Obtain the preserved slide of a bacteria.
2. Use Appendix A (located at the end of this document) and follow the instructions on how to use a Microscope.
3. Observe the slide under 10X, and then 40X objective lenses.
4. Record and describe your observations made using 40X lens.
5. Make sure to note as many details as possible about the size, shape, and color (stain) of bacteria.
6. Use 100X oil immersion lens if needed and record your observations.
Note to Students: Do not forget to use oil with 100X lens only. After completing the observation, clean the oil
immersion lens with lens wipes to remove oil. Be careful to not contaminate other lenses (4X, 10X, 40X) with oil
as this will cause damage to the other lenses.
7. Repeat the steps 1-6 for slides containing bacteria of different shapes as needed. Record your observations in
the lab report.
Exercise 3: Observation of Eukaryotic Cells- Protozoa and Fungi
Materials:
Compound Microscope, prepared slides of Protozoa and fungus
Method:
Protozoa:
1. Obtain the preserved slide of a protozoa.
2. Use Appendix A (located at the end of this document) and follow instructions on how to use a Microscope.
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3. Observe the slide using 10X, and 40X objective lenses.
4. Record and describe your observations made using 40X in the Lab report. Make sure to note as many details as
possible about the size, shape, and color (stain) of protozoa.
Fungi:
1. Obtain the preserved slide of a multicellular fungi.
2. Use Appendix A (located at the end of this document) to follow instructions on using a Microscope.
3. Observe the slide using 10X, and 40X objective lenses.
4. Record and describe your observations made using 40X in the Lab report. Make sure to note as many details as
possible about the size, shape, and color (stain) of fungus.
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BIOS242 Lab 3
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Lab 3 Report: Microscopy
Exercise 1: Parts of Microscope
Table 1: Calculate the total magnification
Type of Objective Lens Magnification of Magnification of Occular Calculate Total
Objective lens lens Magnification
Low Power objective 10 x
High power objective 10 x
Oil immersion 10 x
Label the parts of Microscope:
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Exercise 2: Observe different types of bacteria
Draw your observations inside the circles provided. Label your images appropriately. Write the magnifications at
which the observations were made.
Bacterial Slide of: ________________________
Bacterial Slide of: ________________________
Bacterial Slide of: ________________________
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Questions:
1. What three bacterial shapes did you observe?
2. How does increased magnification affect the field of vision?
3. Why is the oil immersion objective necessary to determine the morphology of prokaryotes?
4. Why is it desirable that microscope objectives be parfocal?
5. Which objective focuses closest to the slide when it is in focus?
6. Which controls on the microscope affect the amount of light reaching the ocular lens?
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Exercise 3: Observe different types of eukaryotes- Protozoa and Fungi
Draw your observations inside the circles provided. Label your images appropriately. Write the magnifications at
which the observations were made.
Eukaryotic Slide of: ________________________
Eukaryotic Slide of: ________________________
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Questions:
1. Did you need oil immersion to observe the shapes of eukaryotes? Why or why not?
2. List and define two features that are found in some, but not all eukaryotes.
Grading Rubric:
Activity Deliverable Points
Experimental Set up Obtain/Set up the slides as directed in the lab or by 6
and Observation instructor; Observe all assigned slides in exercises 2 and 3
using microscope and record observations in lab report
Lab Report and Complete lab report and answer ALL questions 9
Questions
Exercise 1 (4 points)
Exercise 2 Questions (3 points)
Exercise 3 Questions (2 points)
All Lab Deliverables Complete ALL lab work and lab report 15
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Appendix A: General Steps in Using a Compound Light Microscope
1. Take a microscope from the cabinet. Hold the microscope by its arm using one hand and support the base with
the second hand.
2. Plug the microscope cord into an electrical outlet and switch it on.
3. If you need to clean the lens, only use lens paper and not any other types of wipes or towels.
4. Make sure that the light source is set at a low-medium setting.
5. Rotate the nose piece to use the 10X objective lens. You will hear a click when the objective lens comes to the
correct position.
6. Obtain a glass slide with the specimen.
7. Put the slide on the stage and use the adjustment knobs (located right underneath the stage) to move the stage
(left or right or horizontal movement).
8. Orient the slide so that the specimen is directly within the field of light.
9. Adjust the diaphragm to limit the amount of light.
10. Move the stage up using the coarse adjustment knob. When the stage is moved up almost completely, observe
using ocular lenses to bring the specimen in focus.
11. Once the specimen is in focus, use the fine adjustment knob to focus so that the specimen is observed very
clearly.
12. Rotate the nose piece to observe the image under 40X. You may have to use the fine adjustment knob when you
change the Objective lens from 10X to 40X. You should also adjust (increase) the amount of light appropriate for
your eyes when you switch to the higher objective lens. This is achieved by adjusting the diaphragm as well as
the light source knob.
13. Once the observation under 40X is made, record the observations.
14. Check with your instructor if 100X oil immersion lens should be used. If the instructor asks you to use the 100X
lens, follow the steps below.
15. Bring 100X lens in focus by rotating the nose piece. Put a drop of oil on the coverslip so that the surface of 100X
lens touches the oil. You may have to use the fine adjustment knob to clearly observe the image when you
change the Objective lens from 40X to 100X. You should also adjust (increase) the amount of light as suited to
your eyes using the diaphragm as well as light source settings.
16. Record the observations made using the 100 X lens.
17. Once you put the immersion oil on the slide, do not use any objective lens other than the 100X oil immersion
lens. Contaminating 4X, 10X, and 40X objective lenses with oil may damage the lens permanently. Please ask the
instructor if you have any questions.
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Steps to follow after the observations are completed.
1. Lower the stage completely. Rotate the nose piece so the 4X lens remains in focus position.
2. Remove the slide and appropriately discard the slides.
3. Wipe the oil immersion lens to remove any oil using the lens paper and oil cleaner solution.
4. Set the light setting to the minimum and switch it off.
5. Unplug the microscope and wrap the cord around the arm.
6. Cover the microscope and put it back into the cabinet.
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