4 MICROSCOPY AND THE CELL
Learning Objectives
- Test predictions from scientific hypotheses using basic biological laboratory techniques.
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Distinguish living organisms based on the characteristics of life.
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Relate cell structure to function.
Activity
- A microscope has two sets of lenses. What do you think is the purpose of each lens?
- Why do you think scientists use microscopes? Think of useful functions for the microscope and discuss your ideas with your group.
The Use of a Microscope
The hidden world becomes visible with the aid of a microscope. A microscope is used to see items and structures that the human eye cannot visualize. Microbes, cell structures, and minute particulate materials are examples of such objects. The microscopic examination of one-celled organisms as well as the cells and tissues of multicellular organisms are all part of biological research. To properly utilize the microscope, you must first learn the operational modes of each part. In this laboratory exercise, you will learn to use and identify the parts of a compound light microscope.
Parts of a Microscope
A microscope magnifies the image of an object through a series of lenses. The condenser lens focuses the light from the microscope’s lamp onto the specimen. The light then passes through the object and is refracted by the objective lens. The objective lens is the more powerful lens of a microscope and is closest to the object. The light then travels to the ocular lens, which focuses the image onto the user’s eye. Usually, the power of the ocular lens is fixed for a given microscope.
Different microscopes can magnify objects hundreds, thousands, or even millions of times. Magnification refers to how much larger the image is compared to the original object. As magnification increases, the image becomes larger. The total magnification is the power of the objective multiplied by the power of the ocular lenses. Resolution refers to the ability of a microscope to distinguish two points on the image. As resolution increases, objects that are closer together appear as separate points. Contrast refers to the ability of a microscope to distinguish an object from its background. The higher the contrast, the greater the difference in intensity between an object and the background.
Safety Precautions
- Handle microscopes and lenses with care.
- Do not drop or crush slides. Alert your teacher immediately to any broken glass so it can be properly disposed.
For this activity, you will need the following:
- Compound microscope
- Slide with the letter e; if you do not have premade slides, cuttings from a newspaper can be taped to a slide.
For this activity, you will work in pairs.
Structured Inquiry
Step 1: When moving a microscope, grasp the neck firmly with one hand and place your other hand under the microscope’s base. Do not bang the microscope on the desk. Once you have set down your microscope, turn the revolving nosepiece so that the lowest power objective is pointed at the stage.
Step 2: Throughout this lab, handle slides by their edges and do not touch the specimen area to avoid smudges. Place the slide with the letter e on the stage. Make sure it is oriented so that you can read the letter e correctly with the naked eye while standing at the microscope.
Step 3: You will now observe the slide containing the letter e. Center the specimen on the stage and look through the ocular lens. Adjust the coarse and fine focus knobs until the letter comes into focus. Be careful while focusing so that the slide does not touch the lenses.
Step 4: Hypothesize/Predict: From your observations of the letter e, what do you think is the function of each part of the microscope? Write the function of each on your paper.
Step 5: Student-Led Planning: Using your textbook and other resources, research each part of the microscope. Identify each part of the microscope in Figure 2.1. Label each part of the microscope in Table 2.1. Write the function of each part in Table 2.1. Parts and Function of a Light Compound Microscope.
Table 2.1: Parts and Function of a Light Compound Microscope
| Part | Function |
|---|---|
| 1. Ocular lens (eyepiece) | |
| 2. Revolving nosepiece | |
| 3. Objective lens | |
| 4. Coarse adjustment knob | |
| 5. Fine adjustment knob | |
| 6. Stage | |
| 7. Lamp | |
| 8. Condenser | |
| 9. Mechanical stage |
OPTIONAL ACTIVITY
- Visit the BioNetwork’s Virtual Microscope
- Click on “Explore”
- Click on parts “?” to learn their name and function
Step 6: You place a sample on the stage of a microscope, and you look through the ocular lens. All you see is black in the field of view. What would you adjust to fix this problem?
Step 7: Microscope care and maintenance are vitally important. When you are finished using a microscope, make sure the lowest power objective is in place and take the slide off the stage. Use only lens paper to wipe the lenses if they are dirty. Support the microscope by the base when carrying it as described in Step 1. If you are continuing with the other activities in this lab, keep the microscope out.
Guided Inquiry
Step 1: Find the light source on the microscope. Turn on the light source and trace the path of light from the source to the ocular lens.
Step 2: Knowing the function of each part of the microscope, draw how the light path of the microscope bends and changes as it travels from the light source to the observer on Figure 2.1.
Step 3: Using your textbook and other resources, research how light travels through the microscope. Use a piece of paper to block the light path through each component of the microscope to confirm that it is part of the light path.
Step 4: Sometimes, scientists want to study living cells that are kept in dishes. The dishes are very deep and filled with media. The cells are on the bottom of the plate. In order to see these cells on the microscope, the microscope has to be inverted so that the objective lens is below the sample instead of above it. Draw a diagram of how you would design the light path of an inverted microscope.
Magnification[1]
Magnification is the process of enlarging the appearance of an object. We calculate the magnification of an object by indicating the fold change in size. So, if something appears to be doubled the size of the real item, then it is obviously magnified 2x. Because there is a magnification by the eyepiece (ocular lens), as well as the objective lenses, our final magnification of an item is the product of those two lenses.
The lowest magnification objective lens (usually 4x or 5x) is referred to as a scanning lens. There is also usually a low-power lens at 10x and a higher magnification lens at 40x. There may be a higher magnification lens at 100x, but these usually require oil to function properly and are often reserved for microbiology labs.
What is the power of the ocular lens?
We can calculate that as:
Magnificationtotal = Magnificationobjective x Magnificationocular
With this in mind, fill in the following table:
| Objective Magnification | Ocular Magnification | Total Magnification | |
|---|---|---|---|
| Scanning | |||
| Low Power | |||
| High Power | |||
| Oil Immersion | 100X |
Assessment
- What are three precautions you must take to prevent damage to the microscope?
- A scientist uses a 40× objective to observe his specimen. He has a 10× ocular lens. What is the total magnification of the object?
- A magnifying glass is also used to magnify objects. How is a compound microscope different from a magnifying glass?
When you first place a specimen on the microscope’s stage, the image you see in the ocular lens will likely appear blurry. Pull focus by changing the distance between the object and the microscope lens to reduce blurriness. This is because light rays from the object are not reaching your eye at the same time. Focusing is used to improve the image. Focusing moves the stage up and down, which changes the point at which the light rays from the object converge. When the stage is at a position so that the light rays exactly converge on the retina of your eye, the image will appear sharp and clear.
When focusing the specimen, you must be careful! The distance between the objective and the specimen is very small, and it is possible to crush the specimen against the lens if you move the stage too far. The proper distance between the specimen and the object is called the working distance.
When you look at a specimen through the microscope’s lenses, you will likely not see the entire specimen at one time. Because the microscope enlarges the image of the object, only a small portion of the object will be visible at any given time. The field of view refers to the portion of the object that is seen through the ocular lenses (see Figure 2.3).
Safety Precautions
- Handle microscopes and lenses with care.
- Do not drop or crush slides.
For this activity, you will need the following:
- Compound microscope
- Slide with the letter e; if you do not have premade slides, cuttings from a newspaper can be taped to a slide
Structured Inquiry
Step 1: Turn the revolving nosepiece of the microscope so that the lowest power objective is pointed at the stage.
Step 2: Place the slide with the letter e on the stage. Make sure it is oriented so that you can read the letter e correctly with the naked eye while standing at the microscope.
Step 3: Hypothesize/Predict: How do you think the letter e will appear under different magnifications? Draw your predictions in your lab notebook.
Step 4: Student-Led Planning: Change the magnification by turning the revolving nosepiece. Draw your observations
Examining Cells
The specimens you use on your microscope can either be wet-mounted or dry-mounted. A wet-mount refers to living tissues that are placed on a slide with an aqueous solution to keep them wet. Usually, a coverslip is placed on top of the specimen to flatten the specimen onto the slide. A dry-mount refers to preserved tissue that has been fixed and stained on a slide. This technique allows you to preserve specimens for a long time, and it also allows you to add chemicals to increase the contrast of a specimen from its background.
Safety Precautions
- Handle glass slides with care.
- Dispose of specimens as necessary.
- Dispose of coverslips in a broken-glass container.
For this activity, you will need the following:
- Compound microscope
- Slides with crossed colored fibers or crossed strands of hair
- Clean slides
- Coverslips
- Elodea, onion skin, pond water, or other samples
For this activity, you will work in pairs.
Guided Inquiry
Step 1: Hypothesize/Predict: Based on the microscopy you have performed so far, what would be the challenges of viewing living organisms under the microscope?
Step 2: Student-Led Planning: You will now observe pond water or samples of living organisms provided by your teacher. Place a small drop of water from near the bottom of the sample jar on a slide. Place a cover slip at an angle so that one edge touches the drop. Slowly lower the coverslip onto the specimen. Avoid trapping air bubbles under the coverslip.
Record drawings of your observations in your lab notebook. Make sure to record the magnification for each drawing.
Step 3: Critical Analysis: What were some of the differences you observed between your specimens? What were some of the challenges in viewing living organisms that move under the microscope? Be prepared to present your findings to the class.
Procedure
- Prepare a wet mount of a drop of pond water and place a cover slip over the drop (Figure 2.6)
- Swab the inside of your cheek
- Roll the swab across a slide
- Drop some methylene blue onto the slide
- Place a coverslip over the drop
- Visualize and draw your cheek cells
- Document your observations by drawing the cells or by using your phone to snap an image.
Assessment
- A microscope can focus on many fields of different depths. However, thick samples have to be sliced into very thin slices in order to be viewed under the microscope. Why do you suppose this is the case?
- Elodea plants appear as solid green objects to the naked eye, but appear mostly clear under a microscope. Explain this phenomenon.
- A scientist takes a sample from a pond and examines it under their microscope. He thoroughly examines the wet mount and finds no living thing on the slide. He concludes that the pond is unable to support life. Is this a valid conclusion? Why or why not?
- Before the cell theory was developed, two previous theories were prevalent as different historical theories. Explain how the invention of the microscope would lead to the replacement of both of these theories with the cell theory:
- The Miasma Theory, where diseases were thought to be spread by miasma, a poisonous, foul-smelling vapor that can travel through the air.
- The Theory of Spontaneous Generation, where living things could be generated from non-living matter, such as how maggots seem to appear spontaneously on meat left out in the open.
focuses the light from the microscope’s lamp onto the specimen
focuses the light from the microscope’s lamp onto the specimen
focuses the image onto the user’s eye
focuses the image onto the user’s eye
the ability of a microscope to distinguish two points on the image. As resolution increases, objects that are closer together appear as separate points
the ability of a microscope to distinguish an object from its background
area visible through the microscope
the proper distance between the specimen and the object
living tissues that are placed on a slide with an aqueous solution to keep them wet
preserved tissue that has been fixed and stained on a slide
area visible through the microscope
