Microscopes and Cells

TOPIC 2

MICROSCOPES AND CELLS

Objectives:

1. Identify the components of the compound light microscope and know the functions of each. Be able to use the microscope to find and to maintain the image of an object.

2. Understand the following terms: inversion, resolving power,magnification and field diameter.

3. Be able to prepare wet mounts of plant and animal cells. Be able to identify or explain the differences between them. Apply the concept "structure is related to function" to different types of cells.

4. Understand the function of biological stains in microscopy, and be able to apply stains properly to enhance an image.

5. Understand what causes "plasmolysis".

6. Prepare a stomatal peel, and recognize the structure and function of stomates.

7. Understand the function of biological stains in microscopy, and be able to apply stains properly to enhance an image.

8. Prepare a stomatal peel, and recognize the structure and function of stomates.

9. Be able to create a bar graph using Prism.

Pre-lab Questions:

1. Convert 27.5 micrometers to centimeters. Show work below.

Give your answer in scientific notation!

Note: the quiz may give you a different value to convert.

2. Which of the following lengths is the largest? Explain your answer.

a. 12.5 cm c. 0.125 m

b. 125 mm d. 125,000 µm

3. Of the following objective magnifications, which would you use to get the largest field diameter and why? Which would give you the smallest?

a. 4X c. 40X

b. 10X d. 100X

4. What is the function of a “stain” in microscopy?

Web Resources:

One website provides a theoretical overview of how metric system conversions are performed. The other site provides multiple choice questions and answers for conversions using length, mass, and volume.

Introduction:

There are two general types of microscopes used by scientists in the modern biology laboratory: light and electron. The major difference between the two is based on the kind of energy used to illuminate the object. Light microscopes use visible light passing through lenses which produces a magnified image seen by the eye. Electron microscopes send a stream of electrons to bombard the object. Magnetic lenses are able to focus the pattern of reflected and transmitted electrons onto a photographic plate or television screen.

Compound Light Microscope:

This is called "compound" because it contains at least two glass lenses. The lens nearest your eye is called the ocular or eyepiece, and usually has a magnification power of 10X. The lenses closest to the object are called objectives and have varying magnifying abilities. The amount of magnification is printed on the lens itself. Total magnification is the product of the ocular and objective magnifications. Figure 1 shows the major parts of a compound microscope.

Figure 1: Generalized compound light microscope.

Magnification and Resolving Power:

Microscopes have two purposes: to magnify and to resolve images. Magnification alone only makes small objects appear large. Resolving power (or resolution) measures how clearly you can see details of the image. It is usually defined as the ability to view closely adjacent objects or structures as distinct images instead of a fuzzy blur.

The formula for determining resolution is:

RP= 0.6l

n sin a

Most of these details are unnecessary for you to know because the numbers they represent are constant for any type of compound microscope. Notice that resolution is directly related to l, the wavelength (or color) of light used to illuminate the object. The human eye can only see light from 450 nm- 700 nm (see Figure 2). Therefore the resolution of the compound microscope is limited by the human eye, and is approximately 0.23 mm.

Electron microscopes are powerful tools in biology because they use electrons for visualization of the object. Electrons can have a wavelength of up to 0.0037 nm, and therefore have a resolution of 0.2 nm. Remember, this means that electron microscopes may be able to distinguish between two objects that are only 0.2 nm apart.

Purple- Blue Green- Yellow Orange- Red- Far red

Figure 2: The electromagnetic spectrum showing visible wavelengths. Visible spectrum shown in nanometers (nm).

Focusing:

·  On the microscope, click the lowest power objective into place. Raise the objectives as high as they will go using the coarse adjustment knob.

·  Place the slide on the stage, and clip it into place. The E should be oriented so that it is right-side up to you. Using the control knobs, move the E so that it is in the center of the light coming up from below.

·  Viewing the microscope from the side, lower the 4X objective by the coarse adjustment until it is just above the slide.

·  Look through the lens and slowly turn the fine focus knob until the image comes into clear and distinct view. Does the image appear normal or upside-down? Sketch the image as it appears.

·  Move the slide to the right. Which way does the image appear to move?

Inversion refers to the fact that the image is not only flipped, but also reversed.

·  Make sure the E is centered in the field. Move to the 10X objective. Do not adjust the coarse adjustment first.

·  The E should be visible. Ask your instructor if you can’t find it. If any adjustment is needed, use the fine focus knob only. Can you still see all of the E with the 10x objective?

·  Change to the 40X objective. Is all of the E still visible? Did you have to make any dramatic adjustments to the focus?

Compound light microscopes are parfocal. This means that once the object is in focus under low power, it should remain mostly in focus as the objectives are changed.

Field Diameter

Field diameter describes the area you see when looking into the microscope. Field diameter will change as the magnification is changed.

·  Place a transparent ruler on the stage of the compound microscope. Measure the field diameter at 40x and 100x magnification. Record your data in cm and then convert this value to mm and mm (micrometers). If you have problems with the conversions, there is a practice sheet at the end of Topic 2, or use the online resources on the lab webpage.

Field diameter at 40x in cm: in mm: in mm:

Field diameter at 100x in cm: in mm: in mm:

What is the relationship between magnification and field diameter?

Cell Observations:

Often it is necessary to prepare a specimen for observation. In such cases, the object should always be placed in water, forming a wet mount. A wet mount is prepared by placing a drop of water on the clean slide, and placing the specimen in it. Cover the water and specimen with a coverslip.

Living Animal Cells (Human Epidermis):

·  Gently scrape the inside of your cheek with a toothpick and place the scrapings in a drop of water. Cover with a coverslip.

·  Scan for the cells under 40x total magnification. When you find them, raise the power to 100x and 430x. Adjust the iris diaphragm to get the optimum amount of light.

·  Can you clearly see the nucleus? Staining is required to add contrast to the thin and tranparent epithelial cells. Prepare a new slide as before, but this time add a tiny amount of methylene blue to the water and cell mixture. Observe as before. Sketch the cells, and label the nucleus.

Living Plant Cells:

·  Elodea is a common plant of freshwater aquaria. It is an ideal material to use for viewing plant cells because the leaves are only a few cells thick.

·  Place a single leaf in a drop of water on a clean slide, and cover with a coverslip. Do not use stain. Scan at 40x total magnification, and focus on cells near the edge of the leaf. Raise the magnification up to 400x to view details of the cell. Sketch some Elodea cells.

·  Label the chloroplasts and the cell wall.

The chloroplasts are where light is harvested to make carbohydrates for the plant. It appears as though the chloroplasts are attached to the cell walls. This is not true. In fact, the interior of each plant cell is taken up by a large sac, called a vacuole. This is so large that it pushes all other parts of the cell up against the cell wall. You may see some chloroplasts moving around the edge of their cell. They are moving between the vacuole membrane and the cell wall/membrane.

·  Add several drops of concentrated salt solution to the Elodea leaf. Observe the leaf under 400X total magnification.

·  Sketch the new appearance of the cells after the salt solution

Observing and quantifying Stomata:

Stomata are the tiny openings in the epidermis of terrestrial (land) plants (Fig 3). Gases, primarily oxygen and carbon dioxide, can pass through these openings to allow for photosynthesis to occur. Likewise, water will evaporate out of the stomata, leading to potential dehydration.

Stomata typically remain open during the day and close at night, when photosynthesis doesn’t occur, allowing for water conservation. Stomata can also close during especially hot days, during droughts, or as a response to plant growth regulators. Desert plants have a special form of photosynthesis which allows them to open their stomates at night, to perform gas exchange, and close them during the day to conserve water.

Today we will observe and count the stomates on two different types of plants: a monocot and a fern. Monocots are a type of flowering plant which appeared approximately 150 million years ago. Ferns are not flowering plants at all, they actually appeared on Earth about 200 million years before the first flowers. Nevertheless, ferns do have stomates which perform essentially the same functions as in other plant groups.

Fig 3: Stomatal openings in the underside of Polypodium. Seven stomates are visible in this section.

The number, shape and size of stomata will vary from species to species and leaf to leaf! Today we will use a simple technique to observe and quantify the number of stomata found on leaves of different species.

Procedure:

·  You should obtain leaves from a monocot, a dicot and a fern.

·  Using a small amount of clear nail polish, coat the underside of each leaf with a THIN layer. Set the leaves aside for 30+ minutes to dry. Coat the upper side of three additional leaves in the same way.

·  Gently peel away the nail polish, and it will contain a perfect imprint of the cells.

·  Do not use water with these samples!

·  Find the leaf cell imprints at 100X and then move the objective to 400X for counting.

·  For each species, identify the epidermal cells and the stomata. Fix the field of your microscope in place and count the number of stomata visible. Stomates on the edge of the field count! Move the field of view to a new position on the leaf and repeat the procedure. Then do a third count in a new position.

·  Repeat the procedure for each species and each surface, and record your data in Table 1.

Table 1: Number of Stomates observed by surface and species.

Species Surface Number of Stomates

View 1 View 2 View 3

______

upper

lower

upper

lower

upper

lower

This lab was written and revised by:

Tony Botyrius M.S.

References:

Electromagnetic spectrum modified from: http://www.yorku.ca/eye/spectrum.gif

Microscope Image. http://www.towson.edu/~cberkowe/medmicro/315lab1.html.

Stomate Image.

http://acces.ens-lyon.fr/acces/equipes/dyna/travaux/evolbio/phylovegetal/ressources-iconographiques-pour-les-collections-phylogene/photos-lejamble/Polypode%20stomates%20vue%20gle.jpg/view

Metric System and Conversions

In order to easily convert one metric measurement into another, it is essential to understand the metric system divisions and how they relate to the basic unit: the meter, the liter, and the gram. The table below gives this information, and the most often used values are written in bold.

Prefix Division of Metric Unit Scientific Notation

giga (G) 1,000,000,000 109

mega (M) 1,000,000 106

kilo (k) 1,000 103

hecto (h) 100 102

deka (da) 10 101

deci (d) 0.1 10-1

centi (c) 0.01 10-2

milli (m) 0.001 10-3

micro (m) 0.000001 10-6

nano (n) 0.000000001 10-9

pico (p) 0.000000000001 10-12

The most common units used in Biology 1 are the milli-, centi-, and micrometers. They relate to each other in the following way.

Meter

Gram

Liter Centi Milli Micro Nano

1 100 1000 1,000,000 1 x 109

0.1 10 100 100,000 100,000,000

0.01 1 10 10,000 10,000,000

0.001 0.1 1 1000 1,000,000

To convert from one unit to another, you will have to use conversion factors (how many of one unit goes into the other unit). For example, in the conversion of 472 grams into kilograms, it should be clear that you are changing into a larger unit, and therefore the numerical value should be smaller, since you can’t have a larger number of kilograms than grams.

So, to convert set up an equation as below.

Value to Convert Multiplied by Conversion Factor

472 grams x 1 kilogram = 0.472 kg

1000 grams

(Note that grams cancel out of this equation, leaving kilograms)

Of course, it is also possible to go from a larger unit to a smaller one. In this case, your numerical answer should be larger than what you begin with.

Convert 5.5 liters to milliliters.

5.5 L x 1000 ml = 5500 ml

1 L

Convert 15 micrograms to milligrams

15mg x 1mg = 0.015mg

1000 mg

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