Cell Structure and Microscopy

Crosswhite 1 Cell Structure and Microscopy

In the biology lab, you frequently use a compound light microscope. It is called a compound scope because magnification is the result of two lenses: the objective and the ocular.

The objective lenses, located on the rotary nosepiece, provide 4 different degrees of magnification:

Name Characteristics Magnifying power

Scanning power shortest objective, red stripe 4 X

Low power next shortest, yellow stripe 10 X

High-dry power intermediate length, blue stripe 40, 43 or 45 X

Oil immersion longest, black stripe 100 X

The ocular lens, located nearest to your eye, has a magnification power of 10X. The total magnification is determined by multiplying the power of the objective by the power of the ocular. (For example, 4 X times 10 X = 40 X).

Your microscope is referred to as a light microscope because the specimen is observed using visible light. The light source is an incandescent bulb which is turned on by the toggle switch at the side of the base.

There are two ways to adjust the light on your scope. The dial on the base of your scope turns the light level from bright to dim. You can also use the iris diaphragm, just under the stage to control the amount of light that enters the condenser.

Here is a diagram of a compound scope. You will need to become familiar with the different parts and controls of the scope in order to effectively use it. Crosswhite 2

Principles of Microscopy

Resolution

As two small objects are moved closer to each other, a point is reached where the eye is unable to distinguish the objects as separate entities, and only a single object is observed. The smallest distance at which two points can be seen separately is called the resolving power of the lens, and the resolution of highest magnification lens on a light microscope is limited by the shortest wavelength of visible light (400nm).

To be able to distinguish between objects that are closer together than the shortest wavelengths of the visible light, electron microscopes must be used. Electron microscopes, which use a beam of electrons rather than light, are able to resolve very, very small objects such as structures of bacteria (like flagella) and viruses.

Depth-of-Field

The term depth-of-field refers to the vertical distance that is in focus at any one time. Higher magnification lenses have a smaller depth of field – that is, only a thin horizontal slice of your sample may be in focus at any one time. So, at high magnification, very small adjustments to the fine focus can quickly take your sample completely out of focus. Crosswhite 3 Field of View

The term field-of-view refers to how much of the horizontal are of your sample you can see at any one time. The field of view decreases with increased magnification. As you increase the magnification, you are looking at progressively smaller subsections of your sample. That is why you need to be sure to center what you are viewing before increasing the magnification.

Artifact

As you look at things under the scope, you need to be able to distinguish the things that you are supposed to observe from artifacts – things inadvertently introduced (junk).

For example, if you take a sample of your cheek cells and place them on a slide with physiological saline and stain, you will be able to examine this specimen under the scope, but there may be other things in you sample that are not check cells (such as little bits and pieces of things that you have eaten). The cells are what you want to view; the bits of your lunch are the artifacts.

Parfocal

The objective lenses on a microscope are said to be parfocal if you can change from one to another and still have your specimen in focus without having to focus more than a little. This is a very convenient characteristic of a set of lenses, because if you had to find your specimen using a high-magnification objective, it would probably take quite awhile if you had to start focusing ‘from scratch’. With parfocal lenses, if you get an object in focus at low magnification, when you switch to a high magnification lens, your specimen will still be in focus.

Cell Structure: Introduction

In this laboratory activity you will study biology from the viewpoint of the individual cell. Although the cell is considered to be the building block of all organisms, cells differ enormously in shape, size and capability. Prokaryotic cells are less complex, are usually found only in unicellular organisms and have more limited capabilities than eukaryotic cells. Multicellular organisms are made up of highly integrated aggregations of specialized eukaryotic cells, but some complex organisms consist of a single eukaryotic cell.

PURPOSE: To examine the similarities and differences between plant and animal eukaryotic cells and prokaryotic cells using microscopic evidence.

PROCEDURE:

Part A. Basic Plant Cell Structure- Onion Epidermis a. Obtain a piece of a single piece of sliced onion bulb. Snap the piece in half and, using forceps, peel a bit of tissue-like, transparent epidermis from the inner layer. Mount the piece of epidermis in tap water on a glass microscope slide. Be certain that it is flat and not doubled over itself. Add a coverslip and gently press it down with a pencil eraser to remove as many air bubbles as possible. b. Observe the specimen under low power. If you see a confusing mass of overlapping cell parts, you probably do not have a good piece of epidermis. Scan your slide for a single layer of cells that resembles a brick wall. If you can not find such a view, then prepare another slide. Crosswhite 4 c. As you view onion epidermis cells, note that the cell membrane is tight against the cell wall and too thin to be seen with a light microscope. Note also any signs of nuclei. d. Remove the slide from the microscope. Add a drop of iodine stain to the slide immediately next to the coverslip but not on top of it. With a small piece of paper towel, draw the stain under the coverslip by placing the towel on the opposite side of the stain and touching the water. This action should wick the water out from under the coverslip and draw the stain in. e. Observe the epidermis cells again under low power. Note that the cells are now colored, with the cell wall, nucleus and nucleoli stained more darkly than the cytoplasm. Try to distinguish between the cytoplasm and the large central vacuole. f. Observe the cells under high power to help you better distinguish cell structures. g. Make a sketch of a few onion cells. Label the cell wall, cell membrane, cytoplasm, central vacuole, nucleus and nucleolus.

Elodea a. Break off a single leaf near the tip of an Elodea plant and prepare a wet mount. Position the slide so that you are observing the edge of the leaf near the tip. Observe the cells both under low power and high power. If you are viewing at the edge of the leaf you should see only one cell thickness. You should also see two types of cells. One type of cell has a smooth margin facing the environment; the second type, called a spike cell, has a projection out into the environment. b. The thick cell walls and the green chloroplasts are the most conspicuous structures. Sometimes a shadow of a nucleus can be seen. If you are patient and fortunate, you may see movement of the chloroplasts around the periphery of the cell. This movement, called cyclosis, is caused by cytoplasmic streaming. c. Make a labeled drawing of a few Elodea cells to demonstrate your observations. LABEL cell wall, cell membrane, cytoplasm, nucleus, and chloroplast

Part B. Basic Animal Cell Structure- Cheek Cells a. Make a wet mount of cheek cell epithelium by gently scraping the inside of your cheek with a toothpick and transferring the material to the drop of water on the slide. b. Place a coverslip in the edge of the drop of water and gently lower the coverslip down on the slide. If done slowly and carefully, there will not be air bubbles under the coverslip. If air bubbles are present another preparation should be made. Observe the cells under both low and high power. c. Make another wet mount and stain with methylene blue. Do this by placing the stain on one edge of the cover slip and drawing it through with a piece of paper towel held at the other edge of the cover slip. d. Make a labeled drawing of a few check cells to demonstrate your observations. LABEL cell membrane, cytoplasm, and nucleus.

Part C. Bacteria a. Obtain a prepared slide of bacteria. Since these prokaryotic cells are so small you will have great difficulty seeing them, even under high power. While searching for the specimens use the color of the stain to help you locate them. Crosswhite 5 b. After locating and observing the bacteria, observe them using high power. c. Observe that the stained bacterial cells are of three major forms, spherical-shaped (called coccus), rodshaped (called bacillus) and spiral-shaped (called spirillum). d. Sketch a few bacterial cells, showing the three major structural forms. Label the Types.

Note: All drawings MUST be done in the following manner: a. On a piece of blank white paper, draw the specimen as seen under the microscope. b. The drawing must be a minimum 4cm x 4 cm in size. c. Beneath the drawing label the picture identifying the cells and give the magnification, such as "Cheek Cells - 40X" OR Onion Cells with Iodine-100X." d. LABEL all identifiable organelles by printing horizontally. Use a ruler to draw a line to the object. The line MUST TOUCH the object labeled. e. Remember that detail is important in microscopic drawings.