LABORATORY #5: The Microscope and Introduction to the Cell

Required Materials:

  1. Light microscope
  2. Dissection microscope & flower parts
  3. Prepared slides of :
  4. Neisseria gonorrhea - cocci
  5. Treponemapallidum - spiral
  6. Mycobacterium tuberculosis - bacillus
  7. Nostoc – filamentous/colonial cyanobacteria
  8. Human blood smear
  9. Toothpicks for cheek smear
  10. 0.3% methylene blue
  11. Plain slides & cover slips
  12. Pond water & eye dropper
  13. Methyl cellulose
  14. Lens paper
  15. Oil

Introduction

This laboratory assignment introduces you to some of the microscopes used in biology. In your lecture, you learned about the basics of microscopy and the major types of microscopes. Unfortunately, we do not have access to a transmission electron microscope or a scanning electron microscope, but you will become familiar with the simpler (and cheaper) light microscope and dissecting microscope.

Most modern light microscopes use two sets of lenses (i.e. a compound light microscope) to focus and magnify the light. The first set of lenses is the objective, which usually comes in different powers (5X very lower power, 10X low power,20X medium power, 40X high dry & 100 high oil). The microscopes you will be using have 4X, 10X, 40X and 100X objective lenses.The second set is known as the ocular. This usually has a power of 10X. So the actual magnification power is a combination of the two lenses.

A. Identification of parts:Take a look at the microscope on your table and the labeled picture at the end of this lab. Identify these listed parts.

PARTFUNCTION

In-base illuminatorLight source

Iris Diaphragm levelAdjusts the amount of light that passes through the subject

Condenser lensFocuses the light on the subject

StageLocation of the subject

NosepieceHolds and rotates the objective lenses

Objective lensMagnifies the subject based on the power of the lens

Field pointerIdentifies object of interest to a second viewer

Ocular (eyepiece)Contains a 10X power lens

Coarse adjustment knobMoves the stage up and down to coarsely focus on the subject

Fine adjustmentAlso moves the stage to more precisely focus on the subject

B. Focusing: Use your letter ‘e’ slide for the following steps.

Use the following steps to focus the microscope and to increase the magnification on your subject

  1. Raise the nose piece using the coarse adjustment knob and clean the lenses of your objectives with lens paper AND ONLY lens paper to prevent scratching
  2. Place your slide on the stage with the subject centered over the condenser lens (hint: use the location of the focused light to center)
  3. Rotate the nose piece until the lowest power lens is in place. Using the coarse adjustment, lowly move the nose piece down until you see the subject come into approximate focus.
  4. Adjust the iris until the light is correct for viewing the subject. As you increase in lens power, you will need to allow more light to pass through the subject
  5. Use the fine focus to bring your subject into clear focus
  6. Increase you magnification on your subject by rotating the nose piece until the next highest power lens ‘snaps’ into place. Adjust your fine focus with each change of lens power. Observe your subject at 100X and 400X magnification using the 10X and 40X objectives
  7. Replace the “e” slide with that containing three strings and practice focusing on each of these strings.

C. Resolution:

Resolution is the ability of a microscope to distinguish small objects. It is given as a value representing the smallest distance between two points at which they are still distinguishable. Scopes with the highest resolution power have the smallest resolution distances. Resolution is directly related to the wavelength of the illuminating source (i.e. light or electrons) and it is inversely related to the numerical aperture of the lens (i.e. the solid cone of light delivered to the subject and received by the lens.

d = wavelength

2Xnumerical aperture

D. Inversion: Use the slide of the letter ‘e’ for the following

The image of a subject changes when viewed through a compound light microscope. How?? Record your observations below

E. Magnification:

In a compound microscope, magnification is done by the objective lens and the ocular lens. To determine magnification, multiply the power of the two lens together.

Determine the total magnification that the lenses on your microscope would provide. In the space below, list the objective lens and the total magnification associated with each one.

F. Diameter of field: Use a prepared millimeter slide for the following

The field of a scope is the illuminated are seen when an observe looks into the ocular lens. This area is the shape of a large circle. The diameter of the field is any distance which goes across this circle and passes through the subject. If the length of the diameter is known, it can be used to estimate the size of the subject within it. The diameter of field is larger when using lower power objective lenses. In other words, the field diameter decreases as you increase the power of the lens. The amount the diameter of field decreases when using high power lenses is the same as the amount the magnification increases. So if your magnification increases ten-fold, the diameter decreases ten-fold.

low power magnification = diameter field high power

high power magnification diameter field low power

Measure the diameter of field using the ruler slide under 10X, 40X and 100X lenses and record it below.

OBSERVING CELLS:

The Cell Theory states that all living things are composed of cells. During this lab, you will observe an array of different cells types and some of their organelles. From this lab, you should learn that there are a wide variety of cells in many diverse forms.

Record all your scientific observations in your lab notebook. For some cells, you may want to draw what you see. Also, taking photos through the ocular with your smartphones are also possible in most cases. Use these photos to help supplement your observations when studying at home.

  1. Use a pencil for all drawings
  2. Drawing should be large enough for you to add pertinent details
  3. Be sure to properly label all your drawings

Types of cells

Although cells come in many different sizes and shapes, there are a few general categories. There are type types of cells in nature: Prokaryotic (lacking a nucleus and membrane bound organelles) and Eukaryotic (having a nucleus and organelles).

To study these cells, you will use two types of microscope preparations in this course: prepared slides and wet mounts

G. Wet mount: Use the provided pond water and prepare a wet mount by doing the following:

To prepare a wet mount, use a plain slide, water and a coverslip. Place the subject on the slide and add a drop of water on top (if necessary). Place the edge of the coverslip at the edge of the water and slowly lower the cover slip down on the subject. Go slowly to prevent bubbles! To add a substance to the preparation, add it to one edge of the cover slip and remove some water through capillary action by placing a paper towel at the opposite edge of the cover slip. This will draw your substance under the coverslip and over the subject.

Make a wet mount with some pond water and add methyl cellulose to your prep. If you are having difficulty drawing the methyl cellulose under your coverslip, you may add the substance directly to you wet mount and then add the coverslip. But be careful not to add too much methyl cellulose. This laboratory may also provide you with a microscope slide with a shallow depression in the center. You may add your pond water and methyl cellulose to this depression and cover it with a coverslip.

Observe your prep under the microscope using several objective lenses. Record your observations in your lab notebook. Draw some of the subjects you observe in your notebook and guess what they might be. Using your diameter of field measurements for each of your lenses, estimate the average size of some of the objects that you see.

H. Prepared slides: Use the provided prepared slides or prepare the indicated wet mounts to study various prokaryotic and eukaryotic cells

  1. Prokaryotic cells - Eubacteria: Observethe prepared slides of bacteria in class under the 40X and 100X oil immersion lenses. Draw and label what you see in your field of view with respect to cell shape and size.
  1. Prokaryotic cells - Cyanobacteria: For the Nostoc slide, there are two distinct cell types present. Can you observe them? Identify these two cell types in the drawing you made in your lab notebook.
  1. Eukaryotic cells:
  1. Animal cells:

i. Human bloodsmear: Observe the prepared slide of a human blood smear under the 40X and 100X oil immersion lenses Draw and label what you see in your field of view with respect to cell shape and size.

ii. Human cheek smear: The cells on the inside of your cheek are epithelial cells. They are flat, pancake-shaped cells stacked into successive layers, resulting in a tissue called stratified. This stratified epithelial tissue is similar to that found in the epidermis of your skin, with one important difference. The cells on in the inside of your cheeks do not form a water resistant protein called keratin. This means that the cells you scrape from the inside of your cheek are alive.

To prepare a human cheek smear: Obtain some of these cells by gently scraping the inside of your cheek with a wooden applicator or toothpick. Add 1-2 drops of water to a slide, then add the cheek cells to the water by swirling the toothpick in the water. Add a drop of 0.3% methylene blue to the cell suspension. Carefully add a coverslip. Observe the cells using your 40X or 100X objective lenses.

  1. Plant cells – Elodea: Elodea is a multicellular aquatic plant. The leaves of this plant exhibit the general characteristics of plant cells, including a cell wall (for support), a large, central vacuole (for water storage) and many small chloroplasts (for photosynthesis). If present in the lab, prepare and observe a wet mount of a leaf of Elodea. Identify, draw and label the cell wall, central vacuole and chloroplasts (if observable).

USING THE OIL IMMERSION LENS:For each prepared slide, observe your samples using the 100X oil immersion lens. To use the 100X oil immersion lens, rotate the nose piece so that the subject is in between the 40X and 100X lenses. Place a small drop of immersion oil on the coverslip over the subject and rotate the 100X lens into position. The oil will increase the resolution of the subject when at higher magnifications. AFTER YOU ARE DONE, CLEAN THE SLIDES CAREFULLY WITH SOAP AND WATER. DO NOT CLEAN THE OIL IMMERSION LENS!!!!

J. Dissecting scope: Use the provided flower parts

The dissecting scope is for low magnification of larger objects. It is a compound scope, but its magnification varies from 15X to 30X. Obtain some flower parts and place them under the dissecting scope. Using trial error, determine how to use the lights and lenses on this scope. Compare and contrast the images of the light microscope with that of the dissecting scope.

Worksheet exercises

Using the following key, identify what you can from the electron micrographs of the plant and animal cell given below

  1. Nucleus
  2. Nuclear envelope
  3. Mitochondria
  4. Plasma membrane
  5. Cell wall
  6. Central vacuole
  7. Rough endoplasmic reticulum
  8. Chloroplasts

At the end of this lab, please clean up as follows:

  1. Clean all glass microscope slides with soap and water. Dry and return them to the box.
  2. Discard the plastic coverslips in the trash
  3. Return the microscope as follows:
  4. Ensure the objective nosepiece is in the empty position
  5. Ensure the stage is at its lowest position
  6. Ensure the light level is at its lowest level
  7. Blot off excess oil from your oil immersion lens using LENS PAPER ONLY!!!!
  8. Return your prepared slides to the correct trays