Lab 1. Care and Use of Compound Microscope

OBJECTIVES

• Learn how to carry, use, care for and store the compound light microscope.
• Identify and explain the function of the parts of the compound microscope.
• Prepare wet mount slides.
• Understand the terms used in this lab: compound light microscope, stereo or dissecting microscope, objective lens, scanning objective lens, low power objective lens, high-and-dry objective lens, ocular lens or eyepiece, arm, stage, coarse focus adjustment knob, fine focus adjustment knob, condenser, iris or aperture diaphragm lever or control, parfocal, field of view, working distance, light intensity, depth of field, total magnification, cover slip, micrograph.
• Observe some interesting specimens or structures.

PRELAB

Before you come to lab,

1. Read chapter 1, BIOL 160 Lab Information (parts 1-4). This chapter will inform you about your lab responsibility, lab safety, and lab notebook criteria.
2. Read Lab 1 and be ready to begin the exercises. Make sure that your lab notebook is ready for entering the information gained from this lab. In your lab notebook, prepare the Table of Contects (TOC), then on separate page write the title of the lab, date, purpose, and procedure (flow chart or outline).
3. Understand the following terms: compound microscope, dissecting microscope, objective lens, scanning (4X) objective lens, low power (10X) objective lens, high-and-dry (40X) objective lens, ocular lens or eyepiece, arm, stage, coarse focus adjustment knob, fine focus adjustment knob, condenser, iris or aperture diaphragm lever or control, field of view, working distance, light intensity, depth of field, total magnification, cover slip, micrograph. You do not need to define these in your lab notebook!

INTRODUCTION

We humans are highly visual creatures, obtaining much of our information about the world around us by using our eyes.  Not surprisingly, some of the most useful tools in biology are those that allow us to visualize objects, processes, and phenomena.  Powerful computers today allow us to visualize and explore as never before.  But even that technology cannot replace one of the most fundamental tools of biology, the microscope.  With the microscope we can see things that are too small for the naked eye.

There are many different kinds of microscopes.  The one we will be using will be the compound light microscope (brightfield microscope).  In this lab we explore how to use and care for this most basic of biological tools.

General Procedures:

1. Watch the microscopy video from Fresno State: https://www.youtube.com/watch?v=SUo2fHZaZCU
2. Record your work in your Lab Notebook. Do not copy these general procedures in your lab notebook.
3. Work in pairs.

• • If you do not have experience with a compound microscope, pair up with someone who does.
  • If you have experience with a compound microscope, pair up with someone who does not.
  • Please pay careful attention to how you use and care for these instruments. Failure to properly handle the microscopes may lead to a deduction of points from your lab score.  Microscopes are delicate and expensive optical instruments and must be handled properly and carefully.  Prepared slides are also expensive and must be used thoughtfully as well.
  • Do not keep your coats, bags, and other personal belongings (items not used in the lab) on the lab table. You will need room on the lab bench for your lab materials, lab manual, notebook, and microscope

3. Calculating magnification:

• • Multiply the power (magnification) of the eyepiece by the power of the objective lens to obtain the total magnification.
  • The power of the eye piece is 10x.
  • The power of the objective lenses is written on them. It is usually (but not always) followed by an “x”, and is an integer, NOT a decimal number.  An example would be “4x”.
  • Always include in any sketches the total power or magnification under which you observed the specimen. Always include the “x” as part of the magnification (e.g., 10x, 100x, 400x).
  • Note that in BIOL& 160, we will not use the 100x, or oil immersion, lens.

Please copy (or paste) this table into your Lab Notebook and fill it out:

Use lens paper ONLY to clean ocular and objective lenses!

5. When finished, be sure that:

• • the scope is turned off.
  • all slides are removed and returned to their proper container. Prepared slides should be placed in their proper container in the correct orientation.  Slides used for wet mounts should be rinsed clean and put in the dirty slide container.  Cover slips should ONLY be put in the broken glass box.
  • the stage is wiped clean.
  • the scope is set with the lowest power (scanning) objective in place.
  • the stage is lowered (or objectives raised) as much as possible. In other words, there should be the maximum amount of space between the stage and the objectives.
  • the cord is properly wrapped and tied with a rubber band, making certain that the cord is snug and not dangling.
  • the cover is replaced (if it had one).
  • the scope is returned to the proper microscope cabinet. Make certain that the microscope cord is not dangling, and the microscope is well back from the edge of the shelf.

7. If your microscope does not work, tell your instructor, or the lab technician, or put a note on it indicating what you think is wrong with it.
8. If you find a slide on your microscope stage, please give it to your instructor or place it in the lost slide box.

PROCEDURES

A. Obtaining Your Microscope

1. Always carry the microscope in an upright position. The ocular lenses slide into the body tube and can easily fall out, and slides left on the stage can also fall to the floor if the microscope is tilted. With one hand, grasp the arm of the microscope and place the other underneath to support the base. Be patient while you are waiting for students ahead of you to get their microscopes and do not get in the way of individuals carrying microscopes.
2. Carry your microscope carefully to your lab table. Place the microscope on the lab table, making certain that it is several inches from the edge. (Never place a microscope close to the edge of a table, counter or shelf.)
3. Remove the dust cover and set it aside (in the cabinet under the lab table). Unwrap the cord and put the plug through the rubber band before you plug in your microscope (so that you will have the rubber band there when you are finished). Plug in your microscope, making certain that the cord is out of the way and does not loop down on either side of the lab table.  (The cord must never be positioned so that it could catch on anything that would send the microscope crashing to the floor.)

B. Microscope Parts and Function

1. Leave the microscope on low (scanning, 4X) power – that means having the smallest lens in place. It should already be set this way. What magnification is this? (refer to the table above)
2. Determine the answers to the following questions regarding use of your microscope. For the “where” questions, we want you to find the actual location (be able to point to it). You do not need to write the answers to these questions in your Lab Notebook.
   1. How do you turn the microscope on?
   2. What part of the microscope does the light come from?
   3. Where do you look into the microscope?
   4. How do you change the distance between the two lenses that you look into?
   5. Where do you put the slide?
   6. How do you focus?  (Where are the focus knobs?)
   7. What happens on the microscope when you focus (what moves)?
   8. What do the two focus knobs do differently?
   9. How do you increase the amount of light?  How do you decrease it?
   10. How do you move the slide around without touching it?
   11. Does your microscope have a pointer in one of the eyepieces?

C. Higher magnification – Colored Crossed Threads Slide

1. Obtain a “Colored Crossed Threads” prepared slide (from slide boxes).
2. Place the slide on the microscope stage.
   • First observe the slide under the lowest magnification (using the scanning 4X objective). Center the slide on where the threads are crossed. Focus first on any thread (think about the following questions; you do not need to write the answers in your lab notebook).
   • What is the total magnification?
   • Are all three threads simultaneously in focus?
   • If you move the slide to the right, which way does the image in the microscope move?
   • If you move the slide up (away from you), which way does the image move?
3. Increase magnification (power) to the next strongest (this is called the “low power” 10X objective). Center your field of view on where the threads are crossed (think about the following questions; you do not need to write the answers in your lab notebook).
   • What is the total magnification?
   • Are all three threads simultaneously in focus?
   • Change the focus using ONLY THE FINE FOCUS ADJUSTMENT; focus in and out. What do you observe?
   • After you increase magnification, why should you use only the fine focus?
   • After you increase magnification, is the image still in focus?
   • Is it as bright as it was under lower power?
   • What are the names of the parts of the microscope that change the magnification?
4. Now increase the magnification one more time, to the next strongest (this is called the “high power” or “high dry” 40X objective).  Refocus using the fine adjustment knob (think about the following questions; you do not need to write the answers in your lab notebook).
   • What is the total magnification?
   • Change the focus (remember, use only the fine focus knob!): focus in and out. What do you observe?
   • How would focusing in and out help you to determine the three-dimensional structure of a specimen?
5. Make one sketch of the crossed threads slide, at one of the three magnifications, in your lab notebook. These sketches should have a circle around them to indicate your field of view, should be labeled with the name of what you are observing, and should indicate the total magnification underneath, like this:

Title: _______________

Total Magnification: ________________

D. Wet Mount Preparation – Letter e

Prepare a “wet mount” slide of a newspaper letter “e” and observe under the microscope.

1. Obtain a clean glass slide (you may want to clean it again yourself with a lab tissue or Kimwipe).

Hold the slide by the edges to avoid smudging.

2. Put one drop of water on the center of your slide.
3. Wet the tip of a cotton swab and touch it to a cut-out letter “e” from the newspaper. The letter will stick to the swab.
4. Touch the “e” to the drop of water. It should float off. Be sure it is face-up now.
5. Obtain a cover slip. To avoid trapping air bubbles when you place the cover slip on the slide, hold the cover slip at an angle and place one edge of the cover slip at the edge of the drop of water (see figure). Allow the water to “wick” along the edge of the cover slip before slowly lowering the cover slip.
6. Place your wet mount slide on the microscope stage (microscope should be set to the lowest magnification, 4X objective) and focus on the “e.”
7.  Increase magnification (power) to the next strongest (the “low power” 10X objective) and focus on the “e”. Is the image of the “e” right side up or upside down?
8. Now increase the magnification one more time, to the next strongest (the “high power” 40X objective) and observe the “e”. Remember – FINE FOCUS ONLY!
9. Make three (3) sketches of the “e”, one at each magnification, in your Lab Notebook. These sketches should have a circle around them to indicate your field of view, should be labeled with the name of what you are observing, and should indicate the total magnification like this:

Title: _______________

Total Magnification: ________________

E. Prepared Slides of Choice

1. Obtain a box of prepared slides for the BIOL&160 course.
2. In your lab notebook sketch 2 different prepared slides. Do not forget to include titles of specimens/structures and the magnifications. Sketch at a magnification that allows you to see the most detail in your organism. 

Title: _______________

Total Magnification: ________________

Title: _______________

Total Magnification: ________________

F. Definition of Terms

1. “Field of view” The field of view refers to the area that you see when you look through the microscope. As you observe the “letter e” using the three different objective lenses, note how the field of view changes with magnification.
2. “Light intensity” Light intensity refers to the amount of light that you see when you look at the specimen through the microscope. The light intensity changes with magnification.
3. “Contrast” Contrast refers to the degree of difference between dark and light areas of the specimen. High contrast is typically what you strive for, because high contrast brings out detail.
4. “Working distance” Working distance refers to the distance between the tip of the objective lens and the cover slip on the slide. The working distance also changes with magnification.
5. “Depth of field” Depth of field refers to the vertical distance (the thickness or depth) that is in focus at the same time. The depth of field also changes with magnification.

CLEAN UP:

1. When finished, remove the cover slip from the slide and put it in the Broken Glass container.
2. Clean up the microscope and prepare it to be put back in the cabinet, as instructed earlier in this lab. Be sure that the scope
   • is turned off.
   • all slides are removed and returned to their proper container. Prepared slides should be placed in their proper container in the correct orientation. Slides used for wet mounts should be rinsed clean and put in the dirty slide container (or in broken glass box).  Cover slips should ONLY be put in the broken glass box.
   • the stage is wiped clean.
   • for the compound microscope, the scope is set with the lowest power objective in place, and the stage is lowered (objectives raised) as much as possible, so that there is the maximum distance between the stage and the objective.
   • the cord is properly wrapped and tied with a rubber band, making certain that the cord is snug and not dangling.
   • the cover is replaced (if it had one).
3. Return the scope to the proper microscope cabinet. Make certain that the microscope cord is not dangling and the microscope is well back from the edge of the shelf.
   • Replace all other materials (slides, posters, etc.) to their proper location.
   • Replace all chairs under the lab benches.
   • Clean and dry your lab bench (using bench cleaner and paper towels).
   • Remove any debris from the sink(s) and put in the garbage.

THANK YOU FOR LEAVING THE LAB NEAT AND CLEAN!