4.1: Lab 4 Background

Learning Objectives

• Identify the name and function of the microscope parts.
• Focus the microscope in low and high power without damaging the microscope or the slide.
• Adjust the contrast of a specimen.
• Calculate total magnification of a specimen.

Introduction: Microscope Safe Usage & Handling

In biology we make many observations of the world around us using our eyes. In the seventeenth century, the invention of a more powerful microscope by Anton van Leeuwenhoek allowed scientists to see that the living world is smaller than the eye can perceive. Van Leeuwenhoek could see single celled organisms such as bacteria and protists. Using his microscope, he discovered red blood cells and studied insects and almost anything else that could be placed under his lens. Since then, biologists have improved upon this tool and continue to use it to answer questions about the smallest aspects of life.

The microscopes in our lab are very sensitive (and expensive) and need to be handled with care. Please follow the guidelines below every time we use a microscope.

Always hold compound microscopes with two hands! One hand holding the side arm, the other hand should be under the base/bottom. Never tilt the microscope while working with wet mounts (this may cause major damage to the microscope). If the microscope needs to be moved or adjusted on the table, pick it up with two hands and move it; do not try to drag it.

Microscope lenses should be cleaned every time the microscope is used. The only proper way to clean the lenses is with lens paper and the Windex solution. Never use water, paper towels, clothing, or anything else, as these may damage and scratch the lenses.

Avoid smudging the lenses by keeping our eyes a half inch away from the ocular lens, and by keeping fingers away from the objective lenses.

The compound microscopes are stored with the scanning objective in position, the power switch off, the electrical cord loosely wrapped around the base, and the plastic dust cover placed over the microscope. Each time we use a microscope, we need to write our name on the checkout sheet and indicate which cabinet our microscope was in. Match the letter/number on the desk with the corresponding cabinet. Use the key to open the cabinet to obtain the microscope, but then return the key to the wall during lab so as not to lose it. After lab, return the microscope (after cleaning it according to the instructions above) to the same cabinet it was removed from and cross off your name from the checkout sheet.

Slide Cleaning

Prepared slides must be cleaned with lens paper and be fingerprint free. Wet mount slides should be rinsed with water, dried with a paper towel, and returned to the container. Coverslips should be removed from the slide and thrown in the trash. Do not leave any slides or coverslips in or around the sink area.

Prepared slides are issued from the slide boxes at the front of the lab room. They have a label on the left side and an attached coverslip in the middle. These slides must be checked for breakage before starting the lab exercise. Report slides that have breaks in the glass slide or the coverslip over the specimen to the instructor. Do not report slides that have a crack in the coverslip if there are initials on the label. This indicates that the slide is still usable and that the crack has been reported.

Compound Microscope

There are two types of microscopes we have in class. Microscopes are pieces of equipment that allow us to see incredibly small things. A microscope will contain a lens, which is a small piece of glass that reflects light and our eye interprets the image as larger than it really is (i.e. magnified). For a compound microscope, the image of the specimen is magnified by two lenses by the time it reaches our eye. There are the objective lenses in the center of the microscope and the ocular lenses at the top that we look into. To calculate how much the image has been magnified, the objective magnification is multiplied by the ocular magnification using the formula below.

Total Magnification = Objective Magnification x Ocular Magnification

It is important to become familiar with the microscopes used in this course. Below is a list of microscope parts and their functions.

• The base of the microscope is flat and rests on the table.
• A light source is at the base of the microscope and directs light up through the specimen into our eyes.
• The power switch is on the rear of the microscope to turn the light source on and off.
• A brightness control dial changes the brightness of the light source.
• Above the light source is the stage with stage clips that hold the microscope slides in place.
• On the right of the stage are stage control knobs; one knob moves the slide forwards or backwards, and the other moves the slide right or left.
  • Always use the stage control knobs to adjust the position of the slide on the stage. Never use your hands to adjust the slide on the stage.
• Under the stage are the condenser, which focuses light onto the specimen, and condenser/iris diaphragm, which slides left and right to control the contrast and depth of field of the specimen.
  • Opening the condenser/iris diaphragm (sliding it to the left) creates a low contrast image with shallow depth of focus.
    • It may be difficult to see small details, and we will be focused on only a thin section.
    • The image will become very bright, so turn down the brightness control.
  • Closing the condenser/iris diaphragm (sliding it to the right) creates a high contrast image with a deep depth of focus.
    • Small details will be more visible, and a thick layer of the specimen will be in focus.
    • The image will become very dark, so turn up the brightness control.
    • Closing the condenser/iris diaphragm is the best way to find the specimen when we begin: the high contrast and deep depth of focus make it easier to see small objects.
• The eyepiece or ocular lens, which magnifies an image 16 times (16x), is found on the viewing tube.
  • When we look through the ocular lens, we will see a circular area with light called the field of view (see Fig. 1).
• Above the stage are 4 objective lenses on the revolving nosepiece.
  • The scanning objective with the red stripe is the shortest and magnifies 4 times (4x).
    • This is the objective we will use first to focus our specimen before switching to the other objectives.
    • This is also the objective that should be in place when we take out and store the microscope.
  • The low power objective with the yellow stripe magnifies 10 times (10x).
  • The high power objective with the blue stripe magnifies 40 times (40x).
  • The highest power objective with the green stripe magnifies 60 times (60x).
  • We should hear a click when we turn the nosepiece to switch from one objective to the next.
  • Examine Figure 1 and notice the distance between the end of the objective lens and the stage. This is the working distance.
• The arm connects the base to the rest of the microscope.
  • To carry the microscope, grasp the arm with one hand and place the other hand under the base.
• The focus knobs are on the arm. The coarse focus knob is larger and moves the stage up and down by large amounts, which is used to bring the specimen into approximate focus.
  • This knob is only used with the scanning objective!
  • We should NEVER use the coarse focus knob while the low, high, or highest power (10x, 40x, or 60x) objectives are in place. This could damage the lens and break the slide.
• Extending from the coarse focus knob is the smaller fine focus knob which is used to bring the specimen into fine focus and make minor adjustments.
  • Use this knob only when using the low power, high power, and highest power objectives.
• The power switch is on the side of the arm next to the power cord.
  • Always turn the microscope off with the power switch before unplugging it
• Tip: If you wear glasses, remove them before looking into the microscope! It may seem counterintuitive, but the point of the focus knobs is to get the specimen into focus. So, no matter what your prescription is, you can focus the specimen without your glasses.

Figure 1. A.) The working distance between the objective lenses and the stage. B.) Representation of the diameter of the field of view for each objective lens.