Exercise 4: The Light Microscope

Introduction

Bright-field microscopy uses light that is transmitted through a specimen to produce an image. The specimen causes diffraction of the light transmission and appears as a translucent object against a bright background. An unstained specimen is very difficult to see under these conditions. Chromogenic stains can be used to assist in observing the specimens providing color and improving contrast against the background.

Our microscopes are compound, using two lenses to magnify the image viewed. The process begins with a condenser concentrating the light from a light source (bulb) making illumination of the specimen more uniform. As light passes through the objective lens, bending of the light passing through the specimen (refraction) produces magnification of the image. The image is magnified a second time as it passes through the ocular lens producing a virtual image of the specimen. The clarity of the image produced is referred to as resolution. Resolution in bright-field microscopy is limited to approximately 0.2 um. This resolution is sufficient to see bacteria which are typically 1 – 5 um. Any smaller structures will be blurry or undetectable. The amount of magnification that each lens produces is marked on the lenses. The total magnification of the specimen image can be calculated by multiplying the power of the ocular lens (10X) times the power of the objective lens in use: Total Magnification = Ocular X Objective. Your microscope has 4 objective lenses. See table 4-1 for details.

Table 4.1:

The objective lenses on your scopes are parfocal meaning that you can maintain coarse focus when switching between the lenses provided that you do NOT move the focus knobs. The scanning 4X lens (red) is best suited for viewing relatively large eukaryotic specimens such as helminths (worms). It is not effective for viewing bacteria, so students should switch to the next highest objective to begin focusing on bacterial slides. The 10X objective (yellow) will work with all the specimens you will observe during the course, and therefore is the best lens to start viewing your specimens. Use the coarse focus knob to adjust your focus in low power. Bacteria will still look like tiny grains of sand at this magnification (100X total). Once in focus in low power students progressively increase magnification by switching to the next highest lens and adjust the fine focus knob until they have achieved sharp focus. As students switch to higher power lenses the tip of the lens will get closer to the slide. This space between lens and slide is known as working distance. With the 100X objective the working distance will be only about 1-2 mm. The 100X objective should only be used with immersion oil to optimize resolution. The 100X lens should be touching (immersed) the oil if it is properly focused. Be careful to avoid spreading the oil to the other objectives and the stage. Always refer to specific directions from your lab instructor!

 (Fig. 4.1 long description)

 (Fig 4.2 long description)

Course Intended Outcomes

Properly use a compound microscope, identify its parts and describe concepts of microscopy such as magnification and resolution.

Materials Used

Students will work individually but slides can be shared within your group. Each group will need the following materials for this exercise.

• Microscope
• Lens paper and cleaner
• Prepared slides assigned by your instructor

Procedure, Day 1

Initial Focusing / Low Power & High Dry Objectives:

1. Familiarize yourself with the microscope parts and their functions.
2. Place a slide on the stage using the stage clip to secure it from the side or edge of slide. Do NOT use it to clamp slide underneath.
3. Use the X and Y knobs to position the slide so that the specimen is centered over the opening in the stage.
4. Turn on the light source using the control knob. Adjust the brightness to at least 3 LED lights on the arm.
5. Raise the stage all the way to the top using the coarse focus knob. Do NOT continue to turn the focus knob after the stage stops moving.
6. Use the revolving nosepiece to position the 10X objective (yellow) directly over the stage.
7. Check the position of the substage condenser. It should be all the way up, immediately beneath the opening in the stage.
8. Close the iris diaphragm completely using the lever underneath the stage.
9. Now you should begin viewing by looking through the ocular lenses. You may adjust the binocular lenses so they are comfortable for you.
10. Open the iris if you need more light.
11. Use the coarse focus knob to move the stage down while you are viewing through the ocular lenses until you come into focus.
12. Adjust your focus with the coarse knob – then switch to the fine focus knob for “fine tuning” adjustment until the specimen is in sharp focus.
13. Use the revolving nosepiece to change to the 40X objective (blue) WITHOUT moving the stage.
14. Use ONLY the fine focus knob to re-focus the image until it is in sharp focus.

Focusing with the 100X / Oil Immersion Lens:

1. Before switching to the 100X objective (white), you must apply a drop of immersion oil to the surface of the slide.
2. Use the revolving nosepiece to turn half-way between the 40X and 100X objectives. This will provide space for you to add the oil.
3. Add one drop of immersion oil directly to the surface of the slide where you see the beam of light coming through.
4. WITHOUT moving the stage or focus knobs, use the revolving nosepiece to turn the 100X objective into position. If properly focused the lens will slide into the oil when you put it in position.
   • The oil must be in direct contact with both the slide and the 100X objective.
   • If you moved the stage, you lost your focus point. Go back to the 10X objective and start again.
   • If you put the 100X in position and it does NOT touch the oil you are either out of focus or you need to add a larger drop of oil.
5. Open the iris diaphragm to allow more light into the lens.
6. Adjust using ONLY the fine focus knob until the specimen is in sharp focus.
7. Use the X and Y knobs to change your field of view.

Sampling/Inoculation:

1. For this lab students are using only prepared slides. Your instructor will select the preferred slides for your section.
2. It is best to go in order from easiest to most difficult when observing the slides. The most difficult slides are the bacterial specimens. These slides require proper focusing at 1000X total magnification to observe the small cells.
3. It is strongly recommended to clean the slides with a Kim-wipe to remove fingerprints, oil and debris before starting.

Lab Clean-up

Clean your slides and return them to the proper slide trays.

Clean your microscope. Proper cleaning of microscopes is critical to their continued function during the course. View the video provided for proper demonstration.

Cleaning Technique:

1. ONLY use lens tissue paper for cleaning microscope lenses.
2. Start with a dry lens tissue wiping down the oculars 1^st, then the scanning, low power and high dry lenses. If you got oil on the 40X objective remove it completely before moving on to the 100X. Use the dry lens tissue to remove all the oil from the 100X lens.
3. Next use one or two drops of lens cleaner on a NEW piece of lens tissue and repeat the procedure from oculars to 100X objective.
4. Finally, remove any remaining cleaner with a NEW piece of lens tissue.
5. When all lenses are clean and dry position your scope in the way that it was received and return it to the lab prep staff.
6. Never leave your microscope at the door unattended. Wait patiently for one of the lab assistants to check it in.

Results and Interpretation

Record the specimens viewed during the exercise. Check the boxes for the total magnifications used to view the specimen and provide a brief description of what you observed.

In your lab notebook, illustrate the specimens that you saw at the HIGHEST total magnification used.

Critical Thinking Questions

Calculate the total magnification of a light microscope with 5X ocular lenses and a 20X objective.

Why is it better to leave the stage in position without moving it when changing objective lenses?