• Use the terms listed and the figure of the microscope below to complete Part A of your Lab Report.
• Answer the remaining questions in Part A of the Lab Report
• Arm
• Condenser
• Coarse focus knob
• Fine focus knob
• Head
• Iris diaphragm
• Iris diaphragm lever
• Lamp
• Objective lenses
• Ocular lenses
• Rotating nosepiece
• Stage
• Answer questions 16-18 in Part B of the Lab Report.
General Instructions
• The microscope is a precision instrument that must be handled and maintained properly. A few initial instructions are in order.
1. Carry the microscope in the upright position and with both hands. This prevents moveable parts, like the ocular lenses, from falling and breaking.
2. The glass of the lenses is soft and scratches easily. Use only lens paper to clean the lenses. Avoid the use of handkerchiefs, facial tissue, paper towels, or Kimwipes. Avoid touching the ocular or objective lenses as the glass is easily corroded by acids present in fingertips
3. Always cover moist, living, or preserved materials with a cover slip. Always maintain a safe distance between the cover slip and the objective lens in order to avoid damage to the lenses.
4. Always begin an examination of a slide with the scanning-power (4x) objective. Always look at the objective lens when you swing it into position. These precautions will prevent cracked slides and damaged objective lenses.
5. Report any problems (parts missing, loose parts, blurred images when looking through the microscope, burned-out light bulbs) to the instructor immediately. Don't put a problem microscope back on the shelf.
6. At the completion of the lab exercise, please store the microscope properly:
a) Remove all materials from the microscope stage and wipe the stage surface free of dirt or water.
b) Make certain the scanning objective (4x) lens is locked in place over the hole in the stage and lower the stage as far down as it will go.
c) Turn the voltage control dial to 1 and then turn off the light.
d) Wrap the power cord around the microscope base and tuck the power cord plug into the appropriate place.
e) Return the microscope to the numbered slot in the cabinet that corresponds to the number on the back of the microscope.
Setting up the Compound Light Microscope
1. Position the microscope on the table with the ocular lenses pointing toward you. Adjust yourself so that you can look through the ocular lenses easily without having to tilt the microscope towards you.
2. Unwind and insert the plug into the nearest electrical outlet. Wrap and excess cord around the base of the microscope. For safety reasons, do not let the cord hang over the table.
3. Turn on the light and adjust the voltage control dial to 7 or 8. This will give you enough light to view through the scanning-power objective lens without irritating your eyes.
4. Clean the ocular lenses, objective lenses, condenser glass surface showing through the hole in the stage, and the lamp surface with lens paper.
5. Click the scanning-power objective lens in place. You will hear a "click" as the objective snaps in place.
6. Check the position of the condenser. Using the condenser knob, raise the condenser to the top surface of the stage and then back it down a short distance to ensure that it is not protruding above the stage. You don't want the condenser surface scratched by microscope slides.
Obtain a letter "e" slide from your instructor.
• The following exercise uses the printed letter "e" to demonstrate how to center, find and focus a specimen under the microscope and how to achieve the best resolution. As you work, complete Part B in the Lab Report.
1. Place the letter "e" slide on the microscope stage with the labeled surface up. Open the pincer-like clip of the slide holder with your right hand and slide the "e" slide squarely into place. When you release the tension on the pincer-like clip, it should hold the slide firmly in place. Center the "e" over the stage opening using the mechanical stage controls.
2. Lock the scanning (4X) objective lens in place. While watching from the side (not though the ocular lenses), use the coarse adjustment knob to raise the stage as far up as it will go. This is very important. You must bring the specimen near enough to the objective lens so that the objective actually sees the specimen.
3. While looking through the ocular lenses with both eyes, turn the coarse adjustment knob slowly until the letter "e" comes into view. If you cannot find the "e" move the slide right-left or toward-away from you using the mechanical stage control dials until the letter is centered in the field of view.
4. Sharpen the image with the fine focus knob.
Obtaining the best contrast using Kohler Illumination
• Kohler Illumination is a way to adjust the settings on the microscope so effects of aberrations in lenses are minimized and maximum resolution is obtained.
• Examine the condenser below the stage which focuses light from the light source onto the specimen.
• Find the knob that moves the condenser up and down. This moves the focal plane of light. To focus on the correct plane for the specimen once you are in focus at 4X
• Place a piece of cardboard with a hole in it over the light source (lamp) trying to center the hole over the light source. Note - some microscopes have a built-in field diaphragm on the base (built into the light source). If your microscope has one change the diameter of the cone of light using it instead of the cardboard with a hole in it). A small amount of light will still pass through.
• Look through the ocular lenses. You should see the edges of the circle of light with edges that are fuzzy rather than crisp.
• Move the condenser up or down until edges of the circle are in sharp focus. DO NOT change the focus of the microscope with the focus knobs.
• When the edges are sharp the condenser is at the correct height to give the best resolution for THAT objective.
• Remove the cardboard with a hole (or open the field diaphragm until the cone of light just fills the field of view. If you open wider, some resolution is lost).
5. Another important point to remember when trying to find and focus specimens is that these microscopes are parfocal. Parfocal means that when an image is focused with one objective lens, it will be at, or near, focus with the other objective lenses. As long as you are in focus with one objective lens you can move to the next highest magnification without damaging the lens or slide as long as you don't move the stage.
a) Now switch to the low power (10x) objective lens. Use ONLY the fine focus knob to focus the letter "e".
b) Center the image in the field of view using the mechanical stage control dials.
c) Now switch to the high power (40x) objective lens. Use ONLY the fine focus knob to focus the letter "e".
d) Center the image in the field of view.
• Use this procedure for every slide you study. Taking the time to find the specimen with the scanning power, low power and then high power objective lenses will save you time and you won't break the slides or scratch the objective lenses.
• When you are done viewing the letter "e" return to the scanning objective lens, bring the stage down, remove the letter "e" and return it to your instructor.
• Complete question in Part B of the Lab Report.
• Use the definitions in the lab manual and observe the numerical aperture written on each of the objective lenses. Use this information to answer the questions and compete the table in Part C of the Lab Report.
• You will observe protists in pond water to better understand the relationship between resolution and contrast.
• Unlike the letter "e" the living protists in the pond water will be hard to see if we set the microscope for the best resolution. Instead, we need to increase the contrast or we will not be able to see the protists.
• Ponds and puddles contain vast arrays of diverse organisms. In this exercise, you will examine pond water under the microscope to visualize protozoa, which are single-celled eukaryotic organisms, and various small multicellular organisms. Like any ecosystem, a microecosystem has trophic levels and food chains too. You may be able to distinguish the primary photosynthetic producers seeking light at the surface of the pond water from the consumers, who will be eating dead organisms or other living organisms deeper in the pond water.
1. Make a slide of pond water. Take 2-3 drops of the pond water. Be sure to use a cover slip on the slide.
2. Observe the pond water under the microscope. Note: many of the organisms are colorless and you will need to adjust the microscope to maximize the contrast to see the organisms.
3. Use Figure 1.4 to identify the organisms in the pond water samples.
4. When you are done place your slides in the red bucket for disposal.
• Once you have adjusted the lenses on the microscope for the best contrast use Figure 1.4 to help you identify at least two of the organisms in the pond water.
• Then select one of the organisms you identified and observe it in more detail.
• Answer questions in Part D of the Lab Report.
•View both whole mount and a serial section slide provided by your instructor.
•Answer the questions in Part E of the Lab Report.
• Before you leave;
–Cleanup your work area
–Return all materials to the proper location
–Store your scopes correctly
–Hand in completed post lab questions
–Hand in completed pre-lab questions (if you didn’t already)
- Next week –bring in completed pre-lab questions and hand them in BEFORE lab begins.
- Print out and bring Lab Report for next week.