BISC110/S13: Series 1 Lab 2 Tetrahymena Behavior

Objectives: In this lab you will learn:

1. Proper use of the microscope
2. Measurement of specimens using the micrometer

PART I: Microscopy

Most cells measure about 1–100 micrometers (µm) in diameter. This size is smaller than can be detected by the unaided human eye; therefore, microscopes are needed to visualize cells and their component parts. The compound light microscope can magnify to about 1000 times the actual size of the specimen and can resolve details as fine as 0.2µm. In this part of today’s lab, you will learn to use the compound microscope by examining several types of cells.

A. Care of the Microscopes

A compound microscope and a dissecting microscope are available for each student's use. We will only be using the compound microscope today. Remember at all times that your microscope is a precision optical instrument and must be handled carefully. When removing the microscope from the cabinet, do not jar or drop it, always carry it upright with one hand below the base and the other hand on the arm of the microscope. Place the microscope at least 6 inches from the edge of the bench. When returning the microscope to the cabinet, check that:

1. The microscope light is turned off before the microscope is unplugged;
   
2. All lenses have been cleaned with lens paper, especially the oil immersion lens;
   
3. The lowest objective lens is near the stage and the stage itself is lowered; and
   
4. The microscope is covered (if there is a cover available).
   

B. Parts of the Microscope

Figure 1 contains a diagram of a compound microscope and may help you locate some of the parts referred to in the following explanation. The compound microscope derives its name from the two sets of lenses it uses to magnify objects. These lenses are the objective lens, which can be found on the rotating nosepiece near the stage of the microscope, and the ocular lens, which is in the eyepiece. Your microscopes are equipped with several objective lenses, ranging from low to high magnification, including one oil immersion lens. The microscope magnifies by shining light from the light source through the iris diaphragm that limits the diameter of the light beam. The condenser lens focuses the light through the specimen that is on the stage. The stage is movable in order to view different parts of the specimen. The image we see is formed under the ocular lens by the objective lens and is a mirror image of the actual specimen.

C. Regulation of Illumination

1. The illumination intensity knob is located on the right side of the microscope just below the on/off switch. It has a setting range of 1-10 with 10 being the brightest level. This knob should ordinarily be set to 7.
2. Another way of adjusting illumination is by changing the position of the condenser lens. The condenser lens adjustment knob is located below the specimen stage and on the left side. It allows the user to move the condenser lens assembly up or down. As you move the condenser lens up, closer to the specimen, it concentrates (condenses) more light on your specimen. You will need to make this adjustment as you go up in magnification, so that you will have sufficient illumination.
3. The condenser aperture diaphragm is located below the specimen stage on the condenser lens assembly. It is an adjustable opening, which allows you to make fine adjustments in illumination. The lever, which adjusts the size of the aperture, faces the user. By sliding the lever to the left or right, you may adjust the illumination to the correct level for your specimen. Changing the size of this aperture also affects the amount of contrast in the image. Thus, adjusting the condenser aperture involves finding the brightness level, which gives you the best combination of illumination and contrast. This is the method used most often in adjusting illumination in the light microscope.
4. Another method of adjusting illumination is by using the field aperture diaphragm. This is mentioned here for the sake of completeness, as most light microscopes have an adjustable field aperture. However, the microscopes in the 110 lab lack this adjustability, which allows the user to obtain illumination that is uniformly bright and free from glare (Köhler illumination).

D. How to Locate Specimens Using a Compound Light Microscope

1. Place specimen slide on microscope stage and secure with clamping arm. The slide is properly in position if turning each of the the stage adjustment knobs moves the slide appropriately. Position the 4x or 10x objective into place. It will click when it is properly positioned directly over the slide. Make sure that there are several inches of clearance between the glass slide and the lens.
2. Using the stage adjustment knobs, position the edge of the coverslip in the center of the illuminated area. Look into the microscope and adjust the eye pieces so that you can see one image when you use BOTH eyes for viewing. While looking through the oculars and 4x or 10x objective lens, rotate the course focus knob slowly so that the distance between the slide and the objective lens is reduced. When you see the black line that indicates the edge of the coverslip is coming into focus, switch from the course to the fine focus adjustment and bring that "black line" into sharp focus. Switch to the 10x objective and refocus. Use the stage adjustment knob to move away from the edge of the coverslip into the area where your specimen is located. To increase the magnification of the specimen, rotate the nosepiece to the 40X objective lens and focus using ONLY the fine adjustment knob. Never use the coarse focus knob when you have the 40x objective in place.
3. Although you will not use the 100x objective to view your specimens today, be aware that immersion oil must be placed on the slide in order to use the 100X objective lens. After a specimen has been focused sharply using the 40x objective, you would move the 40x objective lenses out of the way and place a small drop of immersion oil onto the slide. Then you would rotate the nosepiece until the 100X (oil immersion) lens is selected and the 100X objective lens is completely immersed in the oil (no air between slide and objective). The specimen could then be focused using the fine adjustment knob only. Never use the course adjustment when focusing a specimen with the oil objective because doing so could result in damage to the 100X objective lens or to the slide. All traces of oil must be removed from the lens before putting away the microscope. Only lens paper should be used to remove oil from the 100X objective.

E. Calculation of Total Magnification

Total magnification of the specimen is determined by multiplying the magnifying power of the ocular and objective lenses. For example, a 10X ocular and a 40X objective together give a total magnification of 400X (Table 1). This means that the specimen appears 400 times larger when viewed with a microscope than its actual size.

F. MEASUREMENT OF SIZE

Cell size can be measured using an ocular micrometer. A micrometer has been installed in one of the ocular lenses of each microscope in the laboratory. It looks like a small ruler with both large and small units. The large units are numbered 1, 2, 3, etc. The small units are subdivisions of the large units and are not numbered. There are 10 small units per large unit. The small units represent different lengths depending on the objective lens in use. You will measure cellular structures in small units only, and then convert to metric units (µm = micrometers) using the conversion values below.

Therefore, if you are observing a cell with the 40X objective, and this cell spans 2.5 small units on the ocular micrometer scale, then the size of the cell is calculated by multiplying 2.5 small units x 2.5µm/small unit = 6.25µm. You should always calculate size of any object that is the focus of a figure in a photomicrograph for a scientific paper. Because digital imaging allows manipulation of size post-viewing, giving the total magnification is sometimes misleading. You should calculate and give the size of any important object in the figure legend.

Figure 1. A Cutaway Diagram showing the beam path of the Nikon Eclipse E200 Compound Light Microscope used in the Biological Sciences 110 Laboratory at Wellesley College. [SOURCE: http://micro.magnet.fsu.edu/primer/anatomy/nikone200cutaway.html]

PART II: Examination of Tetrahymena pyriformis

To help you become familiar with the use of the compound microscope and to prepare for experiments with Tetrahymena pyriformis in Labs 3 and 4, today you will observe live Tetrahymena. Spend some time observing the cells today, take note of their behavior and draw them in your lab notebook. Make a circle to represent the field of view using the bottom of a beaker or petri dish and draw some represenative organisms, labeling all the organelles that you can identify. Be sure to include total magnification by every drawing. Then using the digital cameras take some photographs of your cells.

NOTE: You will not use the oil (100X) objective today to view your Tetrahymena.

Obtain a microcentrifuge tube containing live Tetrahymena. Mix it gently (no vortexing). It is best to obtain a sample from the TOP of the tube, since the cells will be more concentrated in that location. Add 20μL of the live Tetrahymena to the center of a clean glass slide, add a cover slip, and then view the cells using the microscope, starting with the lowest power and moving up to 400x magnification. Carefully observe the behavior of the Tetrahymena and record your observations in your lab notebook. If you have trouble locating the cells, it might help to adjust the field diaphragm.

PART III: Determine Toxicity of Cobalt Chloride or Cupric Chloride on Tetrahymena pyriformis

Each pair of students will obtain the cobalt chloride or copper chloride stock solution and dilutions they made last week to carry out a Tetrahymena toxicity study.

1. Combine 20 μL of live Tetrahymena with 20 μL of 0.11 M copper chloride or 0.26 M cobalt chloride in a clean microfuge tube. What is the concentration of the heavy metal?
2. Wait 1 minute, and then add 20 μL of those Tetrahymena (from the microfuge tube) to a glass slide, add a cover slip, and observe using the microscope.
3. Record your observations and measurements about behavior and morphology changes in Tetrahymena after exposure to the heavy metal.
4. Take pictures with the digital camera.

Is copper chloride or cobalt chloride toxic to Tetrahymena at the concentration used? How do you define toxicity? How can you quantify the effects of toxicity in Tetrahymena? How do you know what is normal behavior and morphology? If you decide to use lack of movement as your criterion for toxicity, how will you determine what is self-propelled movement vs. water flowing on your slide? Should you include a control? YES! What would the appropriate control(s) be? Test the remainder of the dilutions of cobalt or copper chloride solution and determine if concentration is important in the toxicity of your heavy metal. Make a table in your lab notebook to record your data. Don't forget to calculate final concentration (in moles) of the heavy metal every time you use a different dilution and record that information in your lab notebook. Use final (effective) concentration, not dilution factor, in your data table or axis labels in a graph made from the data and in your results analysis.