1.2: Procedures

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Use and Care of the Microscope:

When instructed to do so, obtain the microscope that corresponds to your seat number from the cabinet (power cords are in a plastic box in the drawer near your seat). Familiarize yourself with the major parts and their functions—you may use the information found at the end of this chapter to guide you.

Basic Guidelines for Using the Microscope

1. Always carry the microscope with two hands.
2. Always use the microscope that is assigned to your seat number.
3. Clean the lenses with lens cleaner (Windex) and lens tissue before and after use.
4. Report any problems with the microscope to your instructor immediately.
5. Oil must be cleaned off completely before returning the microscope to the cabinet. If you accidentally get oil on the 40X objective, clean it immediately. Microscopes must always be returned to the cabinet clean.
6. Microscopes should always be put away with a low power objective (4X or 10X) over the stage.
7. Always lift the microscope to reposition it—do not drag it across the surface of the table!

Total Magnification: The microscope you are using has two sets of lenses that both contribute to the total magnification of the image. The ocular lenses magnify your image 10X. There are 4 different objective lenses—each with a different magnification. The total magnification is calculated as follows:

$ocular\: magnification \times objective \:magnification$

Since the ocular magnification of our microscope is 10X, determining the total magnification of an object with this microscope simply requires multiplying the objective magnification by 10. (Note: other microscopes may have ocular lenses with a different magnification, for example, 12X.)

Exercise 1.2.1

Fill in the table below:

 Name of Lens Objective Magnification Total Magnification Scanning Low High-Dry Oil Immersion

Diversity of Cells: There are many types of cells found among the diverse forms of life on the planet. All cells have certain features in common, such as a plasma membrane surrounding the cell, cytoplasm within the plasma membrane, and DNA as the molecule that stores genetic material. However, there is a great deal of diversity among the cells that make up living organisms. Some living organisms are composed of one cell (unicellular); others are composed of many cells (multicellular).

Cells that have a nucleus and other organelles are eukaryotic; bacterial cells do not have these intracellular structures—they are prokaryotic.

Some types of cells (plants, algae and some bacteria) are photosynthetic (capable of using light energy to make organic compounds from inorganic materials.) Others take in organic molecules as a source of energy.

There are several other similarities and differences between cell types that you will learn about throughout the semester. In today’s lab you will use the microscope to look at various types of cells that are often studied in a Microbiology course.

Examination of different cell types under the microscope

In this activity, you will look at some prepared slides, as well as make a few of your own.

Materials:

• Prepared slides: Blue-green algae, mixed protozoa, bacterial types
• Slides and cover slips
• Pond water sample (if available)
• Live Saccharomyces culture
• Methylene blue stain
• Prepared slides (demos): may include Pinworm (Enterobius), pinworm eggs, mosquito, yeast cells (Saccharomyces), bacterial slides showing varying morphologies and arrangements

Instructions: Use the spaces provided at the end of this exercise to draw pictures as you are viewing the slides.

1. Place your first slide (blue-green algae) on the mechanical stage and make sure that it is level and held firmly in place. Your sample /smear should be facing upwards.
2. Use the knobs located below the stage to move the slide left and right, and up and down until the stained area of the slide is centered over the light source.
3. Position the scanning power objective (4X) in place over the slide. (Note: objective will “click” into place).
4. Use the course adjustment knob to bring the stage up as far as it will go.
• When using the scanning or low power objectives the working distance (the distance between the lens and the slide) is large enough so that the slide will never make contact with the lens. This is not the case when using the high-dry and oil immersion lenses, where the working distance is significantly less. This is why the coarse adjustment knob can only be used with the two low-power lenses.
5. Rotate the coarse adjustment knob away from you until the image comes into focus.
• You will not be able to make out much detail at this power—the purpose is to find where your specimen is on the slide so that it is easier to locate when you switch to high power. Low power objectives have a large field of view (the circular area seen when looking through the microscope) and a large depth of field (the thickness of a specimen that is in sharp focus). As magnification increases, both the field of view and depth of field decrease, which is why it is easier to locate your specimen using a low power objective.
6. If needed, use the fine adjustment knob to improve the clarity of the image.
7. After viewing the slide at scanning power, move the slide so that the area you want to focus on is in the center of the field of view.
• Since your microscope is parcentric, when you increase magnification you will be zooming in at the center of the field of view. Objects that are not centered at low power may be out of the field of view at high power.
8. Rotate the objective lens nosepiece so that the 10X objective is in place over the slide. Re-focus and adjust the light (if needed) under the 10X objective.
• The microscope you are using is parfocal—this means that when it is in focus with one lens in place the same stage position will be in focus with all other lenses. Therefore when switching objectives, DO NOT change the position of the stage—just click the objective you wish to use into place.
• Note the differences in the appearance of the cells from when you were using the 4X objective.
9. After focusing at 10X, rotate the objective lens nosepiece so that the 40X objective is positioned over the slide. Re-focus using the fine adjustment knob and adjust the light if needed.
• Note: Remember that you CANNOT use the coarse adjustment knob at high power (40X or 100X objectives). When you are using the high-power lenses the lens is very close to the slide (small working distance) therefore using the coarse adjustment knob at high power could result in damage to the lens, damage to the slide, or both.
• Remember to sketch what you are looking at in the spaces provided.
10. When you are finished looking at the blue-green algae slide, return it to the slide box and proceed to the next slide—Mixed protozoa. Follow the instructions above to view the slide with the 4X, 10X and 40X objectives in place. Draw diagrams of each.
• When viewing the mixed protozoa slide, you should scan different areas of the slide, as there are many types of protozoans and algae present on this slide. Some organisms you might see include Volvox, Amoeba, and Paramecium. Note that in most of these cells the nucleus is clearly visible.
11. Sketch a few areas of this slide with the 10X or 40X objective in place. Use the charts in the lab to identify some of the organisms you see.
12. Return the mixed protozoa slide to the slide box and use a clean slide and cover slip to prepare a wet mount of a live pond sample (see instructions below). View this slide at 4X, 10X and 40X magnification.

Preparation of wet mounts

These slides will be prepared with live microbes on them. Since the liquid is left on the slide, cover slips are needed to prevent this liquid from touching the lenses.

• Live protozoa: use a dropper to place 1-2 drops of a pond water sample onto a clean microscope slide. Take a cover slip and place it down over the water as shown in the diagram below (try to avoid air bubbles).
• Preparation of a live yeast culture: place one drop of the yeast (Saccharomyces) suspension and one drop of methylene blue onto a clean microscope slide, and prepare a wet mount as shown.

Figure 1.2.1: Preparation of a Wet Mount

Observe these slides under the microscope (use the magnifications suggested by your instructor). As time permits, your instructor may have you examine other types of cells that are set up on demonstration scopes.

Observing bacterial cells under the microscope

In this exercise you will learn how to use the oil immersion lens, and observe some common morphologies and arrangements of bacteria.

1. Obtain a bacterial types slide from the slide box and place it on your microscope. Since bacterial cells are very small, you will need to use the highest magnification (100X objective, or 1000X total magnification) to see them clearly—however, as with all slides, you should use a low-power objective lens to focus on the slide before moving to high power.
• The bacterial types slide may be viewed with all 4 objectives—alternatively, your instructor may ask you to skip some (for example, just use the 10X and 100X objectives). Follow the instructions you receive in class.
• There are three areas of bacteria on the slide. The area closest to the slide label is the darkest and therefore the easiest to find: the area on the rightmost side of the slide is very faint and you may not be able to detect any stain with your eyes.
2. . Focus with a low-power objective on the first area of the slide.
• The cells will still be very small at this magnification and you will not see any detail.
3. Once in focus, switch the objectives to 10X and then to 40X (this step may be skipped). Adjust light and focus as needed.
4. You are now ready to use the oil immersion lens. This lens requires the use of immersion oil, which has the same index of refraction as glass, to prevent light from scattering and focus it on your specimen (we need a lot of light to see clearly at this high magnification).
• Immersion oil MUST be used with the 100X lens.
• Immersion oil should NEVER be used with the high dry lens.
5. Rotate the nosepiece so that there is no objective over the stage. Add 1-2 drops of immersion oil to the slide right above where your sample is.
6. Click the 100X objective into place. If done correctly, you should only need to fine focus a little bit to bring the cells into view.
• Remember to NEVER use the coarse adjustment knob when focusing under 40X or 100X objectives.
• If you “get lost,” it’s better (and faster) to go back to low power and refocus, then switch back to 100X.
• Once there is oil on the slide you CANNOT use the 40X lens—rotate the nosepiece the other way to use 4X or 10X to refocus.
7. Use the spaces provided to draw the cells. At this point, your instructor will discuss the morphology (shape) and arrangements of the cells you are looking at. (See Figure 1.4.2. at the end of the exercise for more information.)
8. When you are finished, move the slide to the right to find the second area of cells (middle of the slide), and then to the third area of cells (right side of the slide).
• The third area of cells is very faint and the cells are more spread out. If you “get lost”—try going back to find the second area and then carefully move the slide across to the third area while keeping the slide in focus.
9. When you are finished, clean all oil off of the slide and return it to the slide box.

1.2: Procedures is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Joan Petersen & Susan McLaughlin.