6.2: Exercise

Microscopy Exercise

Use the slides and cultures provided to learn more advanced uses of the microscope and how to prepare wet mount slides.
We will also be introducing phase contrast microscopy.

A. Brightfield Microscopy - Prepared Slides

1. Amoeba proteus

View protozoa (Amoeba proteus) using the 4x, 10x, and 40x objective lenses. Select one organism to sketch using the three objective lenses, making sure you are examining the same Amoeba each time! (Hint: center the organism in your field of view before moving to the next objective.)

Measuring organisms under the microscope:

The “ruler” that you can see in the eyepiece of your microscope can be used to measure the objects you see through the microscope. This ruler is called the ocular micrometer. The units on the ocular micrometer are arbitrary, and depend on the magnification used to view the sample. As the total magnification increases from 40x to 1000x, each mark on the micrometer measures a smaller and smaller area.

The ocular micrometer on our microscopes is designed so that at 1000x (oil immersion), each mark is equal to 1 micrometer (μm). At this magnification, you can simply read the numbers on the scale as μm.

Each mark on the ocular micrometer measures: (show your work)
At 40x = 25 μm
At 100x = _________________
At 400x = _________________
At 1000x = 1 μm

Sketch your view of the Amoeba and the ocular micrometer for each total magnification. Note: the ocular micrometer does not change; however, your organism will appear larger with each objective.

40X Total Magnification:

100X Total Magnification:

400X Total Magnification:

Choose a sample and measure the same object at three different magnifications. Remember, because the object is the same, its size is not changing during this, and therefore the numbers you get should be the same, or at least close.

Data Table for Microorganism Size

Total Magnification	Number of marks on the ocular micrometer	Multiply by:	Measured size & units:
Amoeba at 40x
Amoeba at 100x
Amoeba at 400x
optional: Bacterium at 1000x

It is possible that your measured sizes may not be exactly the same, due to increased precision at higher magnification. For example, you may measure an organism as 100 μm at 40x, 110 μm at 100x, and 108 μm at 400x. This can be considered consistent, and within an appropriate range.

2. Bacteria

View one of the bacteria on the "Three Types Bacteria Slide" under the microscope using the 10x, 40x, and 100x objective lenses. Once you have a clear image under the 40x objective lens, rotate the objective lens until there is no objective lens directly above the specimen. DO NOT change the stage height. Place one drop of immersion oil directly on your slide and slowly push the 100x objective (oil immersion) lens into the oil. Use the FINE adjustment knob to focus your specimen.

Sketch your view of the bacteria and the ocular micrometer for under the 100x objective lens and complete the last row of the Data Table for Microorganism Size (above).

Bacterium 1000X Total Magnification:

Be sure to use lens paper to remove oil from the 100x objective lens after each use. Remove any oil remaining on the microscope slides before storage as well. 

B. Phase Contrast Microscopy - Live Specimens

The purpose of phase contrast microscopy is to see transparent cells; this allows us to see live cells without staining.
Setting up your microscope correctly for phase-contrast depends on the magnification you are using: (refer to the objective lens before turning the phase turret)

• 10x objective (100x total magnification): turn the turret underneath the stage so that the appropriate setting is visible and facing forward.
• 40x objective (400x total): remember to adjust the turret and use the appropriate phase setting.
• 100x objective (1000x total, oil immersion): remember to adjust the turret and use the appropriate phase setting.

Create a wet mount using the live specimens: pond water or hay infusion. If necessary, add 1 or 2 drops of methyl cellulose to slow down the organisms.

View the live organisms under the 10x, 40x, and 100x objective lenses. For each objective lens, view your sample using brightfield microscopy. Then, turn the turret to the appropriate setting and view your organisms under using phase contrast microscopy.

Clean Your Microscope! Please do the following after each lab:

1. Turn the microscope off
2. Wipe off any immersion oil from the lenses. Make sure to check the 40x objective for oil, too!
3. Remove any slides
4. Put the low power (4x) objective in place.

Also, begin using the microscope to measure objects. You can use the Microcopy Part I lab as a reference.

Questions to think about:

1. How do you determine total magnification?
2. How does the size of an organism change as you change magnification? Choose a stationary or very slow organism to focus on as you change magnifications. Use the worksheet on the following page to help you measure the size of the organism.
3. Wet mount: What is the purpose of the phase contrast? Switch back and forth between brightfield and phase contrast. How does your sample look different? Look especially at the smallest organisms on your slide. Describe the differences you see between brightfield and phase contrast under each of the objective lenses.