3.5: Exercise 2 - observing yeast and bacterial cultures

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Students should work in groups of three. Each student should prepare one of the three slides listed below. Students in a group should share their slides. Each group will receive three cultures of S. cerevisiae (SC), S. pombe (SP), and E. coli (EC).

1. Label the three slides. Each slide will contain two samples for comparison. The slides are large enough to accommodate two samples—and two coverslips. Number the slides with a Sharpie (Use the frosted area, if the slide has one.) As you work, be sure to record which of the two samples is closer to the labeled end of the slide. Record your data in the spaces provided below or in your notebook.

Slide 1: compare cultures of S. pombe and S. cerevisiae.
Slide 2: compare cultures of S. cerevisiae and E. coli.
Slide 3: cultures of S. pombe and E. coli with the 100X objective (1000X).

2. Prepare concentrated suspensions of cultured cells. All of the cell cultures appear cloudy, because the cell densities are high. Even so, you will be observing a few microliters of cell suspension under the microscope, and cells may be few and far between. Concentrate
the cultures to ensure that there will be enough cells in the microscope’s field of view for meaningful observations.

Concentrate the cells by centrifuging the test tubes containing the cultures for a count
of 10 in a microcentrifuge set at top speed. Hold down the Quick button on the Labnet microcentrifuges or the button between the two dials on the Eppendorf microcentrifuges.
Remove most of the culture medium with a P1000 micropipette, until the medium just covers the cell pellet.
Resuspend the cells with the vortex mixer.

3. Transfer and stain the cell samples.

• Transfer 2 μL of each cell suspension to the slide, using a P20 micropipette.
• Stain the cells by adding 6 μL of Gram’s Iodine to each cell suspension with a P20 micropipette. Cover each sample with a coverslip.

4. Observe samples of the three species using the 40X and 100X objectives. Each member of your group should observe the two species on the slide that he/she prepared. Work with another member of the group to make observations of the third species. Record your observations and answer the questions in the spaces below.

Note

The oil immersion lens is required for the 100X objective, and this oil can damage the 40X objective. Be careful to follow the instructions below exactly.

The steps must be performed in the correct order to protect the lenses!

Focus the microsope on EITHER an s. cerevisiae or s. pombe culture. Focus first with the 10X objective, then move to the 40X objective and refocus with the fine focus control. Record your observations.
You are now ready to move to the 100X oil immersion objective! Rotate the nosepiece halfway between the 40X and 100X objectives. WITHOUT moving the stage, apply a drop of immersion oil on top of your coverslip where the light is shining through the slide. SLOWLYrotate the 100X objective into place, submerging it into the drop of oil. Use the FINE focus knob to bring the yeast into focus. Record your observations.
Rotate the nosepiece halfway between the 40X and 100X objectives again. Do not attempt to move the stage with the 100X objective in place.
Use the XY controls to move the stage to the second sample on the slide WITHOUT changing the focus.
Rotate the 40X lens into position. Adjust the focus and record your observations.
Repeat step 2 above and record your observations at 100X.
Rotate the 100X objective out of position. Remove the slide and discard it in the glass waste. Clean the 100X lens with lens paper. Check that no oil is present on any of the other lenses.

S. cerevisiae observations
Sketch the cells that you see with the two different lenses in the boxes below.

What fraction of the cells show buds?
Can you detect any subcellular structures at 1000X magnification?

S. pombe observations
Sketch the cells that you see with the two different lenses in the boxes below.

What fraction of the cells show septa?
Can you detect any subcellular structures at 1000X magnification?

E. coli observations
Sketch the cells that you see with the two different lenses in the boxes below.

What fraction of the cells are motile?
How would you describe the structure of an E. coli?