Vacuole Formation and Cytoplasmic Streaming
In this lab you will examine the role of different cytoskeletal elements (tubulin microtubules and actin filaments) in motility, phagocytosis, food vacuole formation, and cytoplasmic streaming. We’ll discuss the cytoskeleton in class on Thursday and phagocytosis in class on Friday.
In the first part of the lab you will use Tetrahymena to study the role of the cytoskeleton, specifically microtubules and actin microfilaments, in motility, phagocytosis, and food vacuole formation. Tetrahymena employs phagocytosis (one form of endocytosis) to ingest bacteria or other small pieces of food from the environment. This process can be studied in Tetrahymena using India ink to study the formation of food vacuoles under different conditions, since the vacuoles will show up clearly as black spots in the cell. We will use cytochalasin B, an inhibitor of actin microfilaments, and colchicine, an inhibitor of microtubules, to determine whether the cytoskeleton is involved in motility or phagocytosis and vacuole formation, and if so, whether actin microfilaments or microtubules are more important.
In the second part of the lab you will design an experiment to examine the effect of inhibitors, and hence, the role of actin and tubulin, on cytoplasmic streaming in one of the test cells: (a) Nitella or Chara internodal cells; (b) Physarum (plasmodial slime mold); or (c) chloroplasts of Elodea mesophyll cells.
Safety concerns: please wear gloves when handling the glutaraldehyde, colchicine, and cytochalasin B. Please pipet glutaraldehyde in the hood. Please exercise care when using these chemicals, they can “fix” (kill) your cells, stop your microtubules, and stop your actin microfibrils. Cytoskeletal elements are your cells’ friends—treat them nicely by protecting them from these dangerous chemicals.
Experiment 1: Preliminary observations
Tetrahymena and counting the ink-containing food vacuoles
(1) Get out your binocular compound microscope, make sure all the lenses are clean and that it is appropriately adjusted for your eyes.
(2) You have been provided with a culture of Tetrahymena grown in 2% proteose peptone. Individually, measure out 0.5 ml of Tetrahymena suspension into a clean 1.5 ml microcentrifuge tube.
(3) Into the tube, add an equal volume (0.5 ml) of 1% India ink
(4) [CAUTION: Try not to inhale the glutaraldehyde fumes - you will fix your nasal epithelium! Pipet the glutaraldehyde in the fume hood. Wear gloves when you handle glutaraldehyde, colchicine, and cytochalasin B.]
Sample the tube 10 min after adding the ink. To do this, remove 20 µl of cells in ink into a clean 1.5 ml microcentrifuge tube containing 3 µl of 3 % glutaraldehyde - this will "fix" (kill) the cells so that they can be observed more easily.
(5) Observe the cells under 40X or 100X (oil immersion) and count the number of black food vacuoles in the Tetrahymena cells.
(a) Tetrahymena without ink. (b) Tetrahymena after being fed 1% ink for 30 minutes: contains12 food vacuoles.
To determine the effect of colchicine and cytochalasin B on motility and phagocytosis in Tetrahymena.
(1) Working in pairs, measure out 2 ml of Tetrahymena suspension into each of three clean test tubes.
(2) Into the first tube add 20 µl of cytochalasin B (5 mg/ml) to give a final concentration of 50 µg/ml, into the second tube add 20 µl of colchicine (400 mg/ml) to give a final concentration of 4 mg/ml. Both the cytochalasin B and the colchicine are diluted in water. The third tube is a control - what do you think you should add to this tube and why?
[CAUTION: If you get either cytochalasin B or colchicine on your hands wash them immediately - you really do not want to inhibit microfilament or microtubule formation in your own cells!]
(3) Incubate the cells for 10 min at room temperature to allow the inhibitors to act before adding ink.
(4) After 10 min and before adding the ink, remove a small sample of Tetrahymena suspension and determine the effect of each inhibitor on ciliary motion and motility Record your observations.
(5) Next, add 2 ml of 1% India ink to each of the three tubes.
(6) Sample each tube at time zero (the moment you add the ink) and fix with 3 µl of glutaraldehyde per 20 µl of cells. Count the number of black food vacuoles in at least 10 cells from each treatment at time zero. [NOTE: you do not have to count them immediately since you have fixed the cells and they will neither gain nor lose food vacuoles, so concentrate on sampling Tetrahymena at the different time periods.]
(7) Sample all three tubes every 10 min for 30 min and quantify vacuole formation – count the number of food vacuoles in 10 cells and take an average.
(8) Plot the mean number of vacuoles (from 10 cells per treatment) against time for each treatment and interpret these data. What were the effects of cytochalasin B and colchicine? Are microfilaments, microtubules, or both required for phagocytosis and vacuole formation in Tetrahymena?
This protocol was modified by M. Wilson and M. Olney, 2002 from: Bozzone, D.M. (2000). Investigating phagocytosis in Tetrahymena: An experimental system suitable for introductory and advanced instruction. The American Biology Teacher 62: 136-139.
Individually, observe and draw cytoplasmic streaming in the different test organisms/cells: (a) Nitella or Chara internodal cells; (b) Physarum plasmodium; and (c) chloroplasts in Elodea mesophyll cells.
At some point, check out the following web sites. These web sites can provide useful background information on the organism you choose to use for your experiment—information you will need for your lab write-up.
To design and conduct an experiment that will determine whether actin filaments, microtubules, or both are involved in cytoplasmic streaming in your chosen test subject. This laboratory exercise is an opportunity to think about the scientific method, experimental design, the importance of controls and replicates, how to analyze data and use to test hypotheses.
As a pair, select a test organism to work with and design an experiment using the two inhibitors cytochalasin B and colchicine to address which cytoskeletal element is involved in cytoplasmic streaming. Your experiment should allow you to take quantitative (i.e. numerical), replicated experimental data.
BY210 Cell Biology
Lab write-ups should be typed (not handwritten) for clarity and neatness. Follow format below.
Note that the Honor Code applies to lab write-ups and you should prepare these on your own, even if the lab itself was conducted as a pair or larger group! Please include a signed HCU on your report.
Title: Brief description of the study or experiment (please do not use sentences here—use standard title format, but make it descriptive of what you did and/or found, including the organism you studied).
Objective: Briefly explain the objective of the lab as you understand it (in your own words—please use past tense and complete sentences).
i. Briefly summarize the methods that you used (a few sentences are enough—please use past tense). Specifically refer to the lab handout as the source of your methods.
ii. If your methods differed from those described in the handout, detail those changes. If there was something you had to decide, please specify what you chose.
iii. Cite the lab handout. (Example: Olney, M. and Wilson, M. 2000-2002. Lab I: Introduction—The Diversity of Cells. Biology 210 Lab Handout. Colorado College, Colorado Springs, CO.)
1. Experiment 1
a. description of your control for (1:3) and your reasoning for choosing it
b. from (1:4): what was the effect of colchicine on ciliary motion and motility?
c. from (1:4); what was the effect of cytochalasin be on ciliary motion and motility?
d. plot of average number of vacuoles against time for each treatment (1:8)
e. your interpretation of your data in part b: What were the effects of cytochalasin B and colchicine? Are microfilaments, microtubules, or both required for phagocytosis and vacuole formation in Tetrahymena?
Conclusions: Your interpretations of your results.
Lab write-up continues on the next page:
Title: Brief description of your experiment: include what you tested and/or found (including the specific organism you worked with and any important chemicals you used).
Introduction (please write in paragraph form using complete sentences and past tense):
i. description of your test-subject (include several sentences about your subject—what is it, how is it classified (domain, kingdom), where does it live, how does it get its energy, etc.)
ii. description of the process you chose to study (what is it, what function does it serve, etc.)
iii. description of the process as it occurs in your test-subject prior to experimentation (what does the process look like, why did you choose this process in this subject—you may include a drawing here if you would like)
iv. explanation of the objective of the experiment you designed and why you designed it that way
v. explanation of your hypothesis(es)
Methods (please write in paragraph form using complete sentences and past tense):
Summarize the methods that you used. Be specific enough that future generations of Cell Biology students could follow your protocol if they wanted to repeat your experiment. Do not include the tiny details that would be common to any experiment (for example, that you used micropipets to deliver the appropriate volumes), but do include the important things that someone would need to know to repeat your experiment.
Results (please write in paragraph form using complete sentences and past tense):
i. Drawings/diagrams of cytoplasmic streaming in the three different organisms. These should be labeled as Figures 1, 2, and 3 with titles that describe the organism and its magnification.
ii. Paragraph(s) describing (not interpreting, see below) your results, include specific references to any tables (Table 1, 2, etc.) or graphs (Figures 4, 5, etc.) of your data. However, do not simply say “the results are shown in Figure 1.” Instead say, “Red light treatment resulted in 2.2% of the cows giving purple milk (see Figure 1).”
ii. Table(s) and/or graph(s) of your data. Be sure that each one is referred to in the text and that the data presented in each is summarized in the text. Each should be labeled (Table 1, Figure 1, etc. Each should have a descriptive title so that each could stand alone (meaning that someone should be able to read and understand your table or graph without referring to the text, including a brief description of the experiment that was conducted). Don’t forget to include all units on your axes’ labels and on your tables’ columns and rows.
Conclusions (please write in paragraph form using complete sentences):
Interpretation and conclusions based on your data. Also include possible sources of error, problems you may have encountered, and a suggestion or two for a future experiment.