Many microscopy activities used in classrooms involve observations of structures in cells and organisms, and in research the microscope is an important and powerful tool for investigating cellular processes. To introduce high school students to experimental science on the cellular level, we developed an exercise based on a fundamental fact about eukaryotic cells and the experiment, performed over 100 years ago, that demonstrated this.
The inside of lysosomes is acidic, and the first demonstration that cells had acidic compartments was a surprisingly simple experiment performed in 1893 by Elie Metchnikoff, who later went on to win the Nobel Prize for discovering phagocytosis, the “cell-eating” process that allows cells to absorb material by engulfing it with their membrane. He fed blue litmus particles to protists and observed that internal compartments became red, which indicated that these compartments were acidic (Metchnikoff 1893). His experiment inspired the inquiry-based activity described in this article in which students determine the pH of the digestive compartment in Paramecia using different pH indicators.
Rationale for the experiment
Although “protist ingestion” activities are not new, in this exercise students must use different indicators in a systematic way and consider the limitations of each reagent in order to determine the pH inside an organelle. This lab activity introduces students to the challenges of research on the cellular level and illustrates one of the primary methods that scientists use to measure the concentrations of molecules inside cells and organelles. Furthermore, as illustrated in this article, students are exposed to the essential features of inquiry because they must generate the evidence, develop explanations based on that evidence, and justify their explanations (NRC 2000).
Figure 1. A Paramecium stained
with methyl red. The pink color
indicates a pH of 4.2.
In this exercise, Paramecia ingest different indicators, and the resulting color in the digestive vacuole (lysosome) provides a quantitative measure of the pH of that compartment (Figure 1). [Details about obtaining and caring for Paramecia are provided in the activity description starting on p. 44.] However, because each indicator is effective only within a limited range, the true value may fall outside the range of that indicator if the observed color is at the limit of the indicator. To test that possibility requires a second trial with another indicator that complements or overlaps the range of the first reagent. The results of the second trial will either confirm the results of the first trial, or indicate that the pH value is still further away, in which case additional trials are required until the pH value is narrowed down. The choice of the first indicator may be based on observation or prior knowledge, but subsequent indicators are selected after analysis of the results from the previous trial.
For example, bromthymol blue may be used first to determine if the lysosome interior is acidic or basic (Figure 2). The color yellow inside the lysosomes of Paramecia indicates a pH of 6 or lower according to Figure 2. To confirm this, a second trial is run with a different reagent. A good choice would be methyl red, which is effective between 6.2 (yellow) and 4.2 (pink). The color pink would indicate pH 4.2 showing that the pH was lower than 6. If a third trial, using ethyl orange [range 3.0 (red)–4.5 (yellow)], produces a yellow color, then it is likely that the pH of the compartment is 4.2, or at least in the range of 4.2–4.5. See Figure 2 (p. 43) for the range and color changes of common indicators.
The protocol for the lab is given in Figure 3. Lens paper is an effective way to deliver small amounts of dye, and it successfully “corrals” the Paramecia so they can be observed. If “slowing” media, such as methyl cellulose, is used, it is important to give the protists a few minutes to ingest the dyes before adding the media and the coverslip. Indicator dyes are very concentrated so only a tiny amount is necessary. Too much dye will result in color so dark that Paramecia cannot be seen or they will be poisoned. Dipping the toothpick once in powdered dye picks up a sufficient amount for the treatments. Spilled powdered dye can be spread over an extensive area and, if not thoroughly wiped up, the dye can find its way into clothing, books, and other items. It is important to spread a paper towel or bench paper over the working area. It is also a good idea to wipe the lab tables at the end of the activity with wet paper towels or sponges and look for signs of dyes.
Figure 3. Determining the pH of the digestive compartment in Paramecia using different pH indicators.
- Lens paper
- Slides and coverslips
- Transfer pipettes (plastic) or eye droppers
- Indicator dyes in powdered form (Figure 2)
- Paper towels and bench paper
- Cups or beakers for waste
- “Slowing” media (optional)
- Cut, tear, or pinch off a piece of lens paper slightly smaller than the coverslip, and place it on a glass slide.
- Dip a toothpick into the powdered indicator dye once and remove. Only a very small amount of dye should be on the toothpick.
- Wipe the dye off on the small piece of lens paper. The amount of dye should be barely visible.
- Add a drop or two of Paramecia culture directly on the lens paper, and gently add a coverslip. Wait a few seconds for the Paramecia to find and ingest the dye molecules.
- Use the lower magnifications to locate Paramecia and higher magnifications to see any color changes.
We avoid using liquid indicator solutions for several reasons. The solutions have to be very dilute. Furthermore, some of the recipes for liquid indicators are complicated or involve other chemicals that might be harmful to the protists or interfere with the experiment. The solubility of indicators varies, but for this activity, only tiny amounts of indicator molecules are needed for the Paramecia, so even low solubility is not a major obstacle when using powder. In our experience, the age of the indicator does not seem to matter. We have used indicators over 20 years old, so this activity is a good way to use up some of those ancient reagents.
Students also gain practice in using different features of the microscope. To see color more clearly, the contrast should be low, so condensers or apertures should be opened wider. Otherwise, the vacuoles will appear dark, and it will be hard to determine color. With incandescent light sources, pale yellow indicators may be difficult to see. A second trial with a different reagent can be used to confirm that result. Students must use the lower magnifications (4x or 10x objectives) to find the Paramecia and then switch to higher magnifications (25x or 40x) to observe the detail of the vacuoles. Because the protists can move around fairly quickly, it may be necessary to move the slide on the stage, so avoid using the slide clips and make sure the stage is horizontal.
Typically, most students will find that the pH of the compartment is slightly above 4, but occasionally, some Paramecia have compartments with a pH slightly below 4. On very rare occasions, a Paramecium will fill completely with dye-stained vacuoles and die soon after. Interestingly, within 30–60 seconds, the entire Paramecium will be colored as the cell begins to degrade. These results demonstrate that not all Paramecia (or cells, or patients) respond the same way to chemicals or drugs. There are also reports, which we did not confirm, that over time, the pH of the compartment increases if the Paramecia can be kept alive (Morholt and Brandwein 1986).
Although any protist that ingests particles is suitable for this lab, Paramecium caudatum is a good choice because of its size and relatively clear cytoplasm. These protists can be purchased from most biological supply companies or grown from kits. Paramecia are not fussy about what they eat, but they are fussy about when they eat. After receiving Paramecia, it is important to let them recover at least overnight, following the vendor’s directions for their care. Be gentle—avoid disturbing the protists too much when moving them or distributing them. Most importantly, avoid temperatures above 78°F (25°C) because the Paramecia will stop feeding. We had firsthand experience with this during a summer workshop. The Paramecia did not ingest any dye particles at all until they were returned to a cooler, air-conditioned room.
To be useful in a controlled experiment, the pH indicators should not interact with other molecules in the cell. Such interactions may place them in more than one microenvironment, which may affect their properties. A literature search indicates that the reagents listed in Figure 2 (p. 43) do not bind membranes or other molecules. Some dyes commonly used in protist ingestion demonstrations are pH sensitive, but they also bind to other molecules and should be avoided. Nile red can bind to lipids, neutral red also binds to membranes, while congo red binds to proteins.
Presentation of the lab
This activity can be presented in different ways depending on how much information the students are given and how much guidance the teacher wishes to provide. This activity can be used at the beginning of the unit on cells to introduce the concept of a cell, but it can also be used in a more traditional way to support other lessons on the cell. One can introduce the concept of pH, connect that with students’ prior knowledge, and then introduce the question of pH on the cellular level. Cells are 70% water—are they at neutral pH? Is the pH of a cellular compartment neutral, basic, or acidic? Are Paramecia little creatures like animals with digestive compartments like the intestine at pH 7, or are these compartments similar to a lysosome (or stomach), which has a low pH?
We like to emphasize the importance of different environments in the cell, that the cell creates these environments, and that membranes play a fundamental role in separating these environments. ATP synthesis, gas exchange, nutrient delivery, digestion, waste removal, action potentials in nerves, and muscle contraction are just some of the processes that require different intracellular environments on either side of a membrane.
One of the best-known examples of how membranes allow cells to create and maintain different environments involves pH. H+ ions are pumped into organelles from the cytoplasm by proteins in the membrane, thereby lowering the pH inside the organelle compartment. Since the digestive enzymes in lysosomes only work in a very acidic environment, the cell is protected if these enzymes should leak out into the pH-neutral cytoplasm, where they will not be at their optimum pH.
The acidic environment inside lysosomes is well-known, but other organelles such as endosomes and the Golgi complex also have acidic interiors, although the pH is not as low as in lysosomes. Cells use several systems to maintain a near-neutral pH in the cytoplasm. Mitochondria pump protons into the intermembrane space, and these protons drive the synthesis of ATP. Many viruses, including influenza, exploit the acidic environment of the endosome to enter a cell. The low pH triggers the entry of the viral nucleic acid into the cytoplasm before the virus reaches the lysosomes, thereby avoiding destruction.
Using microscopes and fluorescent indicators, scientists can not only measure the concentration of different ions, including H+ (pH), inside cells or organelles but they also can measure changes in the concentrations of these ions that occur during cellular processes. These molecules change their properties, like the color changes of pH indicators, when they bind ions, and these changes can be detected and quantified using software programs. These indicators are powerful tools that enable researchers to actually measure things that are very tiny.
This activity is similar in many ways to a fishing trip. Students must “fish” out a few Paramecia from the stock supply. They must add the “bait,” in the form of indicators, to their samples, then search for protists that have “taken the bait.” Like fishing, many things can happen, and there can be varying degrees of success, but it still can be fun and educational. We have found that students enjoy hunting the Paramecia and watching them swim around. Students are excited when they detect the colored organelles and observe variations in color produced by different indicators. There always seems to be one student who knows where all the Paramecia are in the stock container or can find them best under the microscope.
This activity reminds us that facts we now take for granted in science were determined experimentally by scientists such as Metchnikoff. Students experience the challenges and unpredictability of research at the cellular level, the same challenges that Metchnikoff faced. In this way, students are introduced to the authentic nature of science (McComas 2005).
Edward Cluett (email@example.com) is an assistant professor of biology at Ithaca College in Ithaca, NY; Jessica Gould (firstname.lastname@example.org) is a teacher at Oliver Wendell Holmes Middle School in Fairfax, VA.
McComas, W. 2005. Laboratory instruction in the service of science teaching and learning. The Science Teacher 72(7): 24–30.
Metchnikoff, E. 1893. Lectures on the comparative pathology of inflammation. London: Kegan Paul, Trench, Trubner & Co.
Morholt, E., and P.F. Brandwein. 1986. A sourcebook for the biological sciences. 3rd ed. San Diego: Harcourt, Brace, Jovanovich.
National Research Council (NRC). 2000. Inquiry and the national science education standards. Washington, DC: National Academy Press.