Wright Stain and Fluorescence-Based Methods in Tetrahymena for Phenotypic Analysis

Tetrahymena is a model freshwater single-celled protozoan, ideal for investigating the molecular and cellular basis of cell morphology, organelle organization, structure, cell proliferation, and ciliary motion, and can be used to study cell shape, even rate of phagocytosis (Bozzone, 2000; Ruehle et al., 2016). This ciliate also possesses a diploid micronucleus (MIC), mostly silent transcriptionally, and two complete haploid genomes capable of both mitosis and meiosis (Orias, 1998). Tetrahymena thermophila, like other fresh water protozoa and cells of lower fresh water invertebrates, maintains its salt and water homeostasis via contractile vacuoles (Plattner, 2015). This enables the organism to maintain constant potassium and sodium concentrations over wide range of hypo-osmotic environmental conditions. Moreover, investigations using Tetrahymena thermophila led to important discoveries and insights, such as telomeres and telomerase (Gilley & Blackburn, 1996; Chan & Blackburn, 2004), and ribozymes (Cech et al., 1981).

To visualize cellular organelle changes over Tetrahymena life cycle development (Figure 1), there are at least two chromogenic staining methods used before for phenotypic analysis: (1) silver preparation labels cortical basal bodies (Frankel, 2008); and (2) Giemsa stain labels micronucleus and macronucleus (Stone & Cameron, 1964; Gude et al., 1955). The reason for applying Wright stain was to visualize multiple cellular organelles. We wished to apply this stain, routinely used in medical laboratory settings, as a tool to explore and define multiple organelle labeling for phenotypic analysis. The traditional Wright stain dates from 1890s; it is also a modified version of the Romanowsky method. The original Wright stain was an alcohol-based solution of methylene blue and eosin Y, used primarily to stain peripheral blood smears, urine samples, and bone marrow aspirates to be examined under a light microscope (Wright, 1902). The acidic or basic nature of cellular structure determines their staining for the components of Wright's polychromatic dye.

We applied Wright stain as a method to label heat-fixed and live Tetrahymena as a tool used for phenotypic analysis. Sytox green is a high-affinity nucleic acid stain that easily penetrates cells with a compromised plasma membrane. In these cells, which are dying or undergoing cell death, Sytox green dye binds to DNA inside the nuclei, which brightly fluoresces green with over 500-fold enhancement. Stained cells will generally have bright green nuclei as well as some low-level cytoplasmic staining (Lebaron et al., 1998). In addition to using the compound light microscope, we utilized both the portable low-cost fluorescence microscope (Miller et al., 2010) for fluorescence detection, along with Cellcams (Martin & Shin, 2016) for videos. In the following sections, we provide step-by-step methods of Wright stain and Sytox green fluorescence that can be adapted to lab inquiry research and educational settings.

1. Tetrahymena standing (non-shaking) culture at room temperature, grown in 2% pentose peptone from Carolina Labs (Tetrahymena supplied with Phagocytosis and Vacuole Formation in Tetrahymena Kit, Catalogue Item # 131182B). Note: this kit also contains India ink.

2. Compound microscope (Olympus) with 4x, 10x, and 40x air objectives.

3. Portable battery-operated, low-cost, bright field, and fluorescence microscope (gift from Dr. Rebecca Richards-Kortum, Rice University; cited in Miller et al., 2010).

4. Alcohol lamp from Carolina Labs (Catalogue # 706604) and matches (Catalogue #12-075 from Fisher Scientific).

5. 10P, 20P, 100P, 200P, and 1000P hand-held pipettors (Gilson^® Pipetman^® Classic, Fisher Scientific). 10P (Catalogue # FA10002PG); 20P (Catalogue # FA10003MG); 100P (Catalogue # FA10004MG); 200P (Catalogue # FA10005PG); and 1000P (Catalogue # FA10006PG).

6. Eppendorf safe-lock micro centrifuge tubes (Part # 022363638 from Eppendorf).

7. Glass slides (Catalogue # 12-544-2, Fisher Scientific) and cover slips (Catalogue # 10-016-24, ThermoScientific). Note that glass slides were not pre-coated with gelatin, nor with poly-D lysine.

8. Wright stain (Fisher Scientific brand, SureStain^TM Wright Stain, CS-432, opened 08/27/2015), placed in a glass bottle with dropper (Catalogue # 12-000-158, Fisher Scientific).

9. Methylene blue stain (Millipore Sigma R0310174), placed into a bottle with dropper (Catalogue # 12-000-158, Fisher Scientific).

10. Sytox green nucleic acid stain, 100µM stock solution diluted in distilled water (use stock SYTOX^™Green Nucleic Acid Stain , 5mM Solution, Invitrogen™ S7020).

11. Pentose peptone media, 2% (Stewart & Giannini, 2016).

12. Wash bottle with distilled water.

13. Stain rack holder (handmade, shown in Figure 3, panels A–C).

14. Parafilm paper (Parafilm M Sealing Film, #7315D11, Thomas Scientific).

15. Number 5 forceps (Model # IMS-JF5, Premium High Precision Jeweler Style Forceps, #5 Tweezers, Fine Point Tips, Stainless Steel, 4.5″ L) used to make holes in parafilm to cover Eppendorf tubes.

To preserve and adhere Tetrahymena cells onto the glass slide, use this step-by-step heat-fixation procedure:

1. Place 50 microliters of the sample in a glass slide.

2. Fix Tetrahymena onto the glass slide by intermittent exposure to heat using flame lamp onto the sample. For lab safety purposes, wear gloves and use tweezers to hold one end of the glass slide.

3. Pass the slide quickly through flame several times, sample side up, until completely dry. Allow 5 to 10 seconds to cool at room temperature away from the flame.

4. Carefully track the temperature by touching your arm with the glass slide to make sure it will not burn the sample.

1. Prepare the sample by pouring 25 microliters of untreated Tetrahymena placed on a glass slide.

2. Fix the sample using the heat–fixation procedure described above.

3. Place the slide sample side up on the staining rack.

4. Apply Wright stain to the slide using dropper bottles or pipettes.

5. Wait 5 seconds, the add an equal volume of distilled water.

6. Mix the stain and water, and allow it to incubate at room temperature for 5 minutes.

7. Pour the stain and water mixture off the slide.

8. The slide may be rinsed with distilled water until the stain is removed, or it may be washed using a wash bottle.

9. Wipe the back of the slide.

10. Dry the slide in a vertical position, on an absorbent surface (bibulous paper).

11. Examine cells using a compound microscope or bright field in low-cost portable microscope.

1. Referring to Table 1, use either 20P or 100P or 200P micropippetor and add the appropriate solutions for the negative control, experimental groups (Wright stain at different concentrations ranging from 0.5%, 1%, and 5% vol/vol), and positive control using 1% (vol/vol) India ink diluted in distilled water.

2. When each component has been added and mixed, use a table-top centrifuge to pool all the solution at the bottom of the 1.5mL Eppendorf tube.

3. Use a parafilm and aluminum foil setup (Figure 2D–2G), and observe at 4 hours and 24 hours. Students can decide observation timepoints for their experiments. Tables 2—4 highlight example data that can be collected by students. Key frames from 1% (vol/vol) Wright stain from time-lapse experiment (Figures 3C–3E and 4).

Wright stain allows visualization of different stages of Tetrahymena throughout its life cycle. It labels the cortical grooves, the oral cavity, and cilia (Figure 3A–3C). This stain can either be used in heat-fixed samples, or be modified to monitor cellular events in living cultures as mentioned below. Based on the collected data from the above pilot experiment in living cells, we determined that the 24-hour pre-incubation of 1% Wright stain (vol/vol) highlights the following organelles: (1) several food vacuoles and vesicles labeled in ranges of color from light violet to dark purple; (2) the activity of the contractile vacuole; (3) and ciliary motion.

1. Make a dilution of 20µM by mixing 5µL of Sytox green dye from the original stock concentration of 100µM with 20µL of Tetrahymena (cultured for three days) to a final volume of 25µL. Follow the set-up of reagents listed in Table 5 with appropriate micropippetors.

2. Use No. 5 forceps to make seven holes into Parafilm paper, and cover the Tetrahymena culture in the Eppendorf tube. Let the sample incubate for three hours.

3. Use Tetrahymena untreated as a negative control, following the same incubation steps.

4. Place 20µL of the untreated Tetrahymena on a glass slide carefully; then place 20µL of the experimental group on a different glass slide, and cover it with a cover slip, making sure of not let the air bubbles to form on any of these samples.

5. Use an inverted battery-operated fluorescence microscope (Miller et al., 2010).

1. Take 50 microliters of pre-diluted Tetrahymena in Sytox green already incubated for three hours (from the previous procedure).

2. Distribute it onto a glass slide

3. Heat-fix the sample carefully.

4. Examine by inverted low-cost fluorescence microscope (Miller et al., 2010).

After three hours of incubation, we observed that labeled nuclei in cells with plasma membrane were compromised, and several Tetrahymena cells were moving while observed with an inverted bright field fluorescence microscope (Figure 5A–5D). Heat-fixed pre-incubated Tetrahymena with Sytox green dye labels macronucleus (MAC) and some visible cytoplasmic stain (Figure 5E–5G).

In summary, we developed new, useful low-cost application tools for phenotypic analysis in Tetrahymena. In combination with Cellcams (Martin & Shin, 2016) and a portable low-cost fluorescence microscope (Miller et al., 2010), we demonstrated the utility of the following methods: heat fixation, Wright stain, and Sytox green fluorescence dye labeling. The versatility of the Wright stain in heat-fixed and live cell approaches allows the observation of cellular organelles, such as cilia, oral cavity, and some of the subcellular organelles (e.g., vacuoles and vesicles) during the life cycle of Tetrahymena. In future lab-inquiry and research settings, Wright stain and Sytox green dye staining approaches will provide a platform for phenotypic analysis of cellular decision-making processes in the changing microenvironment of Tetrahymena.

We thank Dr. Rebecca Richards-Kortum at Rice University for her gift of low-cost fluorescence microscopy; Dr. Terese Abreu Director of MLS program at Heritage University (HU) for her gift of Wright stain; Alejandra B. Cruz Adjunct English Teacher at HU for support in writing process; anonymous reviewers for their comments; and support through HU's NSF REU grant DBI #1460733.