The Use of Tetrahymena Stained with Acridine Orange to view Inner Cellular Changes

John Giannini

Biology Department, St. Olaf College

giannini@stolaf.edu

Introduction

The ciliated protozoan Tetrahymena is an excellent organism to study cellular functions. It is easy to grow and there is a wealth of information known about its physiology and genetics. It is especially good for student labs due to the many experiments already available (see the Tetrahymena experiment lab manual at http://pages.stolaf.edu/opn-lab/). It is often studied using standard brightfield microscopy techniques. One way to increase the information available to students is to stain the cells with a fluorescent molecule and to view the cells with fluorescence microscopy. Until recently the cost of a good fluorescence microscope ($20,000 dollars and up) has been prohibitive to most schools. Recently our lab (OPN LAB) has designed and built a low cost epi-fluorescence microscope ($250.00 ) that gives excellent images at a fraction of the usual cost (http://pages.stolaf.edu/opn-lab/). In combination with a fluorescent dye like acridine orange you can identify many cellular components not visible with a compound brightfield microscope.

Acridine orange is a cell permeable cationic dye that will sequester in acidic organelles such as lysosomes or acidic vacuoles (1). The pH of the compartment determines the emission wavelength (color) seen under the fluorescence microscope. As the compartments become more acidic their color changes from green to red. It is also nucleic acid selective often used to stain the macronucleus and micronucleus of Tetrahymena (2, 3) with a green emission. Typically you use a blue excitation filter followed by a green emission filter ( we used a NikonB-2A GFP blue /green epi-fluorescence cube). The dye is easy to mix and can be added to the cells without cellular fixation.

Materials:

- glass microscope slides- glass cover slips- acridine orange solution (50mg /L of water)- nail polish- pipette that can measure 5µl of solution- Pipette tips- fluorescence microscope (such as the OPN epi-fluorescence microscope, Nikon B -

2A ,blue / green, GFP epi-fluorescence cube)- 24 hour Tetrahymena culture grown in modified Neff media (see the Tetrahymena

experiment manual at http://pages.stolaf.edu/opn-lab/ for details on how to grow Tetrahymena)

Methods and Results

Step 1. To view Tetrahymena cells with acridine orange they must be alive, but they are mobile and hard to view and photograph while moving. You can make a simple device to trap them and still keep them alive. Begin by placing a very thin layer of nail polish in a line across the end of a microscope slide (Figure1). Let it dry. Now you can place a small amount of Tetrahymena (no more than 10 µl) next to the dry nail polish line (Figure2). Place a cover slip over the top of the cells making sure one end is on the nail polish line (Figure3). You can now find live trapped Tetrahymena under the cover slip. Search the area farthest away from the nail polish line first. We call this device our Tetrahymena trap.

Step 2. On your Tetrahymena trap slide place 5µl of your Tetrahymena culture. Next add 5ul of acridine orange (50mg/L) to the Tetrahymena. Gently mix then cover with a cover slip and view under a fluorescence microscope (such as the OPN Epi-fluorescence microscope on the OPN LAB web site (http://pages.stolaf.edu/opn-lab/).

Step 3. You can now place your Tetrahymena cells that are under stress (age, environmental, chemical, etc.) on a slide and observe the changes to the nucleus, stained vacuoles and acidic organelles inside the cell. The series of photos below illustrates the cellular changes due to the aging of a Tetrahymena culture (Figure 4,5,6). The experiment took place over an 8 day period where a Tetrahymena culture (31ᵒc,75 RPM on a shaker in Neff media) was sampled every day until the cells died (day 9). Photos were taken at 1000x (oil immersion) with the OPN epi-fluorescence microscope (Nicon B-2A, GFP cube) and an Omax A35140U digital camera.

Figure 1. Paint a thin layer of nail polish onto a microscope slide. Be careful to make the layer as thin as possible by wiping the excess polish off the brush before you paint.

Figure 2. Pipette 5 microliters of Tetrahymena sample and 5 µl of acridine orange onto the slide next to the nail polish line.

Figure 3. Place a coverslip over the sample with one edge of the slip resting on the nail polish strip.

Figure 4. A one-day-old Tetrahymena cell stained with acridine orange. In this photo you can clearly see the macronucleus, the micronucleus and a number of acid compartments (with various pH and sizes) inside the cell. You can also see the contractile vacuole at the posterior of the cell (top right in the photo) as a large dark vacuole.

Figure 5. A four-day-old Tetrahymena cell stained with acridine orange. After four days you can see an increase in the number of acidic compartments. There are more orange vacuoles and the average size of the vacuoles has decreased. You can also see the macro-nucleus.

Figure 6. Eight day old Tetrahymena cells stained with acridine orange. After eight days you can see many small acidic compartments that are dark orange or red (very acidic). The macronucleus and the micronucleus are visible with the macro-nucleus showing enlargement and distortion. The contractile vacuole is visible and enlarged.

Conclusions

As you can see, Tetrahymena stained with acridine orange can reveal a lot of information about the cell’s internal organelles and physiological condition. These results are similar to what others have observed when staining cells with acridine orange (1, 2, 3). In all three of the photos we can see nuclei, contractile vacuoles and numerous acidic compartments. Also the size and shape of the acidic compartments shows tremendous variation. The cytoplasm stains green which makes it easy to see the complete outline of the cell. It is also apparent as the cells age numerous changes can be observed. There seems to be an increase in the number and acidity of the acidic compartments with a concurrent reduction in size. The macronucleus was misshapen by day 8 and the cells appeared smaller. You can also see the enlargement of the contractile vacuole by day 8. These results demonstrate how acridine-orange stained cells can be used to investigate intercellular changes due to stress.

This simple procedure allows students to ask questions about the effect of stressors on Tetrahymena and follow the progression of the cellular changes that take place. An example might be looking at the effect of aging as demonstrated in this manual or investigating the effect an environmental pollutant such as Round-Up (a herbicide) on inner cellular structures. The possible questions are endless and they allow students to do investigative research that gives real data that can be used to write a paper or lab report.

Safety

Acridine orange is an eye and skin irritant. It should not be inhaled or ingested. Be sure to read The MSDS for details.

References

1. Wikipedia.https://en.wikipedia.org/wiki/acridine orange (accessed March 2017).

2. Mpoke S. and Wolfe J. (1997) Differential Staining of Apoptotic Nuclei in Living Cells: Application to macronuclear Elimination in Tetrahymena. 45(5): 675-683.

3. Lu E. and Wolfe J. (2001) Lysosomal enzymes in the Macronucleus of Tetrahymena During its Apoptosis-Degradation. 8: 289-297.