ETHIDIUM BROMIDE/ACRIDINE ORANGE VIABILITY STAINING METHOD FOR COUNTING CELL POPULATIONS OF TETRAHYMENA PYRIFORMIS

A Report of a Senior Study by Abigail Elizabeth Ogle

Major: Biology

Maryville College Fall, 2010

Date Approved

, by Faculty Supervisor

Date Approved

, by Editor

ABSTRACT

Fast, inexpensive, reliable techniques to quantify cell populations in culture are crucial for laboratory experiments. Currently, standardized methods for cell counting of culture populations include hand counting with a hemacytometer, digital measurement with a Coulter Counter, or spectrophotometric absorbance. In this study, the objective was to investigate a new method for enumeration of Tetrahymena pyriformis (ciliated protozoa) using an ethidium bromide/acridine orange (EB/AO) viability stain. Initial subcultures from dense cultures were diluted to a concentration of 5000 cells/mL. For the three days of linear population growth, daily subsamples of T. pyriformis cultures (n=27) were analyzed via standard hand counting with a hemacytometer and with EB/AO staining. The stained cultures were plated in triplicate and absorbances at 495nm were read by a microplate reader. The data were standardized by z-scores and the slope of the regression of the average hand count versus day was compared to the slope of the regression of the average absorbance versus day. A Z-test indicated that there no significant difference between the two slopes (p=0.8258). The line equation generated from the linear regression of average daily absorbance as a function of average density, y=1540.4x+1532.3, can be used to determine the population density of cultures. It is concluded that the ethidium bromide/acridine orange cell counting method is recommended for further validation studies. iii

TABLE OF CONTENTS

Chapter I II

Page Introduction.............................................................................1 Materials and Methods............................................................12 Preparation of Phosphate Buffered Saline..........................12 Preparation of EB/AO stain................................................12 Preparation of 2% Proteose-peptone..................................13 Innoculation of cultures......................................................13 Establishment of a population growth curve of T. pyriformis with hemacytometer counting..............14 Enumeration of T. pyriformis with EB/AO stain...............14

III IV V

Results....................................................................................17 Discussion..............................................................................22 Works Cited...........................................................................27

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LIST OF FIGURES

Page Figure 1. Light microscopy image of Tetrahymena pyriformis Figure 2. A comparison of the average density (cells/mL) and average absorbance at 495nm as a function of time Figure 3. A comparison of the average density (cells/mL) and average absorbance multiplied by 100,000 at 495nm as a function of time. Figure 4. A linear regression of the calculated Z-scores for average daily hand counts and average absorbance at 495nm as a function of time Figure 5. A line graph displaying the average daily absorbance multiplied by 100,000 at 495nm as a function of average daily density (cells/mL) with linear regression. 21 20 19 18 3

ACKNOWLEDGEMENTS

I would like to thank Dr. Angelia Gibson for generously loaning me a centrifuge for the duration of my experiment. I would also like to thank Dr. Jeff Bay for his patience with me and for his insight into statistics. Finally, I would like to thank Dr. Jerilyn Swann for all of the support, encouragement and instruction that enabled me to complete this study.

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CHAPTER I

INTRODUCTION

The most diverse kingdom of Eukaryotes is kingdom Protista which includes a vast number of organisms all of which are very different. The one characteristic that all protozoa share is that they are neither fungi, plants, nor animals (Raven & Johnson 1999). Protozoa are single celled organisms that have existed on earth for millions of years. These organisms are indeed primitive; however they are complex in the sense that in one cell they are able to perform all of the necessary mechanisms that are characteristic of multicellular organisms and vital for life (Caulkins 2007). The kingdom Protista is generally divided up into 3 different categories: slime molds, algae, and protozoa (Milewski 2004). Of kingdom Protista, protozoa are considered to be animal like because they are able to move about and find food. Protozoa are able to live in a wide range of environments, including ocean water and freshwater. The distribution of protozoa in a habitat is influenced by a number of factors including temperature, light, chemical composition of water, and food availability (Kudo 1971). They are able to occupy and

survive unfavorable conditions by formation of cysts which are dormant cells that have a strong outer-layer that protects them (Raven & Johnson 1999). Protozoa are suitable and important for scientific research in a number of different fields. Geneticists use them to study inheritance and variance protozoa have a short generation time so changes in the genetics are quickly and easily observable (Kudo 1971). In cytology, studies of protozoa have also provided insight into the relationship between the cytoplasm and nucleus, because they have numerous modes of cellular division (Kudo 1971). Protozoa can be divided into smaller subgroups by their mechanism of movement. These subgroups are flagellate, pseudopodiate, or ciliated protozoa (Raven & Johnson 1999). Ciliated protozoa are highly complex cells with many unique features. The ultrastructure of these organisms is incredibly intricate for a single cell. The cell body of a ciliated protozoan is covered with hundreds of cilia, in kinetosomes, which are arranged in organized rows called kineties (Lynn 2003). Cilia movement is synchronized, and enables protozoa to navigate through water and find food. The majority of ciliates also have a buccal cavity that is highly ciliated. As far as internal structure goes, ciliates are heterokaryotic, meaning that they have two nuclei; a macronucleus and a micronucleus. The macronucleus is larger and is responsible for the fundamental processes of the cell that keep it alive, such as metabolic processes (Elliott 1959). The micronucleus is both smaller than the macronucleus and not an essential component of a cell. It is concerned with regulating the sexual cycle and the exchange of genetic material among two cells (Elliott 1959). Tetrahymena also have mitochondria, golgi and endoplasmic reticulum

present in their cytoplasm. These organisms are amazingly complex in contrast to their small size.

Figure 1. Light microscopy image of Tetrahymena pyriformis.

Tetrahymena are ciliated protozoa that belong to the order Hymenostomatida of subclass Ciliophora (Corliss 1961). The majority of ciliates possess a peristome, or a functional mouth, often associated with a well-defined buccal or peristomal cavity equipped with compound ciliary organs (Corliss 1961). The oral cavity is composed of four membranes that are extremely specialized. On one side of the oral cavity, there are three membranes that are highly ciliated and collectively are called the undulating membrane, opposite the undulating membrane are oral ribs which extend to the cytostome (Sattler and Staehelin 1974). When Tetrahymena find food particles, their cilia act to move the food into their mouth and towards their cytostome. Tetrahymena and other unicellular organisms feed by phagocytizing bacteria and particulate matter present in their environment. Tetrahymena are commonly 3

characterized as grazers, they are very non-selective about the food they ingest they will phagocytose anything, from bacteria to latex beads, to india ink. The mechanism of phagocytosis in Tetrahymena is a complex process which is dependent upon the rearrangement of the actin cytoskeleton (Rosales 2003). Calcium and gelosin act together to sever the actin filaments of the cytoskeleton and allow a phagocytotic vesicle to enter the cell (Serrander et al. 2000). Once phagocytized, the ingested material are held in membranous vacuoles called phagosomes (Rogers and Fosters 2007). The phagosome travels through the cell and moves towards the cytoproct, or anus, at the anterior end of the cell and is eventually released via exocytosis (Jacobs et al. 2006). Ciliated protozoa, such as Tetrahymena, are particularly important in the sewage treatment process. Communities of Tetrahymena and other ciliated protozoa act as grazers and are able to remove a substantial amount of particulate matter and bacteria present in the sewage tanks to aid in the purification process. One study on the ability of ciliates to purify sewage indicated that ciliates account for the removal, by predation, of mass quantities of bacteria present in sewage (Curds and Cockburn 1968). These communities of protozoa are particularly susceptible to pollutants and toxins, and their growth is an indicator of the poor quality of water in the treatment plants (Nikolau et al. 1999). Tetrahymena is a particularly useful organism for determining the effects of pollutants and toxins on cells. Toxicity tests are used to determine if a chemical or a substance will have a negative effect on an environment and the populations within it. Tetrahymena have been used to investigate the toxicity of thousands of substances. A few of the types of substances that Tetrahymena have been exposed to for toxicity tests 4

are carcinogens, insecticides, radiation, drugs, and detergents (Lukacinova & Mojzis 2007). Most toxicity research has been investigating population growth rather than cell viability; researchers believe that the impairment of population growth is a better indicator of toxicity than cell death. The effects of toxic substances on ciliated protozoa is useful as an indicator of how the substance will affect other animals. It is thought that ciliated protists may be able to serve as a model for much more complex organisms like mammals because mammals contain cilia in their respiratory tract. Therefore, if a substance damages the cilia of a protist, it may be possible to infer that the cilia of the mammalian respiratory tract could be harmed as well by exposure to the substance. Tetrahymena are also used in production of biopharmaceuticals. They are able to produce a high yield of the desired product at low costs. One of the advantages of using Tetrahymena in biopharmaceuticals is that the product can be produced in bioreactors in large amounts. The generation time of the organisms is short so a stationary production phase can be reached easily (Nanney and McCoy 1976). Tetrahymena pyriformis naturally occur in lakes, ponds, and virtually anywhere with freshwater. It has been documented in habitats ranging in temperature from 4 to 37C, and even at elevations as high as 10,000 feet (Elliottt 1959). T. pyriformis is pyriform, pear shaped, and possesses 17-21 rows of cilia (Elliott 1959). Cell length ranges from 30 to 60m in length but is variable depending on the environmental conditions that the cell is experiencing (Elliott 1959). Naturally, they are bacteria feeders, but can be grown in the laboratory in nutrient rich liquid media. Tetrahymena are able to be grown rapidly and in large quantities their doubling time is about two hours and they can grow and survive in high densities. The established standard growth 5

curves for T. pyriformis have a characteristic shape of a flat line that slowly begins to increase then there is a dramatic spike and peak followed by a sudden crash of the population. The phases of the growth cycle are evident in the growth curve. During the culturing of Tetrahymena there are four different steps of growth that have been identified. The first step encompasses the inoculation of a culture to the lag growth phase and through the acceleration growth phase. Step two includes the first half of the logarithmic growth phase and step three consists of the second half of the logarithmic growth phase and the peak of the population density. Once the population of Tetrahymena exhausts the resources of its environment, the population crashes, this is step four of the growth cycle (Satir and Dirksen 1971). This rapid expansion and crash of the growth cycle is characteristic of populations of T. pyriformis. The life cycle of Tetrahymena is characterized by alternation of haploid and diploid generations. Reproduction in Tetrahymena can be either asexual or sexual depending on existing environmental conditions. Tetrahymena reproduce asexually by binary fission during vegetative growth when sufficient nutrients are available and environmental conditions are favorable. The micronucleus is inactive during asexual reproduction and the macronucleus is active. During asexual reproduction, the macronucleus elongates, cinches in the center and each of the two halves are passed on to daughter cells (Kerrer 2000). There are no functional centromeres present in the macronucleus of Tetrahymena, and the macronuclear envelope remains intact throughout cell division (Kerrer 2000). Tetrahymena will continue to reproduce asexually as long as they are maintained in nutrient rich media.

Sexual reproduction in Tetrahymena is by conjugation between two different mating types and two cells that are sexually mature. Seven different mating types have been identified in Tetrahymena (Kerrer 2000). The mating type of a cell is under genetic control and is determined during sexual reproduction in a newly produced macronucleus. The micronucleus is functional only during sexual reproduction and it is involved in the transfer of genetic material between two cells of different mating types. In sexual reproduction, genetic information is exchanged though a process called nuclear exchange. Nuclear exchange in Tetrahymena is a unique process diploid cells form a fusion junction anterior to their oral apparatus, and emerge from conjugation 12 hours later having exchanged haploid nuclei, while reestablishing cortical integrity (Cole 2006). Conjugation results in the formation of four cells which are called karyonides (Kerrer 2000). Research has revealed that Tetrahymena can have their growth cycles synchronized by inducing conjugation (Gardonio et al. 1973). Conjugation can be induced in Tetrahymena by removing the cells from their nutrient rich media and starving them. When Tetrahymena are starved and then introduced to cells of complementary mating types, they will undergo conjugation (Kerrer 2000). The synchronization of growth cycles enables genetic research to be performed on these organisms. Tetrahymena are microbial models for research. Culturing and maintaining them in a lab is relatively easy and inexpensive. These organisms have the fastest growth and doubling time of any microbial model. Tetrahymena can be grown anexically, in pure culture, or with other bacteria for it to feed on, therefore the researcher can choose whether or not phagocytosis is needed for an experiment and completely control the culturing environment. They can also be grown on a wide range of scales, from just a 7

few drops to large reactor vats (Orias 2001). These characteristics of Tetrahymena make it ideal for use in research in various scientific studies. While it is relatively easy to culture T. pyriformis, it is difficult to find a method to enumerate them that is low-cost, easy, and accurate. A reliable technique to quantify the number of cells is necessary for many experiments performed in laboratory settings. Currently, the well recognized methods and instruments used to count the number of Tetrahymena are a hemacytometer, Coulter Counter, and a Spectrophotometer 20. Counting cells using a hemacytometer is one of the most widely used methods of counting cells used today. It is often regarded as the gold standard by which cells are counted. A hemacytometer is a specially designed glass slide that has two chambers that are each divided into smaller grids. A specific volume of the cells to be counted are loaded into the chambers and the cells are counted manually. The number of cells counted in the squares is multiplied by the dilution factor to calculate the original number of cells in the sample (Invitrogen 2010). Benefits of using a hemacytometer are that it is relatively easy to use and it has a low cost. While this method is widely used, there is also a significant amount of error that comes with using a hemacytometer. Error can come from improper loading of the hemacytometer, inadequate dilution of the samples, and human error in counting the cells. Another issue with hemacytometer counts is that this method is extremely tedious, time consuming and it is difficult to distinguish between viable and nonviable cells (Invitrogen 2010). A Coulter counter is another method for enumerating cells in a sample. This machine employs a much more advanced method of calculating cell numbers. A Coutler

counter has two electrodes that are submerged in a conductive liquid. There is an opening of a fixed size that a current flows through. Cells are forced through the opening and there is a voltage pulse per cell that flows through (Heaney et al. 1994). The number of charges that occur are calculated and the number of cells can be determined. The Coulter counting method is rapid and reliable in determining the total number of cells, however there are a number of drawbacks that arise when using a Coulter counter. Primarily, the Coulter counter is a very delicate device and is prone to become clogged with cells or particles if they are too big to fit through the aperture. Also, the counter is unable to distinguish between cells and debris, so if a piece of debris is small enough to fit though the hole, then it would be counted as a cell. The Coulter counters also cannot distinguish between a living and a dead cell, so it does not give an accurate viable cell count. A final issue with the Coulter counter is that it is a very expensive piece of equipment and is not feasible for use in undergraduate laboratories because of the high cost. The final method, a Spectrophotometer 20 (Spec20), measures the turbidity of the sample. A Spec 20 works by measuring the amount of light that passes through a sample, which is directly proportional to the number of cells in the sample. The amount of light transmitted is dependent upon the shape, mass, and opacity of the cells suspended in the culture (Knox & Rodriguez-Zas 2010). A Spec 20 is not an ideal method to count cells because the machine itself requires a lot of supplies including a filter that absorbs at a specific wavelength (Kaiser 2010). Also, the machine itself limits the number of samples that can be counted at a time. A Spec 20 is able to analyze only one sample at a time

which makes it a very inefficient and time consuming process to count a large number of samples, and collect data. In my senior study I want to develop a method for counting Tetrahymena pyriformis that is accurate, relatively easy to perform, and low-cost. I specifically want to be able to enumerate viable cells in a culture. In order to do this, an ethidium bromide/ acridine orange stain will be used in combination with a microplate fluorescence assay. The ethidium bromide/acridine orange stain (EB/AO stain) is a viability stain that detects apoptotic cells. Viability stains determine the membrane integrity of a cell based on the uptake or exclusion of a dye from the cell (Park et al 2000). Ethidium bromide is a dye that is only able to pass through the membrane of a dead or dying cell. Acridine orange is a membrane-permeable dye that will stain all cells in the sample. Each dye that is taken up by a cell fluoresces- AO makes a cell green, and EB makes a cell red (Baskic et al 2006). This stain will allow for the cultures of T. pyriformis to be visualized and enumerated by microplate fluorescence. Live cultures of T. pyriformis will be put into wells of a microplate and then read in a microplate reader with filters at 495nm and 515nm to visualize the fluorescence of the cells. To determine the number of cells per sample in relation to the amount of florescence emitted by cells, a log growth curve must first be established. Liquid cultures of T. pyriformis will be grown and counted using a hemacytometer. Samples with known numbers of cells will be stained and read by the microplate reader so baseline numbers can be established for the fluorescence of the cells and a standard curve can be made. The growth curves that are established by the hemacytometer counts will

10

be compared to the curve established by the microplate reader and tested for significant differences.

11

CHAPTER II

MATERIALS AND METHODS

Preparation of phosphate buffered saline A liter of 10X phosphate buffered saline (PBS) was prepared with 10.9g of anhydrous Na2HPO4 weighed out in a weigh boat and put in a 2000mL beaker. Next, 3. 2g of anhydrous NaH2PO4 and 90g of NaCl were weighed out and added to the 2000mL beaker containing the Na2HPO4. Distilled water (1000mL) was added to the beaker and the mixture was stirred into solution with a stirbar on a stirplate. Once dissolved, the pH of the solution was adjusted to 7.4 and stored in a screw cap bottle at room temperature. Preparation of EB/AO stain An amber bottle containing 1g of powdered Ethidium Bromide was obtained from Sigma-Aldrich (E8751-1G). A 100X stock solution of Ethidium Bromide/Acridine Orange stain was prepared and frozen according to the protocol provided in the Manual of immunological methods (Brousseau et al. 1999). A 1X working solution was prepared from a thawed 1mL aliquot of the 100x stock solution according to the protocol and stored in an amber bottle at 4C.

12

Preparation of 2% proteose-peptone Proteose-peptone (20g) (Fisher Cat no. 661852) was added to a 2000mL beaker containing 1000mL of distilled water. The mixture was stirred into solution using a magnetic stir-bar and stir-plate. Once in solution, the 2% proteose-peptone was divided equally among (5) 500-mL glass bottles. Each bottle was labeled as 2% proteosepeptone, the date, and my name. The 2% proteose-peptone was autoclaved for steriliziation. The sterilized 2% proteose-peptone was stored at 4C until needed. Innoculation of cultures An axenic culture of T. pyriformis was ordered and obtained from Wards Science (Cat no. 87.v 1400). The proteose-peptone was removed from the refrigerator and allowed to warm up to room temperature. Before inoculation of cultures, the concentration of cells/mL in the stock culture was determined by hand counting with a hemacytometer. A total of 5 sterile glass test tubes were set up in a wire test tube rack. The desired concentration for subcultures in test tubes 1-3 was approximately 5000cells/mL. Because the two stock cultures had different concentrations of cells the ratio of mL of stock culture to fresh 2% proteose-peptone was adjusted to allow the three subcultures to have the desired concentration. The caps were placed loosely on top of each tube to allow airflow. The inoculated cultures were placed in an incubator and incubated overnight at 22C.

13

Establishment of a population growth curve of T. pyriformis with hemacytometer counting Samples were taken from tubes 1-3 daily and counted to establish a growth curve. From each test tube, a 100-1000L micropippete was used to sample 250L of culture. Before the sample was taken, the test tube was gently agitated to break up any aggregations of cells. The sample was taken from just below the top of the culture, and deposited into a microfuge tube labeled with the date and tube number. Next, a 20200L micropipette was used to measure out 50L of 3% glutaraldehyde and placed in the microfuge tube containing the Tetrahymena. The tube was agitated on a vortexer to mix the cells thoroughly. Then the cells were sampled using a Pasteur pipette. Before loading the hemacytometer, the first 2 drops of T. pyriformis were discarded. Each side of the hemacytometer was loaded by capillary action until the chamber was filled completely, but not overflowing. The hemacytometer was placed under the low power objective of a microscope and the entire center grid was counted on each chamber of the hemacytometer. Then, the average between the two values for each grid was taken and multiplied by 2000 to get the number of cells per mL of culture. This method was repeated for each culture every day. Enumeration of T. pyriformis with EB/AO stain At the same time that the sample for enumeration by hemacytometer was taken, a second 200L sample of the culture was stained with EB/AO stain. Each 140L of culture was placed in a microfuge tube labeled with the tube number, date and EB/AO. Then 560L of EB/AO stain was added to the microfuge tube using a micropipette. The

14

microfuge tube was gently agitated and then placed in an incubator for 15 minutes 22C. After incubating, the microfuge tube was placed in the centrifuge and spun at 14.6 rpm for 5 minutes. Once spun, a micropipette was used to remove and discard the supernatant taking great care to avoid disturbing the pellet and remove any T. pyriformis. The sample was then spun again at 14.6 rpm for 2 minutes and the remaining supernatant was carefully extracted and discarded. Then the sample was re-suspended in 700L of fresh media. This technique was repeated for each sample tube. Once all cultures had been subjected to the stain and were re-suspended in fresh media, 225uL of each sample was placed into separate microplate wells of a 96-well microplate, to fill a total of 3 wells per sample. The location of each individual sample was noted. Also, 3 additional wells were loaded with cell-free 2% proteose-peptone to serve as blanks. Once the microplate was loaded, the microplate reader was turned on and the plate was placed into the microplate reader. On the computer, the program Microplate Manager 5.2 was opened up. The reader was set to read through a 495nm filter and the sample was read. The data were copied and pasted into Excel. Each day, the average absorbance for each tube was calculated and compared to the density (cells/mL) of the culture determined by hemacytometer. To observe trends, the daily absorbance and density for all tubes were averaged, and plotted in a scatter plot with absorbance on the primary y-axis, density on the secondary y axis, and time (days) on the x axis. The data were then analyzed using Minitab software to determine if the slope of the hand count data differed from the slope of the microplate data. Each data point was

15

standardized by converting it to their z-score. This was done using the following formula: Z-score= (value-mean)/standard deviation,

where the value was an individual data point for either the hand count, or microplate. Then the mean and standard deviation were the mean value and standard deviation of the appropriate data set. A simple linear regression model was fit for the z-score of microplate vs. day, and for hand count vs. day. The slope and standard error of each slope was determined and a Z-test was performed along with a 95% confidence interval to establish if the data were statistically significant.

16

CHAPTER III

RESULTS

The graphical comparison between the average absorbance at 495nm and average density (cells/mL) of cultures (n=27) was plotted as a function of time (days) is shown in Figure 2. Both trend lines increase as time increases, but have line equations that are on different scales. The line equation for the daily microplate absorbance from day 0 to day 2 is y = 0.0154x + 0.0153. The line equation for daily hand count from day 0 to day 2 is y=3884.6x+1174.4. To standardize the data, and compare the line equations when on the same scale, the average daily microplate absorbance was multiplied by 100,000 and the graph is displayed in Figure 3. Again, both trend lines increase as time increases, however the line equations are on the same scale and very similar. The line equation for the average daily hand count remained the same, y=3884.6x+1174.4, because the data were not multiplied. The line equation for the scaled daily average microplate is y=1540.5x+1523.3.

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14000 13000 12000

0.065 0.06 0.055 0.05 0.045 y = 0.0154x + 0.0153 R_ = 0.8275 y = 3884.6x + 1174.4 R_ = 0.9993 0.04 0.035 0.03 0.025 0.02 0 1 Time (Days) 2 handcount microplate

Absorbance at 495nm

Density (cells/mL)

11000 10000 9000 8000 7000 6000 5000 4000

Figure 2. A line graph comparing the average density (cells/mL) to the average absorbance at 495nm of cultures (n=27) both plotted as a function of time (days).

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14000

7000 6000 5000 y = 1540.4x + 1532.3 R = 0.8275 y = 3884.6x + 1174.4 R_= 0.9993
2

Absorbance x 100,000 at 495nm

Cell Density (cells/mL)

12000 10000 8000 6000 4000 2000 0 0 1 Time (Days) 2

4000 3000 2000 1000 0

handcount microplate

Figure 3. A line graph comparing the average density (cells/mL) to the average absorbance multiplied by 100,000 at 495nm of cultures (n=27) both plotted as a function of time (days).

However, the portion of data collected that was of interest was during the period of exponential growth in the cultures, so only days 0-2 were analyzed in Minitab. The data were stacked into columns and the z-score was calculated for the average hand count and microplate absorbance from days 0-2. The fitted line plot comparing the z-scores of the average hand counts and microplate absorbance versus day is displayed in Figure 4.

19

Figure 4. The fitted line plot of the linear regression of the calculated Z-scores for average daily hand counts and average absorbance at 495nm as a function of time (days).

Using the slopes of the z-scores, the calculated test statistic, Z= -0.216, lead to a two-sided p-value of 0.8258. The p-value was larger than the significance value (=0.05), therefore the average daily hand counts do not differ statistically from the daily average microplate absorbance. The 95% confidence interval (-0.9058, 0.7258) contains zero, therefore fails to reject the null hypothesis that the daily average hand count does not differ from the daily average microplate absorbance.

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Once it was determined that the daily average density (cells/mL) does not differ from the daily average absorbance at 495nm, an equation needed to be generated in order to be able to use the EB/AO staining method to determine the density of a culture from measured absorbance. The daily average absorbance at 495nm multiplied by 100,000 was then plotted as a function of daily average density (cells/mL) by hemacytometer as seen in Figure 5. The resultant line equation of the linear regression is y=1540.4x+1532.3.

6500

Absorbance at 495nm*100,000

6000 5500 5000 4500 4000 3500 3000 2500 2000 5000 9061.54 Density (cells/mL) 12769.23 y = 1540.4x + 1532.3 R2 = 0.8275

Figure 5. A line graph displaying the average daily absorbance at 495nm*100,000 as a function of average daily density (cells/mL) with linear regression.

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CHAPTER IV

DISCUSSION

The comparison between the slope of average hand count vs. day and average absorbance vs. day indicated that there was a lack of statistical evidence to support a claim of difference between the slopes (p=0.8258). This supports the null hypothesis that the slopes are similar, and that the absorbance of cultures subjected to EB/AO staining increases as the density of the culture increases. However, it is key to note that typical statistical tests are set up to show inequality among samples as opposed to equality, so the indication that there is a lack of statistical evidence to support the alternative hypothesis that the slopes were significantly different does not necessarily mean that the slopes were equal. A larger sample size would need to be tested to provide more concrete results. In comparing the average daily hand count to average absorbance per day, it cannot be definitively concluded that the absorbance measure for a culture could be used to predict the density of the culture with complete accuracy. This is because within each data set, the individual samples showed no significant correlation between the hand count and microplate absorbance recorded. However, from the graphical and statistical analysis of the averages of the data it can be said that there are similar trends in the hand count

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data and in the microplate data. The trends in the average daily density and absorbance indicate that over time, there is an increase in both absorbance and density. The linear regression of the average absorbance multiplied by 100,000 at 495nm plotted as a function of average density (cells/mL) resulted in the production of a line equation that can be used to determine the density of a culture from the average absorbance. The equation is y=1540.4x+1532.3, where Y represents the average absorbance at 495 and X represents the density of the culture. Using the EB/AO method, the average absorbance x 100,000 of a culture can be substituted in for Y and then the equation can be solved for X, and the density of the culture can be determined. There were several possible sources of error in this experiment, and several suggestions for improvement and future research. The wide range of absorbance values among each individual day could be attributed to pipetting errors and insufficient removal of the entire supernatant containing the stain. For future research, it may be beneficial to wash the cells and resuspend them twice in fresh 2% proteose-peptone to seek to eliminate the influence of excess stain being present in the culture. The presence of unabsorbed stain could potentially increase the absorbance measured by the microplate reader. Another factor which may have resulted in the presence of stain in the culture once resuspended into fresh media could be the result of diffusion. Acridine orange is a dye that initially enters the cell by diffusion across the membrane and binds to nucleic acids, therefore when the cells are resuspended in fresh media, some of the stain may diffuse across the membrane and back into the media (Robbins and Marcus 1963). Also, insufficient centrifugation of the cultures incubated with the stain may have resulted in varied results. Even though extreme care was taken to avoid removal of any T. 23

pyriformis while removing the excess stain, a number of cells may have been extracted on accident. This could be because T. pyriformis do not remain in the pellet for very long after centrifugation, so if the supernatant was not removed rapidly enough, some cells may have escaped the pellet and been removed as well. For future research, a longer centrifugation time may be beneficial in achieving a dense cellular pellet which stays intact for a longer time. Despite numerous experimental trials where the cell to dye ratio and the incubation time for stained cells was varied it is possible that the cell to dye ratio that was determined to be the most effective was not the best ratio possible. Also, the period of incubation of culture samples in the EB/AO stain may not have been the ideal incubation period. An improper cell to dye ratio and incubation period could prevent the cells from absorbing the stain and could alter the absorbance measured by the microplate reader. A final suggestion for further research would be to increase the sample size of cultures tested. An increase in sample size would decrease the influence of each individual data point on the overall averages, and decrease the standard deviation among the samples. The EB/AO staining method holds potential to be a very effective method in determining the density of cultures of T. pyriformis in laboratories of modest means. The materials and reagents required for the EB/AO staining method are inexpensive and readily available in most laboratories. The EB/AO stain is significantly lower priced than current machines used to count cultures, like the Coulter Counter, which requires a significant monetary investment which is more suited for commercial cell culturing (Stegemann et al. 2000). Additionally, the Coutler Counter is prone to becoming clogged with particulate matter in the culture and cannot distinguish living cells from dead cells 24

and debris (Stegemann et al. 2000). Also, the EB/AO method employs a viability stain in which AO diffuses into all cells and EB is not able to diffuse across a cell membrane unless it is compromised or the cell is apoptotic. Cells with only AO present inside fluoresce green, cells that are dead or have compromised membranes absorb EB which dominates over AO and results in the cells fluorescing red (Ribble et al. 2005). The wavelength measured by the microplate reader is 495nm, which is the optimal absorbance for AO, so only living cells and no apoptotic cells or particulate matter are quantified. The EB/AO staining method is also more efficient than the traditional method of cell enumeration by hemacytometer. Using a hemacytometer, only a single culture can be counted at a time. With the EB/AO stain, up to 32 cultures can be counted at a time if the samples are plated in triplicate on a 96-well microplate. In addition, hemacytometer counting is complicated by the fact that a person counts the number of cells in a determined area; this introduces subjectivity and potential human error (Stegemann et al. 2000). The use of a microplate reader eliminates the possibility of human error being influential because the absorbance is measured by a machine and then quantified to determine the density instead of individual cells being counted and multiplied to determine the density. An older spectrophotometric device that some labs use to quantify cell cultures is the Spec 20. This device, however, poses several hindrances for practical use in a laboratory setting which requires mass batches of cultures to be enumerated. Primarily, it requires a large volume of cultures and a filter that absorbs a specific wavelength of light (Knox et al. 2010). In addition, only one sample can be analyzed at a time in the 25

machine. This results in a time consuming process if mass batches of cultures need to be quantified for data collection. The EB/AO method does not require a wide range of materials or large volumes of individual cultures which leads to lower cost and ease of performance. This increased efficiency allows for faster data collection and quantification at a significantly lower cost. In conclusion, the usage of EB/AO viability stain as a potential method of enumerating cell cultures is recommended for further investigation. Preliminary research indicates that there is indeed a positive relationship between the measured microplate absorbance (at 495nm) of cultures exposed to the EB/AO stains and hemacytometer counts of cellular density (cells/mL) of the culture. Current data indicate a trend that as the density of the culture increases, the measured absorbance increases. This new method is rapid and relatively easy to perform and is economically feasible for modest laboratories.

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