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.
Hometown: Memphis, Tenn.
Senior Study Title: “Ethidium Bromide/Acridine Orange Viability Staining Method for Counting Cell Populations of Tetrahymena pyriformis”
Advisor: Dr. Jerilyn Swann
“Don’t get your hopes up” isn’t advice Dr. Jerilyn Swann gives all of her Senior Study advisees, but for the research biology major Abby Ogle was about to undertake, Swann knew it was appropriate.
“I told her from the start not to expect any positive results from her study – that most experiments like this don’t work,” the associate professor of biology explained. “I told her that she probably would be laying the groundwork for other students to continue what she did, and that eventually we’d figure out a protocol that worked. I really believed that.”
Ogle’s research was ambitious. Devising a method for accurately counting cell cultures of the ciliated protozoa, Tetrahymena pyriformis, was one that Swann had needed in her laboratory that operated on a modest budget and lacked some cutting-edge equipment.
“This freshwater protozoa is important to study because it models what happens in humans and other mammals,” Swann explained. “Cell counting is important in toxicology tests – the data can tell us how a population is going to react to toxins and chemicals.”
But counting cells for this type of research was no easy matter, unless a laboratory was equipped with a state-of-the-art Coulter Counter, which uses an electric charge to determine the number and concentration of particles flowing through a tiny hole in a tube. Other means include hand counting with a hemacytometer or assaying cultures with spectrophotometric absorbance.
Swann’s lab included a spectrophotometric device called a microplate reader, which, compared to the hand-counting method, yielded data in record time, but Swann was unable to find any literature on how to stain Tetrahymena pyriformis cells and assay them with the microplate reader.
Swann believed that Ogle, who had laboratory experience dating back to her freshman year in high school, could lead such an investigation. Ogle said she was excited by Swann’s suggestion for a Senior Study topic and wasted no time digging into published literature to broaden her understanding of protozoa and stains and dyes.
In the laboratory, she experimented with dozens of different kinds of stains. The research involved figuring out what ratio of stains to use, how long and how fast to centrifuge cultures (so as to acquire all the cells without killing them) and determining at what wavelength maximal absorption is obtained with the microplate reader.
“Viability stains determine the membrane integrity of a cell based on the uptake or exclusion of a dye from the cell,” Ogle wrote in her study. “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. This stain will allow for the cultures of T. pyriformis to be visualized and enumerated by microplate fluorescence.”
Swann said Ogle’s research required long hours in the laboratory, adding that her colleagues jokingly teased the biology major by suggesting that she must have a cot set up in the biology laboratory to be able to gather the amount of data she was collecting.
“She acquired the spectrophotometric data for numerous cultures, and for those same cultures, she also hand-counted the cell populations by microscopic observation to have a standard set of known values to compare with the spectrophotometric data,” the associate professor explained.
Admitting that the laboratory work was challenging, Ogle said she was also challenged by the statistical analysis needed to validate her hypothesis that ethidium bromide/acridine orange was the stain that could be used to accurately count cell cultures using the microplate reader.
Dr. Jeff Bay, associate professor of statistics, helped her to look at two data sets that have different measurement scales and plot them on a line graph. From the analysis, Ogle determined there was no statistical difference in data the two methods of counting yielded.
Ogle said she “got lucky” in her findings, but Swann said success was due to her advisee’s strong work ethic, great attitude and extraordinary laboratory research techniques.
She was so impressed with Ogle and her study that she submitted an abstract of the research to a panel of the American Society for Cell Biology (ASCB), which determined who would present at the society’s global conference in Philadelphia in December.
“Acceptances of abstracts is not guaranteed at this event – they are screened by a panel, and some people’s abstracts are rejected,” Swann explained. “We were notified in November that Abby’s abstract was accepted. And she was not accepted for the undergraduate poster session held the afternoon before the official meeting starts – her research was accepted for the main poster session, alongside graduate students, post-docs and principal investigators.
“I was so proud of her,” she continued, adding that she has attended the ASCB’s conference regularly as a member but has never taken a student with her to present.
Ogle said presenting at the conference was a “nice culmination” of her work back on the campus.
“I didn’t think [my process] would work,” she said, “so being able to contribute to the field meant a lot.”
While in Philadelphia, Ogle explained her research to numerous scientists who attended the poster presentation. One professor from the Midwest sought her out specifically after reading the conference abstracts.
“He was excited to find out what I had done,” Ogle said. “I think he wanted to replicate it in his own laboratory.”
Swann and Ogle are now working on a manuscript to submit to Bios, a scholarly publication for undergraduate research in biology. The associate professor expects Ogle’s procedure to be further tested and refined by future students, eventually resulting in a formula that will determine cell population density from the microplate reader data. Swann expects to use Ogle’s procedure in future cell biology courses.
Ogle said she isn’t sure if she has a future with Tetrahymena pyriformis, but after completing the study, she is more open to a career in the laboratory. (“I think the hands-on experience of the lab is really fun,” she said.) After a year, she plans to attend medical school and possibly earn an M.D./Ph.D. degree.
According to Swann, having presented at the ASCB main poster session and possibly being published in Bios, Ogle will be an impressive medical school candidate.
“Her Senior Study was exceptional for its process – and its product,” she said.