Written By: Madison Calvert, Amanda Hernandez, Natalie Sorial, Sarina Spolidoro, Benjamin Spenciner

Transcription

1 Written By: Madison Calvert, Amanda Hernandez, Natalie Sorial, Sarina Spolidoro, Benjamin Spenciner

2 A t Foxborough Regional Charter School (FRCS), a group of students began conducting tests to develop fixation techniques for other high school students to use that are simple to replicate and that use easily available chemicals. Normally, fixation techniques are complex and require chemicals that are difficult to acquire. We set up this experiment to find if ethanol (which is easy to acquire) will work very well in fixating organic specimens to be used in a scanning electron microscope (SEM). To use a SEM, the sample is placed on the stage and the door is closed. The machine then pulls a vacuum inside the chamber. At the top of the machine, electricity is sent through a filament made of tungsten, in a similar fashion to how a light bulb works. The filament heats up and starts to throw off electrons into the chamber. An anode focuses the electrons into a beam, which is fired at the sample. The electrons bounce off of the sample and their directions are picked up by three or four detectors, depending on the machine s style. Before using the SEM to view samples, another machine must be first used. The sample first has to be coated in metal to allow the electrons to bounce off of the sample instead of going through the sample. The machine that accomplishes this is called a sputter coater. This machine works by first drawing a vacuum inside the chamber, similar to how the SEM started. Then, the machine fills the chamber with argon gas, preventing the risk of explosion. There are magnets inside the machine that direct the flow of a bunch of gold-palladium atoms, making them stick to the sample. After about a minute, the sample comes out, coated in a gold-palladium alloy.

3 Kidney Samples: 10% at 492x, 10% at 1230x, and 10% at 720x (L to R)

4 Fixation Methods and Techniques: In this experiment we used Different concentrations of ethanol and distilled water as our main fixation technique. In essence we used the ethanol because it is a systematic chemical used to "dehydrate" samples and also because our experiment revolved around which percentages of ethanol used to fixate a biological matter, would make the image on a Scanning Electron Microscope (SEM) the clearest. Each member of our team was accountable for their own percentage of Ethanol. 50% Ethanol (the largest amount), 20% Ethanol, 35% Ethanol, 10% Ethanol, and 5% Ethanol. We also included a control solution which was the perfect solution. We soaked a total of six heart samples and six kidney samples, one for each solution. We utilized the heart and kidney of a rat. After allowing the ethanol solution to soak for 55 hours, we took the specimens out and placed each one on a petri dish to dry for hours. Once fully dry we sputter coated the samples as our last fixation method. This step enabled the sample to be covered with a layer of metal so that the tissue could be observed under the SEM through the use of bouncing electrons from the central electron beam of the microscope. What the experiment is and why it was conducted: Heart 10% at 136x, Heart 35% at 1230x, Heart Control at 135x (top to bottom) This project was started with the intent of determining which percentages of a particular fixation method are most efficient for getting the clearest image on a scanning electron microscope. At the commencement of the experiment we established that every group member would take a percentage of ethanol (fixation liquid) under their wing which they would measure out, soak sample in, allow organ to dry, sputter coat their specimen, and take pictures on the Scanning electron microscope to determine which fixation was able to take the clearest image on the microscope. We hoped this lab would be extremely revealing because we needed to find the most efficient fixation percentage that would develop the best picture. Because ethanol dries out biological matter and the dryer a sample is the better resolution you get we believed the percentage that would take the best picture would be the 50% since it was the highest level of ethanol present in any of the samples.

5 Procedure: First choose a solution that will act as your fixation solution. In this case that would be the chemical, ethanol. Next choose a total of five different percentages of your fixation solution (ethanol). To follow our exact experiment you will need a 50%, 35%, 20%, 10%, and 5% solution of ethanol and distilled water along with your control (perfect solution) solution to soak your specimens in for hours. Dissect a rat and harvest its heart and kidney. Remove the organs from the animal. Cut each organ into six equal parts- 5 for Heart 10% at 1220x your ethanol solution and 1 for the perfect solution. Label each of your tubes, where you have let your samples soak in their solutions, with their percentages of ethanol so that your experiment will not be compromised. After the hour time period remove the samples from their individual tubes. Allow the specimens to dry fully on petri dishes for hours. Again label each petri dish with the percentages of ethanol that corresponded with each organ portion. Sputter coat your fully dry specimens. Insert each of the samples into the Scanning Electron Microscope and take a total of five images of each sample at various magnifications to later analyze the quality of the image the microscope was able to take of the specific specimen. Colorize each picture on Adobe Photoshop and analyze which percentage of ethanol was able to result in taking the best picture by drying out the specimens at different strengths. Draw a conclusion based on your observations and conclude whether your hypothesis was correct or incorrect. Kidney 35% at 1230x, Kidney 50% at 135x (L to R)

6 Heart 5% at 135x, Kidney control at 715x

7 Results: Through analyzing the data, we came to the conclusion that the results were inconclusive. We believed before conducting the experiment that samples left in solutions with higher amounts of ethanol, would be more effectively dried out. Each solution that was prepared resulted in varied and unexpected outcomes. Overall and as seen in the pictures below, the heart sample reacted more effectively to the fixation solutions than the kidney sample. To obtain these results we determined the highest magnification that could be reached with clarity for each ethanol percentage, and used it to measure the effectiveness of the solutions. The kidney samples were put into the microscope in increasing order of percentage of ethanol. After the pictures were captured and colorized, as a group, we chose the picture, from each category, with the best clarity at the highest possible magnification. For the 10, 20, and 35, it was found that the highest magnification we were able to achieve was 1230x. The control group and 50 percent samples were only able to be magnified up to 492x, and the 5 percent sample could only be seen clearly at 135x. We repeated these exact steps for the heart samples, but received extremely different results. Each specimen that was soaked in the 10, 20, 35, and 50 percent solutions was able to clearly be magnified up to 1230x. The 5% solution was able to be magnified up to 715x with exceptional Heart 5% at 715x (top) vs. Kidney 5% at 715x (bottom) results, but the most unexpected result was the fact that control sample actually worked the best with the microscope after being dried out and sputter coated- it was able to be magnified 3320x. As mentioned before, the heart samples reacted in a more positive way than the kidney samples. The pictures to the side exemplify these conclusions. The top picture is of the 5% heart sample at 715x, and the bottom of the 5% kidney at 715x. The pictures show the vast difference in results. The heart sample was able to produce a crisp image, while the kidney produced a slightly unfocused, fuzzy, and low resolution image. This heart control sample was left in the perfect solution that was inside of the rat specimen from which the organ was taken out of. It was left in the same conditions as the other samples and received no other attention. This sample was meant to be a baseline to compare results with of the experimental groups, but instead it outshone the

8 performance of the other samples. Ultimately, our hypothesis was incorrect, in that the solutions that contained higher amounts of ethanol did not help to create better image. Sources of Error: There were many factors that could have altered the results of this laboratory experiment. First, it must be acknowledged that the students of FRCS were not able to control the quality of the specimen. They were ordered through Carolina Biological Company. Results concluded are based on the assumption that all biological samples are prepped in the same conditions before the animal is dissected. Second, the dilutions were measured and made by different students from FRCS using possibly imprecise Heart Control at 3320x measurements (a 10ml graduated cylinder). It is very possible that each solution could be 2-3% off what we calculated it to be. These minor changes in solution could have possibly affected the image quality produced by the scanning electron microscope. The samples were cut by hand by the same students, and cut by estimating the sample sizes to be equal. This presents two possibilities that could have also impacted the images. First, although the organ samples were taken from the same organ, they were not the same segment of the organ. Each organ has specific functions that take place in localized areas of the organ, therefore, we were comparing different parts of the organ: hence different cellular functions, and possibly a different ability to produce quality images under the scanning electron microscope. Second, since the organs were cut with a scalpel and estimated by eye to be the same size- they could have dried differently due to the variation in volumes of the samples. The samples were left in a laboratory preparation room to dry. This room is open to all science teachers and students in our school, anyone could have affected the results, whether through traces of chemicals left from another experiment in the room or by handling it incorrectly. Any altercation leaves trace residue on the samples, even if it is a minuscule impact. The sputter coater used in this experiment is at least 40 years old, and has had a history of acting up or not working. It is highly probable that the metal coatings on the samples are different because of: the different surface areas of the samples, possible remaining moisture in the samples, and faults in the older machine manually run by high school students. Like the sputter coater, the scanning electron microscope has a history of being difficult to work with, especially since we are high school students with a lack of expertise managing the machine. There were some days when it would not work at all. There were moments when it would not focus on a low magnification. Many pictures of the samples were taken under situations like these This experiment took the course of many weeks, some samples from the same organ were viewed several days apart from each other. This gap in time of data collection allows for possible variation in the samples itself. Although all of these considerations may seem minor, when considered with respect to each other, it gives a high probability of error in the results we collected.