Objective:

The main goal of this experiment was to be able to continue to find ciliates in our soil samples. During the lecture portion of this lab, phylogenic tree examples were presented and explained in order to better our understanding of relationships between different types of ciliates. These trees and other images of ciliate morphology allowed us to try and identify the ciliates we have found in the soil samples. During this lab, we also analyzed our soil textures that were prepared in the previous lab in order to better understand the type of environment the ciliates live in.

Purpose:

It is important to understand ciliate morphology and phylogenic trees because it helps uncover the various amounts of ciliate species within the soil ecosystem. Morphology and phylogenic trees allow us to compare the ciliates in the soil samples to other ciliates already known to see if they’re related, the same, or an entirely different type of ciliate. It is important to know and understand the certain types of ciliates within our soil samples because the ciliates effect the nutrients of the soil which in turn effect the plant growth and various other organisms in the environment.

Materials:

-dissecting microscope

-compound microscope

-concavity slides

-methyl cellulose

-iodine

-micropipettes

Procedure:

1-Soil Texture

-obtain the falcon tube from last lab

-hold the tube next to a ruler and measure the total amount of millimeters of soil is in the tube

-measure the number of millimeters for each layer of the soil

-divide each layer by the total to determine the percent composition of the specific type of soil in the sample

-record values in Table 1

2-Identifying Ciliates

-first, look at the non-flooded plate under the dissecting microscope to locate ciliates

-after locating ciliates, pipette out 2 µl and place on a concavity slide

-add 2 µl of methyl cellulose if the ciliates are moving too fast to observe

-take videos and pictures as needed before attempting to stain with iodine

-add 1 µl of iodine to stain and take more pictures to have for ciliate power point in later labs

-observe and record ciliate characteristics in Table 2

-repeat as many times as needed to find and record as many ciliates as possible

Data/Results:

Table 1: Soil Texture

Overall Soil Texture: Fine Sand

Table 2: Ciliate Observations

Conclusion:

The results show that there are two different kinds of ciliates between the two drops, but the two ciliates are very different in morphology and movement characteristics. These characteristics will later allow for identification of these two ciliates and further DNA sequencing can solidify the identity. The results also show that the soil texture is fine sand, which gives us insight into the type of soil environment in which these two ciliates live.

Future Steps:

In future labs, these ciliates genomes will be analyzed to further identify the type of ciliate and the relationships they have to other ciliates within the sample soil sample. This will further allow us to determine the make-up of ciliates within the soil ecosystem. The make-up of the ciliates and other protists within the soil ecosystem is important to know because they determine the health of the soil and other organisms involved in the soil ecosystem.

Objective:

The main goals of this experiment were to be able to understand phylogenic trees, prepare our soil samples for preparation, and continue to find and identify key characteristics of ciliates in our non-flooded plates. In the lecture and pre-lab portion of this experiment, various phylogenic trees were provided to better help us understand how different species in various classes and super groups are related to each other. They are identified and classified by both morphological features and DNA sequencing. To better understand the type of environment these types of ciliates live in, we prepared our soil samples by mixing in a separating solution to determine the composition of soil types. This may later help us further identify the ciliates we find within the soil sample, along with dichotomous keys and phylogenic trees.

Purpose:

It is important to understand phylogenic trees and know the soil composition because these both help us identify the ciliates within the soil samples. Little progress has been made for the identification of the various soil ciliates, but these experiments allow for further results and hypotheses to be made about how one ciliate that lives in a certain environment may be related to another ciliate in another completely different environment. It is also important to see how the different soil compositions support certain ciliate populations because soil ciliates provide nutrient for the soil and vice versa. If the soil environment were to change due to climate change or some other natural event, it could negatively impact one population of ciliates which could in turn negatively impact plants and other organisms in the soil ecosystem.

Materials:

-dissecting microscope

-compound microscope

-concavity slides

-methyl cellulose

-micropipettes

-falcon tubes

-dispersing agent

Procedure:

1- Soil Texture Preparation

-place about 4ml of the soil sample into the falcon tube

-fill the tube up to 8ml with DI water

-mix vigorously

-add one drop of dispersing agent and mix again

-leave in a test tube rack to settle until next lab

2- Identifying ciliates

-first, look at the non-flooded plate under the dissecting microscope to locate ciliates

-after locating ciliates, pipette out 10 µl and place on a concavity slide

-add 2 µl of methyl cellulose if the ciliates are moving too fast to observe

-observe and record ciliate characteristics in Table 1

-take videos and pictures as needed

-repeat as many times as needed to find and record as many ciliates as possible

Data/Results:

Table 1: Ciliate Observations

Conclusion:

From the observations, it seems as if there are two different types of ciliates between the two drops. The smaller, oval shaped ciliate appears to be more abundant than the larger ciliate. Although not fully identified as of now, these characteristics will help identify what type of ciliate they are and will allow us to see which other types of protists they are related to using the phylogenic trees and, in later labs, DNA sequencing. Once the soil sample composition is calculated and the ciliates are identified in later labs, we will have a full picture about the small environment in our soil samples.

Future Steps:

In future labs, DNA sequencing will further solidify the identity of the ciliates from our soil samples. This will further allow us to understand the make-up of protists within our soil environment which will in turn allow others to make hypotheses and run new tests about how these certain types of ciliates are only found in certain soil types and how these ciliates affect the soil nutrients. DNA sequencing will also allow us to better compare these ciliates to other ciliate genomes in order to determine their relationship. This will also allow us to better understand the soil environment composition.

Objective:

The main goals for this lab were to calculate percent water concentration, pH of the soil, and find a variety of ciliates in the soil. The calculation of water concentration in the original soil sample was found by subtracting the original soil mass from the mass of the now “dry” soil and then dividing that value by the original mass of the soil. After multiplying this new value by 100, you will have the percent water concentration from the original soil sample. The pH of the soil sample was determined simply by adding pH paper to the water in the sample and seeing what color the paper changes into. To find the ciliates within the soil sample, we used both the dissecting and compound microscopes as well as methyl cellulose to slow any ciliates or organisms down in order to make it easier to find them.

Purpose:

It is important to calculate the percent water concentration, pH, and number/kind of ciliates in the soil because this soil sample reflects the overall soil near the river from where the sample was taken. These calculations allow us to further understand the soil ecosystem by the river and what kinds of ciliates are present. The types of ciliates can also indicate the health of the soil and lead to insights as to why some soils are better to grow crops in than others. Also, identifying these ciliates just by looking at morphology and structures of each individual one will enable us to later solidify this identity through DNA sequencing.

Materials:

-compound microscope

-dissecting microscope

-various sizes of pipettes

-soil samples in a non-flooded plate

-methyl cellulose

-flat slides

Procedure:

A-Calculating Water Concentration

-use the original soil sample mass from Lab 5

-find the mass of the dry soil sample found on the top of the lid

-subtract the dry mass from the original mass

-divide the above value by the original mass and multiply this by 100

-record this value in Table 1

B- Finding pH

-since the soil samples are now non-flooded plates, pipette water out of this plate and into a small test tube with a lid

Note: Non-flooded plates are when the dry soil is saturated with water in order for the encysted ciliates to reemerge. These plates have been sitting out overnight before the ciliates reemerge

-place a small segment of pH paper into the test tube and allow it to sit for at least one minute

-after a minute has passed, take out the pH paper and compare it to the colors on the inside of the pH box

-record this value in Table 2

C- Discovering Ciliates

-first, look at the whole non-flooded plate under the dissecting microscope

-if the ciliates are found here, pipette segments of water out from the sport in the late in which they were seen

-pipette out about 400 µl and place into four different drops on a flat microscope slide (about 100 µl each)

-add methyl cellulose to each drop if needed (2 µl at a time)

-observe and record ciliate characteristics in Table 3

-take videos and pictures as needed

Data/Results:

Table 1: Percent Water Content

Table 2: pH of Soil

Table 3: Ciliate Characteristics

Conclusion:

For the final results of this experiment, the pH of the soil sample was 7.5, the percent water content was 20.65%, and there were at least two different types of ciliates found in the soil sample. Although these ciliates were not yet identified, in the next lab we will have the chance to view them again and compare them to known ciliates and their characteristics. It is important to analyze these soil samples to better understand the soil environment we live with because little is known about the protist make-up of the soil ecosystem. These results will allow others to make hypotheses about why one pH or one calculated water content would be different from another.

Future Steps:

In the future, these ciliates will be further observed and identified to see if they’re known or unknown common protists found in the soil. Also, a presentation will be made in later labs with this useful data and we will come up with different ideas as to why the data resulted in these values. Further DNA sequencing will then solidify the identity of these ciliates. This will further help us understand and try to help solve the complex environment of the soil protists and how they affect the soil nutrients and health.

Reflection of Lab Report Writing

Abstract:

In the first section of the lab report is the Abstract. The abstract is the main summary of the lab report, including sentences that talk about each of the other sections of the report. One should be able to read the abstract and know the basic important findings and significance of the experiment. There should be a couple sentences about the Introduction, such as background information, and a couple sentences about the Methods and Materials, Results, and Discussion sections. The abstract should be no more than around 250 words and should be clear and concise. There should be no abbreviations and no references to other pieces of scientific literature. The reason for all of this is so that the abstract provides the reader with a brief overview of what happened in the experiment and what the significant findings were.  If the abstract were too long and didn’t include significant details, the reader would be confused about the experiment and not obtain the most relevant information for what they’re researching for.

Introduction:

In the introduction, the second section in a lab report, background needs to be included. It is common for the introduction to move from general background information to specifics about the experiment conducted. The introduction also presents the question about why this type of research is being conducted and how other experiments relate to this one. After presenting the question about why this experiment is occurring, explain to the readers that more research needs to be done in this area and state the reasons why. Provide a quick explanation of the main methods and results of the experiment but do not elaborate. Finally, tie in another research article that is similar to this experiment and show how they’re related and the purpose for both experiments. The introduction is important because it educates the reader about the research already done on this topic and the background information needed to understand the inputs of the experiment.

Methods and Materials:

Methods and Materials includes everything needed for someone to know in order to recreate the experiment exactly how it was initially conducted. This section includes all of the materials needed for the experiment as well as a step by step process on how it was done. This is meant to be explanatory and not in a bullet point or numbered list. Also include how the samples were prepared and stored each time after use. It is important to explain the process and materials in detail because the experiment needs to be able to be recreated by other scientists who want to validate or confirm the results or test another hypothesis.

Results:

The results section details the statistics and data recorded from the experiment without explaining what the data means (this is for the discussion section). The results section includes data in the form of paragraphs as well as tables or graphs. There should at least be a few graphs or visual representations of ones data because being able to view the data in multiple ways allows for a better understanding of the data overall. It is important to represent the data in many ways and outline which values are the most significant because the data values are why one would conduct the experiment. These values show the difference between the tested groups, providing insight to whether the hypothesis was rejected or accepted.

Discussion:

In this section of the lab report, one would provide the purpose of the study as well as the possible explanations for the results section. When providing explanations, use other references to support your claims and provide details as to why you think this is a plausible explanation. Also include how this explanation contributes to the overall context of the study and suggestions for future studies. It is important to compare results with other studies, provide explanations, and suggest ideas for future studies because provides alternative hypothesis to be created by readers who want to test the hypotheses again.

References:

The references section should include APA style citations from all of your outside resources, such as journal articles, books, websites, etc. These should be listed in alphabetical order and numbered according to when they were used for comparison or background information in the report. It is very important to include the citations because if they were not included, the author of this lab report would not be giving other authors and scientists credit for their work, which is plagiarizing.

Objective:

The main goal of this experiment was to organize our data into an Excel sheet and find a meaning between the numbers of the control group and the numbers of the treatment group. The significance between these numbers is important to find because it allows us to either accept or reject our null hypothesis and make an alternative hypothesis. The significance between the two groups can be determined on the Excel sheet by using the data analysis tool pack. This pack contains multiple tests, such as the descriptive statistics test, F-test, histograms, and various T-tests. All of these tests were run on our data to determine the relationship, if any, between the control group and the treatment group.

Purpose:

The purpose of finding the significance between these two groups is to determine whether or not the Tetrahymena were actually affected by the treatment of microplastics. This is important for us to know and understand because if the Tetrahymena are being affected by the microplastic solution, then we need to understand how they’re being affected and why. These tests in the data analysis tool pack allow us to hypothesize and analyze these numbers to try and figure out the details of how polypropylene is affecting the ciliates.

Materials:

-laptop or desktop with Excel that has an installed Data Analysis tool pack

Procedure:

1- Record all data on the excel sheet

-make sure all data for each behavioral assay is uploaded correctly to the excel sheet for everyone in the class to use as a part of their experimental data

-copy data onto your own excel sheet to run tests on

2- Conduct a descriptive statistics test on your data

-once you have organized your data into cell counts (control and treatment) and swim speed assay (control and treatment), you can then run a descriptive statistics test on each set of data

-for this test, make sure all data for each group (cell counts-control, cell counts-treatment, speed-control, and speed-treatment) are all in one column

-input this data into the descriptive statistics boxes and select an output box

-make sure only have one mean per column

3- Conduct an F-test on your data

-after completing the descriptive statistics, an F-test needs to be conducted to determine if the means are significant and if the variances of the population are equal or unequal

-input the needed data for the test (a mean from the control column and a mean for the treatment column) and allow it to calculate

-if the variances are equal, use the t-test that assumes equal variances

-if the variances are unequal, use the t-test that assumes unequal variances

4-Condcut a T-test on your data:

-once the F-test determines equal or unequal variances, you can then decide which t-test to run on your data

-this test determines if the means of each experiment (the means between the control and the treatment of the cell counts and the means between the controls and the treatments of the swim speed assay) are equal or unequal

-input the data needed in order to run the test

5-make a histogram

-finally, make a histogram, which is a chart showing the natural curve or pattern of the data

-first, look at your maximum and minimum values in the descriptive statistics test

-these two values should be your lowest bin number (x-axis point) and your highest bin number

-after this, determine the intermediate values you wish to be displayed as your x-axis on your histogram

-finally, input this data into the histogram test under the data analysis tool pack and allow it to formulate a frequency and a chart

Data/Results:

Data for Speed Assay

Control Descriptive Statistics

Treatment Descriptive Statistics

Control Histogram

Treatment Histogram

Data for Cell Counts

Control Histogram

Treatment Histogram

Conclusion:

The analysis of these numbers indicates that the Tetrahymena are being affected by the polypropylene. The cell counts indicate that the Tetrahymena in the polypropylene solution reproduce more than those in the control group because the counts for the treatment are immensely higher than those in the control group. For the speed assay, there was not a very large difference like the cell counts but there was a significant difference between the speeds for the control group and the experimental group. The control group speeds were slightly higher than the speeds of the experimental group. These results are indicating that the Tetrahymena are being affected by the PP in some way.

Future Steps:

Since it is clear that the Tetrahymena are being affected by the PP microplastics, alternative hypotheses now need to be thought of in place or the rejected null hypotheses, which states that the cell counts, and the speed of the ciliates would be unaffected by PP. New hypotheses need to be created because we need to further understand what exactly is happening to the Tetrahymena in the PP solution compared to the control group. This is important to all of science because it could indicate how the microplastics in our environment are affecting humans as well as other living organisms on Earth.

Objective:

The mail goal of this experiment was to establish our data for the Tetrahymena being treated with polypropylene and for the control group of Tetrahymena. Multiple behavioral assays were conducted, including speed, directional changes, vacuole formation, and reproductive rates. Data was recorded for all of these assays for both the control group and the experimental group in the solution containing polypropylene (PP). Also, the Optical Density (OD) has been recorded for both the control group and experimental group for comparison and analysis. Another goal of this lab was to practice using a serological pipette and further practice our micropipetting and our calculations involving mean and serial dilutions.

Purpose:

The main purpose of this experiment is to compare the results of our control data and the experimental data for each behavioral assay to determine if PP has any affect on Tetrahymena behavior. These results will allow us to either reject or accept our hypothesis that PP has a negative effect on Tetrahymena behavior. Also, with the various behavioral assays, one will be able to determine if most of the Tetrahymena’s normal behaviors are effected or if only some aspects of the behaviors are affected. This data is important overall because if the Tetrahymena become affected by the intake of microplastics, then humans and other animals will also be affected in some way.

Materials:

-serological pipette

-flask of Tetrahymena stock solution

-flask of PP solution

-small test tubes

-micropipettes

-Iodine

-dissecting microscope

-compound microscope

-metric ruler

-flat microscope slides

Procedure:

1. Preparation of Polypropylene Media for this experiment

-cut the twine into very small pieces that weigh a total of 0.5g

-place into a Tetrahymena media (this contains no Tetrahymena, just the solution that the  

ciliates would go in later)

-this media contains 5.0g proteose peptone, 5.0g tryptone, 0.2g of K2HPO4, and 0.1L of distilled water

-proteose peptone: helps keep solution at a regular pH balance

-over the course of  next 2 days:

-place in microwave for 1 hour

-provide 55*C water bath overnight

-filter and autoclave (make sure pH is around 7.2 before autoclaving)

-leave in autoclave for 15 minutes at 121*C and high pressure

-on Tuesday at 5pm, after cooling and sitting overnight, 5ml of a 6.1X10^4 cells/ml Tetrahymena culture was added to 45 ml of media (1:10 dilution)

1. Sample Preparation

-have 50 ml of both the PP solution and the control solution in the back of the classroom

-each group will transfer 5ml of both medias into different sterile glass tubules using a serological pipette

-swirl flask before pipetting and pipette from the top

1. Cell Counts

-pipette three different drops each with 2µl of Tetrahymena control culture and 1µl of Iodine

-observe the three different drops under a compound microscope and count the number of cells per drop

-record this in Table 1 and repeat steps with the PP Tetrahymena experimental group

1. Measure of Optical Density

-measure the optical density of each solution in Table 2 at a wavelength of 600nm

-record the absorption value in Table 2

1- PPT solution (clean Tetrahymena media with no Tetrahymena)

2- PPT and PP solution (still no Tetrahymena added)

3- PPT Tetrahymena solution (PPT + Tetrahymena)

4- PPT, PP solution, and Tetrahymena (all three)

1. Behavioral Speed Assay

-take 20µl of stock solution and place on a flat microscope slide

-set the slide on top of a metric ruler so that you can see the Tetrahymena swimming over the  

millimeter side of the ruler through a dissecting microscope

-pick a cell to watch and line it up with the inside of the millimeter mark

-start the stopwatch and end the stopwatch when the ciliate reaches the next millimeter mark

-try to only record cells swimming in a straight line

-record the time and repeat for at least ten cells

-repeat for experimental group

-calculate the average and record all data in Table 3

Comments:

Although one behavioral assay is stated in the procedure, three other behavioral assays were conducted by others in the lab. The data and observations for these other behavioral assays were recorded in a class Excel sheet. All behavioral assays were observed and data was recorded for both the control group and the experimental group.

Data/Results:

Table 1: Cell Counts

Table 2: OD Measurements

Table 3: Behavioral Assay-Swim Speed

Average Speed for Control: 0.52mm/s

Average Speed for Experimental Group: 0.34mm/s

Conclusion:

After analyzing the data provided, it is concluded that the Tetrahymena in the PP solution increased in amount (cells/ml) and are slower than those in the control group. Also, the OD values indicate that Polypropylene absorbs almost three times the amount of photons than a PPT media containing Tetrahymena. These differences in values between the control groups and the experimental groups for the behavioral assays and the spectrophotometer indicate that the Tetrahymena are being affected by the PP. Since Tetrahymena are model organisms for other organisms, these results could give us insight into how PP will affect humans and other animals that ingest this type of microplastic.

Future Steps:

Since this experiment helped establish the data for the control groups and experimental groups in various behavioral assays, the next step for next lab is to analyze the data and come up with hypotheses as to why the PP is affecting the Tetrahymena and how to try and fix the problem. These thoughts and analyses will be put into a lab report, showing all of the data as well as the materials, methods, background, information, and anything else to help others understand what we studied and observed in our experiment. This will allow others to replicate the experiment and provide their own input and analyses about how the Tetrahymena are being affected and how to fix the problem before it becomes detrimental to other organisms.

Objectives: 

The main goal of this experiment was to further our practices with serial dilutions, pipetting, and cell counts to prepare us for future labs. These practices along with the calculations of molar solutions, percent solutions, and volume in our pre-lab allow us to expand our knowledge and be better suited to conduct an experiment on our own.  We also began to consider the control behavior and lysosomal activity for Tetrahymena. This is an important objective because for our experiment, we plan to observe how the Tetrahymena’s behaviors are affected by the microplastic polypropylene found in hay bailing twine. Without establishing a control for the movement and speed of these ciliates, it would be hard for us later to then observe and see how the behavior is different. 

Purpose:  

The purpose of furthering our skill practices and becoming familiar with the natural behavior of the Tetrahymena are to lessen our chances of making mistakes in our future lab experiments. Practice allows us to make mistakes and understand what we did wrong to fix the problem, otherwise known as metacognitive regulation. This allows us to grow in knowledge and expand our problem-solving skills to lessen the chances of systematic error as well as random errors in our future experiment.  

Materials: 

-compound microscope 

-dissecting microscope 

-stock culture of Tetrahymena 

-Iodine stain 

-micropipettes 

-concavity and flat slides 

-India Ink 

-metric ruler 

-stop watch 

Procedure: 

1. A) Cell Counts with Iodine:

-add 20µl of Tetrahymena stock culture to 5µl of Iodine stain 

-take 5µl of this solution and put on a concavity slide and view under a compound microscope 

-if this amount is too much to count, dilute the 20µl of stock and Iodine solution by adding  

180µl of media to the solution 

-this should make the solution a 1:10 dilution and the dilution factor for this solution is 10^-1 

-place three separate drops, each 5µl, onto a flat microscope slide and view with a compound  

microscope 

-count the number of stained cells in each drop and record the average 

-calculate the number of stained cells in the stock solution 

-record in Table 1 

1. B) Behavioral Assay- Simple Speed:

-dilute a 20µl solution of stock culture by adding 180µl of media to the solution 

-take 20µl of this diluted solution and place on a flat microscope slide 

-set the slide on top of a metric ruler so that you can see the Tetrahymena swimming over the  

millimeter side of the ruler through a dissecting microscope 

-pick a cell to watch and line it up with the inside o the millimeter mark 

-start the stop watch and end the stop watch when the ciliate reaches the next millimeter mark 

-try to only record cells swimming in a straight line 

-record the time and repeat for at least ten cells 

-calculate the average and record all data in Table 2 

1. C) Behavioral Assay- Directional Changes:

-add 5µl drop of culture to clean flat microscope slide 

-view under dissecting microscope 

-focus on the black plate using the reflected light 

-choose a cell to follow for ten seconds 

-keep track of how many times this cell changes direction over ten seconds 

-record number of directional changes in Table 3 

-repeat for at least 10 cells 

-calculate the average of directional changes 

1. D) Lysosomal Assay- Vacuole Formation:

-place 20µl of Tetrahymena culture on a concavity slide 

-add 1-5µl of India Ink to the drop of cells and pipette up and down in order to mix 

-quickly add a cover slide and focus the slide on 400x magnification on the compound  

microscope and start your stop watch 

-scan the slide for 10 cells and count any vacuoles. Record this as your Time 0 

-after 10 minutes, count the number of vacuoles for 10 more cells 

-repeat the procedure for 20 minutes and 30 minutes 

-record all data in Table 4 

1. E) Twine Preparation and Storage:

-cut the twine into very small pieces that weigh a total of 0.5g 

-place into a Tetrahymena media (this contains no Tetrahymena, just the solution that the  

ciliates would go in later) 

-this media contains 5.0g proteose peptone, 5.0g tryptone, 0.2g of K2HPO4, and 0.1L of distilled water 

-proteose peptone: helps keep solution at a regular pH balance 

-over the course of  next 2 days: 

-place in microwave for 1 hour 

-provide 55*C water bath overnight 

-filter and autoclave (make sure pH is around 7.2 before autoclaving)

-store in autoclave until use in experiment

Data/Results: 

Table 1: Cell Counts with Iodine Stain 

Average cells/ml in undiluted stock: 220 cells/ml 

Table 2: Behavioral Assay- Simple Speed 

Average Time: 1.68 seconds 

Average Speed: 0.6 mm/s 

Table 3: Behavioral Assay- Directional Changes 

Average number of directional changes: 6 

Table 4: Lysosomal Assay- Vacuole Formation 

Average Number of Vacuoles for 0 minutes: 2 

Average Number of Vacuoles for 10 minutes: 5 

Average Number of Vacuoles for 20 minutes: 7 

Average Number of Vacuoles for 30 minutes: 8 

Comments: 

For this lab experiment, my group completed all the procedures, but we did not have time for each of us to do all four procedures. I completed the cell count and both behavioral assays, but I was not able to complete the lysosomal assay due to lack of time. Therefore, the data collected for the lysosomal assay is from my partner Marci Jordan who completed the procedure. For the cell counts, I believe that my data is systematically biased. My counts for each drop differed dramatically, showing that I most likely pulled each of the 5µl drops from different areas in the solution. These protocols helped us establish a control for the Tetrahymena behavior and eating patterns, but I think they can be improved. These protocols only sought to identify controls for behavior and lysosomal activity. I think the lab should also include a control observation protocol for reproductive rates and a protocol to find the specific pH balance of the media in which Tetrahymena are stored.  

Conclusion: 

These different experimental protocols allowed us to further practice our techniques using serial dilutions, pipettes, and volume calculations. These skills are important to practice because they help minimize our chances of systematic biased errors and random mathematical errors when we need to calculate for solutions and cell counts. Observing the different behavioral assays and the lysosomal assays gave us the opportunity to have a control group for our experiment and the variables we want to test on the Tetrahymena. Overall, we now have very valuable data to use in our research to determine how microplastics affect the soil ecosystem.  

Future Steps: 

Now that we have established a control group and our basic skills for lab, we can move forward with our experiment to test how the microplastics in bailing hay twine affect the behavior of Tetrahymena in the soil. After experimenting on these ciliates, we will have valuable data to provide for others to do research with, which is important because microplastic pollution in soil is a relatively undocumented area in biological studies. It is important to do this research on soil ciliates because if the soil ecosystem is becoming polluted and is changing, it will affect the lives of animals and plants on Earth.  

Objective:

The main goal of this experiment was to further our skills with pipettes, serial dilutions, and our abilities to research topics needed for later labs. Utilizing various pipettes, we made a 10-fold serial dilution. This helped us practice with pipettes because we had to make sure that we used the correct one for the correct amount of liquid and change out the tips every time we transferred culture from one well to another. This experiment also helped us evolve our skills with 10-fold serial dilutions and our ability to count the number of Tetrahymena in a specific well. Finally, the last goal of this experiment was to further our research on microplastics and Tetrahymena to develop ideas about our experimental design and what we will be testing on the Tetrahymena from our soil samples in future labs.

Purpose:

The purpose of becoming familiar with pipettes, serial dilutions, and primary research is to improve our overall ability to create and perform an experiment on our own while making sure that measurements are as accurate and precise as they can be. In our future experiment, we need to lessen the chances of an error occurring and in order to lessen it, we need to be skilled in pipetting, serial dilutions, and research.

Materials:

-compound microscope

-varying sizes of micropipettes

-concavity slides

-cover slips

-computer or laptop

-a well with Tetrahymena

Procedure:

1. Preparing a 10-fold serial dilution:

-label four wells of the well plate 10^-1, 10^-2, 10^-3, and 10^-4.

-add 900µL of Tetrahymena culture media to the 4 wells

-add 100µL of 10^0 (undiluted) stock culture to the -1 well, mix briefly by slowly pipetting up and down. Change tips.

-note: avoid pipetting from the bottom of the well. Tetrahymena tend to stay towards the surface and dead Tetrahymena as well as debris are on the bottom.

-add 100µL of 10^-1 sample to -2 well, mix briefly and change tips.

-add 100µL of 10^-2 sample to -3 well, mix briefly and change tips.

-add 100µL of 10^-3 sample to -4 well, mix briefly and take off tips.

       1. Cell Counts:

-observe each under the dissecting microscope and determine which has a countable number of Tetrahymena

-record the optimal dilution for cell counting and transfer 5µL of that dilution onto a concavity slide with a cover slip

-count the cells using the 10x objective

-if cells are moving too fast, use methyl cellulose to slow them down

-if there are too many to count, repeat the steps using the next dilution

-record counts for as many 5µL drops as you are able to complete before end of lab session

1. Experimental Ideas

-collaborate as a team to fill out the worksheet handed to you about how you want to design your experiment and what you plan to do

Comments:

I was not able to complete the lab due to lack of time and retrying trials. My group found varying numbers of Tetrahymena in each step of the dilution, but I was not able to find any in all of my dilutions. Therefore, the numbers in the tables belong to both of my partners. We had to retry each solution multiple times to try to find a dilution that had Tetrahymena present within it. The dilution of 10^-4 is not included within our results because none of us found Tetrahymena in 10^-4.

Data/Results:

Table 1: Tetrahymena Cell Count (Lou Gilbert’s Numbers)

Table 2: Tetrahymena Cell Count (Marci Jordan’s numbers)

Conclusion:

These Tetrahymena are important to study and analyze because through experimentation, scientists can understand the effects of microplastics on Tetrahymena and in turn, humans. It is important to look at Tetrahymena because if these ciliates are being affected by the microplastics in our environment, then the organisms that eat Tetrahymena will be affected and so on down the food chain. In order to make these experiments as meaningful as they can be, the results need to be minimized, which is why this lab is important. Practicing these basic lab skills like dilutions and pipetting is important to our future results so that they have small amounts of error instead of large amounts due to mistakes in basic lab techniques.

Future Steps:

When we later analyze our soil samples taken from the rhizosphere around cypress trees next to a semi-polluted water way, we will have the skills learned from this lab to better experiment on the Tetrahymena. In order to completely design our experiments, we need to incorporate the techniques of pipetting, diluting, and research to obtain accurate and precise results that will allow others to recreate the experiment and see the same results.

Objectives: 

The main goal of this experiment was to become familiarized with a ciliate classified as Tetrahymena. Utilizing both the compound microscope and the dissecting microscope, we had the chance to observe Tetrahymena as it would naturally move about in the well. We would observe and record the basics about this ciliate for later reference in future classes. Another main objective of this experiment was to become familiarized with the primary scientific sources found on Baylor research engines. The main piece of literature we had to find was a primary source that relates Tetrahymena to be a model organism for the effects of microplastics. Based upon these articles, we would then start formulating ideas about how to test these microplastic effects on Tetrahymena ourselves. A third objective of this experiment is to better know how to handle and use micropipettes.  

Purpose: 

The purpose of becoming familiar with Tetrahymena and primary literature is to better prepare us for future classes. Later in the year, we will have to research soil ciliates and design an experiment ourselves to observe and record the effects of microplastics on Tetrahymena and other common ciliates. Along with designing an experiment, we will also have to write a scientific paper, which is why we need to familiarize ourselves with primary literature early in the course.  

Materials: 

1- a well with Tetrahymena 

2- a micropipette 

3- a compound microscope 

4- a dissecting microscope 

5- a computer with access to Baylor’s databases 

Procedure: 

1. A) Tetrahymena Activity:

– Transfer 100µl of Tetrahymena stock culture to a clean well using the correct micropipettor 

– Observe the Tetrahymena through the dissecting microscope 

– After observing, pick 5µl of cells from the well using a P-10 micropipettor 

– Transfer the 5µl of the cells to a concavity slide and observe on 4x and 10x using the compound microscope 

– Estimate the number of cells and the size of these Tetrahymena using the FOV measurements from last lab 

– Record these traits in Table 1 

1. B) Scientific Literature Activity:

– go onto Baylor’s database  

– type in key words to find a primary source related to Tetrahymena and microplastics 

– record the title and the author on the worksheet provided 

Comments: 

Our group was not able to complete the following procedure due to lack of time. Due to the knowledge gained from previous interactions with stains and information from the pre-lab power point, we could predict that both stains would have either slowed the Tetrahymena or killed them completely, allowing us to see the distinct shape of the whole Tetrahymena as well as the nuclei while it is not in motion.  

1. C) Staining Technique 1:

-pick 5µl of Tetrahymena using the P-10 micropipettor 

-transfer this amount to a clean, flat/regular microscope slide 

-observe on 4x to make sure there are cells present 

-add 2µl of MG-PY or K-Iodine to the cells 

-carefully lower coverslip over the culture to make sure there are no bubbles 

-observe the cells using 4x, 10x, and 40x objectives 

-record observations in a table 

1. D) Staining Technique 2:

-prepare a wet mount slide 

-collect a drop of stain with a pipette 

-add the stain to the corner of the cover slip while holding a paper towel on the other corner, helping to draw the stain across the whole cover slip 

-you may need to add another drop for full coverage 

Data/Observations: 

Table 1: Observation of Tetrahymena 

Conclusion and Future Steps: 

These Tetrahymena are important to analyze because they are commonly used in labs as model organisms, meaning they’re easy to replicate, buy, and observe. Also, these are common ciliates, so it is important to be able to identify them as we may see them in future lab experiments or our soil samples. It is also important to be familiar with primary research and micropipettes as these will also be used in future lab experiments and projects. Practice with using micropipettes and identifying true primary sources are important skills a scientist would need to create an experiment and carry it out in the future.