Sheridan Mikhail

11/9/18

Lab 12-Ciliate Classification

Objective: The objective of this Ciliate Classification lab is to allow students to practice skills needed to identify specimen and develop their own procedures on how to isolate specimen.

Purpose: The purpose of this lab was to give students time to find, isolate, and eventually identify a ciliate in their soil sample.

Methods:

Soil separation

1. Using a plastic Falcon tube with a cap, place 4 mL of dry soil into the tube.
2. Fill the tube to the 10mL line with tap water.
3. Add 1 drop of texture dispersing liquid.
4. Spin the tube on a vortex mixer for about 30 seconds.
5. Label the tube with identification number.
6. Leave in a test tube rack to observe next class.

Ciliate Isolation

1. To find a ciliate take 10 drops of 5ul water from the non-flooded plate and place on a slide.
2. Scan each of the drops to see if there are any ciliates.
3. When a ciliate add 2 ul of water to dilute the drop so that the ciliate can be isolated.
4. Draw up the ciliate into a pipette and move to a concavity slide.  
5. Video the ciliate swimming with an IPhone or Moticam.
6. Add 2ul of iodine to the drop on the concavity slide.
7. Place 4 drops of vaseline to the corners of the slide cover and place over the ciliate.
8. Photograph the ciliate at 40x (Figure 1 ).

Results:

Figure 2. Picture taken with 40x using the Moticam, the ciliate was stained with iodine.

Conclusion:

The ciliate moved very quickly, obtaining a good picture was very hard. To isolate the ciliate, it was necessary to dilute the drop with water continually until there was very little dirt left. I have not identified my ciliate yet, however Dr. Adair suggested that it is a Halteria. Students focused of ciliate morphology and taxonomy earlier in the class. To identify the ciliate, one must focus on the morphology as well as general size and movement. Soil texture will be used to identify what type of soil harbors which type of ciliates.

Future Use: The compiled class information will be helpful to build a database for ciliate classification and location around campus.

Storage: Falcon tubes are stored in test tube racks at room temperature for next lab. Slides were washed and microscopes were stored.

Lab 6: Twine Juice, Cell Count, Assays

Sheridan Mikhail

9/28/18

Objective: The objective of Lab 6 was to identify experimental procedure and give students an opportunity to finalize the procedure for trials to be used in the class experiment. Students also had the chance to practice new assays testing, speed, directional change, and vacuole formation. 

Purpose: Twine Juice was made to emulate the wear and tear on utilized twine and extract the microplastics so that the Tetrahymena will be able to ingest the pollutants. The purpose of the Cell Count was to practice preforming the skill as well as collect data for the control group. Different assays were preformed to begin a data set for normal Tetrahymena behavior so that there is a control to test against.

Materials:

Twine juice-

• 50 ml Proteose-peptone-tryptone (PPT) media
• 0.5 g Green Polypropylene Twine (PT)
• Weigh boat
• Balance
• Glass jar
• Graduated Cylinder
• Compound microscope
• Scissors

Cell Count-

• Petri dish bottom
• Iodine
• P-20 Micropipettor
• P-10 Micropipettor
• Concavity slide
• Compound microscope
• Tetrahymena stock culture

Direction Change Assay-

• P-10 Micropipettor
• P-200 Micropipettor
• Timer
• Concavity slide
• Dissecting microscope
• Tetrahymena stock culture

Procedure:

Twine juice-

1. Students cut up Twine into pieces around 3mm long
2. After placing a weigh boat on a balance and zeroing the balance, students measured out 0.5 g of the cut twine.
3. 50 mL of PPT was measured in a graduated cylinder.
4. Then in a sterilized glass jar 0.5g of twine and 50 ml PPT were combine.
5. Lab assistants boiled the jars for 1 hour and let sit in a 55ºC water bath overnight.
6. In open lab, the jars were autoclaved then filtered through a 5µm filter paper into a 50ml sterile tube.

Cell Count-

1. Pipet 20 µl of Tetrahymena onto the bottom of a petri dish. 
2. Add 5µl of Iodine to stain the Tetrahymena.
3. Take 5µl of from the combination above and pipet 3 different drops onto a concavity slide.
4. Observe under compound microscope at 4x.
5. Pictures of each sample were taken through microscope (see Table 2) 
6. Count number of cells per drop and average
7. Calculate Cells per milliliter using the equation below:
   1. # of cells/ 5µl x (dilution factor)x 1000µl/mL
8. 67/5x5x1000= 67,000 cells/ mL

Direction Assay-

1. Pipet 5 µl of Tetrahymena and 45 µl using P-200 micropipettor onto a concavity slide (1:10 dilution)
2. Using the black plate and reflective light, focus the directing microscope the sample.
3. Once sample can be seen, choose one Tetrahymena to track for 10 seconds
4. Record how many times the Tetrahymena turns (more than 17 degrees).
5. Collect data for 10 cells, recorded in data Table 2
6. Standard deviation would be 1.58
   

Data:

Table 1:

Table 2:

Results:

Cell counts determined that there are roughly 67,000 Tetrahymena in every 1 ml of media. Direction Change Assay suggests that Tetrahymena either swim relatively straight or turn around several times. The Speed Assay tested the speed at which a Tetrahymena cell swam in a certain time. Lysosomal Assays would be used to calculated the amount of vacuoles normal in India Ink digestion.

Conclusion:

The class’s average cell count would be used to determine control cell/mL of stock culture. This will be used to help determine the effect that polypropylene pollution has on the Tetrahymena. To determine direction of Tetrahymena it was necessary to add 20µl of media so that the ciliate could be tracked. The 1:9 dilution allowed the ciliates enough room to move freely. With the original stock culture, the drop was over crowed with Tetrahymena and the specimen was impossible to track. All assay data will be collected and averaged to use as a control in the future experiment. 

Storage: Any slide used was bleached and left to dry. Information was collected and stored on Google Documents for collaboration.

Future goals: All techniques in this lab will be used in the larger experiment to determine the effect of polypropylene on Tetrahymena. Data collected will be used as a control for the experiment.

Lab 5: Serial Dilutions and Cell Count

Sheridan Mikhail

9/21/18

Objective: The objective of Lab 5 was to allow students to practice sample collection, preform serial dilution, and experience experimental procedure set up.

Purpose: Soil samples were collected for future use. The purpose of the Serial Dilution and Cell Count lab was to give students another tool to use in the experiments they would design in testing microplastics and Tetrahymena. Specifically the ability to preform a serial dilution in order to count Tetrahymena.  The computer lab time was used to finalize experiment process and create a feasible materials list.

Soil Collection:

Materials

• Plastic Bag
• Spoon
• Soil
• Phone
• Petri dish
• Balance

Procedure

1. Students collect soil from designated areas
2. Using a spoon to remove the top layer of dirt students filled 1/2 of a plastic bag.
3. The plastic bag was then labeled with designated ID number SGM32F18
4. In class, a petri dish was placed on a balance and the mass was recorded in grams.
5. Then the petri dish bottom and allocated amount of soil was placed on the balance and mass recorded in the Table 1 below.

Table 1:

Serial Dilution:

Materials

• P-10 Micropipettor
• P-200 Micropipettor
• P-1000 Micropipettor
• 24 Well plate
• Concavity slides
• Compound Microscope
• Dissecting Microscope

Procedure

1. Students were given a 24 well plate with a preprepared stock solution with Tetrahymena in well 4C
2. The stock solution was observed under a dissecting microscope, the solution looked like moving dust particles and slightly cloudy.
3. Students then chose a column of wells to preform a 10-fold serial dilution. Fig.2 
4. 900µl of Tetrahymena culture media was placed in the wells of the column using a P-1000 micropipettor
5. 100µl of 10⁰ Tetrahymena stock culture was added to well  3A with a P-100 micropipettor and mixed by pipetting up and down 5 times and the tip was disposed of making a 10^-1 dilution. Fig.1  
6. Then 100µl of 10-¹ was added to 3B, mixed by pipetting up and down 5 times making a dilution of 10^-2 and the tip was then disposed of (see Fig. 1) 
7. 100µl of 10^-2 was then added to 3C, mixed, and the tip was disposed of. 
8. 100µl of 10^-3 was then added to 3D, mixed, and the tip was disposed of. 
9. After the serial dilution was complete the different dilutions were observed under a dissecting microscope. The well with a reasonable amount of cells, 30-40, was chosen to prepare a wet mount.
10. Using concavity slides, students placed 3 5µl drops onto the slide with a P-10 micropipettor. Then they counted how many cells were present under the compound microscope. Table 2 shows cell count.

Figure 1:

Figure 2:

Table 2:

Practice Problems for Serial Dilution and Cell Count

1. 0.5 ml= 500µl
2. There would be 5000 Tetrahymena in a 10^-3 mL of the 20mL water sample.
3. .1 g of India ink should be added to 10mL of water to make a 1% solution.
4. 4 mL of cells and 16 mL of cell culture media would make
5. The solution of 100% ethanol diluted 1:10 would be 9.09% ethanol
6. To make a 1/10^6 dilution one would have to preform 6 1/10 serial dilutions (Figure 1).
7. 4700 cells per 1mL would be found in a sample of 10µl of culture with 42 cells.

Computer Lab:

Procedure:

*See below for proposed experiment

1. Students used pre-prepared questions to come up with a falsifiable hypothesis
2. Students finalized their hypothesis for their experiment
3. Students also identified their realistic reason for the type of microplastic chosen
4. A list of materials was created for the experiment

Data:

Question:

What effects do microplastics have on Tetrahymena survival rates?

Hypothesis: Increased amount of microplastics will show decreased survival rates in Tetrahymena.

Reason for microplastic chosen: Polyethelene Microbeads have been used as an exfoliant in face wash and body wash. Microplastics between 1-5µl can be eaten by Tetrahymena therefore the microplastics are ensured to be eaten by the ciliates.

Experimental procedure:

1. 4 treatments would be preformed by 2 groups each.
2. Groups 1-2 would add 0.025g of Polyethelene Microbeads to 900µl of media and 100µl of 10⁰ stock solution.
3. After 24 hours, the each person in the group would preform 1 10-fold serial dilution and count the surviving cells.
4. The same procedure would be preformed for the remaining groups however the amount of pollutant added would change.
5. Group 7 would preform a serial dilution and cell count on a non polluted sample which would act as the control.

Conclusion: The serial dilution and cell count portion of this lab allowed students to develop another tool for their experimental design.  This lab allowed students to develop real life experimental procedure. Using knowledge gained from the pre-lab about experimental procedure, students proposed a hypothesis and experiment on Tetrahymena and Microplastics uses.

Storage: Students stored collected soil samples in petri dishes under the fume hood. Wet mounts made in the serial dilution experiment were bleached and laid out to dry. All information for possible experimental procedure was saved to Google Documents.

Future goals: The future goals of this lab is to have students develop and preform their own experiments using the techniques taught.

Objective: The objective of Meet Tetrahymena Activity was to allow students to further develop lab skills such as micropipetting, determining size and estimating the specimen count.

Purpose: The purpose of this lab was to allow students to practice microscopy, micropipettors, wet mounts, field of view, and specimen count. These skills will be used in student designed experiments in the future. Students also became more acquainted with Tetrahymena. 

Materials:

-100 µl of Tetrahymena stock culture

-24 welled plates     

-Dissecting microscope 

-Compound Microscope   

-5 Concavity slides

-P-10 micropipettor

Procedure:

1. Students first practiced micropipetting, becoming comfortable with the process by transferring water to empty petri dishes.
2. Students were then given a 24 welled plate, with 100µl of Tetrahymena stock solution.
3. The 24 welled plate was placed on a dissecting microscope with a black stage plate using the reflected light. This allowed students to see the ciliate.
4. Using the P-10 micropipettor students took 5µl of the Tetrahymena stock solution and made a wet mount by placing the specimen on a concavity slide.
5. Students observed the ciliate under the compound microscope at 4x and 10x.
6. Students then estimated the number of ciliates in the sample by sectioning the field of view (determined in an earlier lab), counting the ciliates then accounting for rest of sample size.
   • Ex. In 1/10 of the FOV at 4x there were roughly 40 ciliates. To account for the rest of the sample size multiply 40×10=400 ciliates in 5µl of stock solution.
7. Students also recorded the size of the ciliates by estimating how many ciliates would fit in the field of view end to end.
   • Ex. 100 ciliate would fit in FOV at 4x. FOV= 4000µl.                       4000µl/100= 40µl (approximate size of ciliate).
8. This process was repeated 5 times

Data Table 1:

Data Table 2:

Figure 1: 10x magnification

Conclusion: In this lab students learned how to work with micropipettors, estimate size of specimen and determine amount of ciliates in sample taken. To obtain these results students used previously determined FOV and new lab skills such as micropipetting and using a wet mount. Students determined for how to determine size and count by their own technique (listed in step 6 and 7 of procedure). 

Storage: Students washed the concavity slides with bleach. The tip of the micropipettor was disposed of and both microscopes were covered. 

Future goals: Techniques used in this lab will be used in future labs, specifically labs students design themselves regarding microplastics and Tetrahymena reproduction.