11/1/18

Marci Jordan

Bio Lab 1105-31

Pre-Lab and Lecture

Learning Theory:

The purpose of this course is to intertwine the cognitive science of metacognition with the content of ciliate discovery. We must be able to transfer our ability to reflect, discuss, propose, analyze, fail, dig deeper, work together, and so on from this experience to future courses and to situations beyond formal education. Some goals to reflect on include to understand that constructivist learning requires the learner to take responsibility for constructing their knowledge by being actively involved in the process of learning. To move closer to a growth mindset in which you believe that abilities can be developed through dedication and hard work. To understand that creativity powers science and increases with diverse collaborations. To be willing to fail to succeed and take the intellectual risks required for discovery.

Background Information:

The discovery and characterization of soil ciliates is an extremely important area of biological research. The biodiversity of life in the soil is important for a healthy planet.  We depend on soil for the air we breath and the food we eat.  Even so, we are just learning about the complex ecosystems involved in nutrient recycling that are driven by the microbiota of the soil.  Very little is known about the roles of ciliates in this foodweb or the diversity and abundance of ciliates in soils. The study of ciliates is a topic that will allow us to explore the core concepts of biology: evolution, cell structure and function, genetics, biochemical pathways, and interactions in living systems.

Among the least known soil organisms are protists. Soil protists are, however, the most diverse soil eukaryotes. Their functional importance expands well beyond being bacterivorous, including their role as keystone organisms of soil ecosystems and regulators of essential processes of soil fertility such as nutrient cycling and plant growth.New sequencing methods are now available to disentangle the composition and function of the protist community. Our goal is to isolate ciliates using the non-flooded plate method and ultimately culture our isolates for DNA sequencing.

Bioskills:

Soil pH Protocol

An essential part of understanding the ciliates we collect is having a detailed understanding of their environment – the soil. While there are many biotic and abiotic factors that influence the soil microbiome, the biotic factors in these environments can be highly variable and sometimes difficult to precisely quantify. Thus, our initial soil assessment will evaluate several abiotic factors that will be helpful in evaluating the genomic data produced in this lab.

Soil acidity

Acidity is measured on the pH scale, which ranks the concentration of H+ ions on a scale from 1 (very acidic) to 14 (very basic). A pH below 7 is considered acidic and a pH above 7 is considered basic.  pH can be determined by using a pH meter or pH paper.

In this lab we will use pH paper in the range of 5.0-9.0.

1. Place a small amount of soil in a glass vial and add deionized water. Mix thoroughly for 3-5 minutes and then let the soil settle to the bottom.
2. Remove about 1 ml of liquid from the top of the tube and transfer it to a clean Petri dish lid.
3. In order to test the pH of your soil water, remove a small strip of pH paper and place it in the drop of soil water. Add the clear soil water so that you submerge the strip.
4. Wait 1 minute and record the result after comparing the color.
5. Record the pH in your notebook.

Non-flooded Plate

A non-flooded plate is a simple technique used to observe and maintain microscopic life in a collected soil sample. Since ciliates congregate along the water-air interface, using the non-flooded plate allows life to be easily maintained and observed.

As the name for this technique indicates, non-flooded plates involve adding enough water to soil in a petri dish to saturate but not flood the plate! As water evaporates from the plate over time, you may need to add additional water as you see the soil drying.

Protocol

1. Put 10-50 g of fresh or air-dried soil in a petri dish. You just need enough to cover the bottom of the plate on one side.
2. Saturate but do not flood the sample with distilled water.  Add water until about 5 ml will drain off when the petri dish is tilted.  You may need to give the soil time to be saturated.
3. Observe your soil using the dissecting microscope and record your observations.
4. In order to observe the abundance and diversity and capture a ciliate from the soil, use your micropipette and remove 100 µl or so of liquid sample.  Transfer this to a concavity slide, watch glass, or small glass petri dish in order to observe and further isolate.  Record the number of ciliates observed.
5. Isolation may be done by serial dilution (if there is more than one type of ciliate in your sample).
6. Once you have captured a ciliate, transfer it to 500 µl of media in a clean 24 well plate.

**You may need to experiment with types of culture media.

7. Put the lid on the non-flooded plate to decrease evaporation, but still allow for airflow and gas exchange.
8. Continue to check your plates after flooding as often as possible up to 30 days.

Note: There is a predictable succession of ciliates in the plate over time.  Your challenge is to observe and describe the abundance and diversity of ciliates in your sample and isolate and culture a single type of ciliate.

The process on air dried soil (in which the ciliates enter into their encysted form)  may isolate more individuals and species than a fresh sample, probably due to the ability of ciliates to tolerate this change to a greater degree than other organisms (microbiostasis).

Purpose

This purpose of lab this week is to begin part three of CILI-CURE, soil ciliate discovery. The short term purpose of this is to gain observations and data to present to the class in three weeks. Within each lab group, members will identify ciliates and discuss their finding through presentation. Long term the purpose is to continue the soil ciliates into next semester. Next semester in CILI-CURE students will use the same samples but will work with DNA sequencing of the PCR product for barcoding.

Objective

The research objective of this weeks lab meeting is to determine the soil ciliate biodiversity within soil samples collected earlier this semester. Students measured soil characteristics and ciliate discovery to later present in group scientific presentation. The plan for lab this week was to calculate the & water content, determine pH of soil extract, and observe the non-flooded plate for ciliates.

Hypothesis

I think that students will be excited about part three of CILI-CURE. It is nice to take a break from the research paper and be back in the lab. Discovering living organisms on a microscope is much more fun than searching for primary sources on a computer.

Procedures

Percent Water Content

1. Retrieve your soil samples
2. Weigh the samples and calculate the mass of water that has evaporated from the soil
3. Convert this to a percent

Wet soil-dry soil/wet soil x 100= % water content

pH of Soil Extract

1. Place about 1.5mL of liquid from soil sample and insert into a microfuge tube
2. Spin the tube in centrifuge fir 1 minute to pellet the soil
3. Remove a small strip of pH paper and place it into the liquid, not touching the soil.
4. Wait 1 minute and record the results after comparing the color.

Non-Flooded Plate Observation

1. Push soil to one side of petri dish and let water collect on the opposite side
2. Place petri dish under dissecting scope to observe any bigger ciliates
3. Take a micropipette and attempt to collect some ciliates and transfer onto a microscope slide
4. Observe cells under a compound microscope and try to collect photos or videos

Data

Percent Water Content

((17.01-13.8)/17.01) X 100 = 18.87

18.9% Water Content

pH of Soil Extract

pH: 6.0

Non-Flooded Plate Observation

So both my partner Lou and I had a unique situation. Our petri dishes did not get flooded 24 hours before like everyone else’s. Therefore, our ciliates still remained in their cysts and nothing could be seen besides rocks and dirt. However, our third partner Riley did have one ciliate and one worm found from her sample.

Conclusion

Overall this weeks lab was a great introduction to our third and final portion of CILI-CURE for this semester. It was nice to get our soil samples back and find information about them. It was unique to find the water content that was already in the soil when I collected it. Also it was interesting to see that most people had the same pH level but others differed. I’m excited for the weeks to come to see what my group will produce.

Next Step

After this lab I have a lot of thoughts for the next coming weeks. First, I a little worried that not having my dish already flooded set me back. Two out of three group members could not record observations as our ciliates were still in cysts. I hope next week there is time to catch up as I want as much material to present as possible since our last group presentation did not go well. I wonder what characteristics we will be observing next week and we still have two more lab meeting before presentations. Overall, I’m excited to see what everyone discoveries but I feel like I have some catching up to do.