Respiration Lab

Abstract

The purpose of this experiment is to observe cellular respiration using a closed-cell respirometer. By placing seeds, both germinating and nongerminating, into the respirometer with potassium hydroxide and submerging the system into water, oxygen consumed can be calculated. Comparing seed maturity and water temperature in the final results show correlation in cellular respiration. Through observation of manometer fluid in the capillary tubes of the respirometer, it was concluded that germinating seeds respire much more than idle seeds.

Introduction

Cellular respiration, in simple terms, is the process in which cells break down sugars for energy, specifically glucose. This process occurs in the mitochondria in eukaryotes such as protists, animals, plants, and fungi. Cellular respiration doesn’t take place in prokaryotes, since they don’t have mitochondria, but some prokaryotes do break down sugars in a type of oxygen-using respiration (90). This process is divided into 3 stages with a series of chemical reactions. The first stage consists of glycolysis, which occurs in the cytosol of the cell and starts the process by breaking glucose into 2 molecules of pyruvates- a three-carbon compound. The production of ATP in this stage is a result of substrate-level phosphorylation, in which “an enzyme transfers a phosphate group from a substrate molecule directly to ADP, forming ATP.” (94) Then pyruvate oxidation occurs in the mitochondria in the second stage, in which pyruvate is oxidized to a two-carbon compound. After which the citric acid takes place also in the mitochondria and finishes breaking down glucose into carbon-dioxide. (96) In the third stage, oxidative phosphorylation takes place involving electron transport and chemiosmosis- when the electron transport chain pumps hydrogen ions across the inner mitochondrial membrane. The potential energy of the concentration gradient resulting from this process leads to the production of ATP. (92) In this lab, we will be using the following concepts to determine the effect of temperature on cellular respiration and the difference in respiratory rates of germinating seeds versus non-germinating seeds.

Methods

This lab was completed in the New Tech @ Coppell Biology Lab over a week in April of 2016. Before the baseline protocol, a week or so ahead of time, the prep work needs to be done. In this experiment we used barley seeds(Figure 1.0) but other easily germinated seeds can be used. Place 20 of your chosen seeds (of the same variety) inside 4 wet paper towels. Once wrapped around put the paper towels into a plastic bag, keep the plastic bag open to avoid mold. Place the bag in a sunny spot to germinate until the baseline protocol is started(Figure 1.1).

Also in the prep work two respirometers need to be constructed. The materials needed are 2 capillary tubes, 4 hex nuts, 2 plungers, and 2 syringes. Combining these materials will be a two person job and will require hot glue. Place the plunger all the way into the syringe. Secure a hex nut at the base of the plunger with hot glue. After it has cooled place the capillary tube into the syringe on the opposite side of the plunger. Inside the syringe it will should rest on the plunger. Keeping it in place coming straight out of the syringe place a single sealing drop of hot glue. Quickly place a hex nut over this and place more hot glue to secure it. Hold the capillary tube in place and let the glue cool. Make sure the syringe is not plugged and the plungers work in the syringe. Continue to finish making both respirometers (Figure 1.2).

After approximately a week it is time to begin the baseline activity. Fill a 16 ounce cup with room temperature water (70º F or 20º C)(Figure 3.1). To ensure the temperature or any changes place a thermometer in the cup. In the following steps remember basic safety procedures. To start the preparation of the respirometers draw some red manometer fluid through the capillary tube of the syringes. Then pushing it back out to prepare it for the manometer fluid that will later be put in it. After this place half a cotton ball in respirometers on the side of the capillary tubes. To get it to this location use a glass stirring rod. Next with a graduated pipet place 0.5 mL of 15% potassium hydroxide on the cotton ball. This chemical is highly corrosive so be careful and use the proper safety procedures. Then again with the glass stirring rod push a nonabsorbent fiberfill into each syringe, this is to protect the seeds from the corrosive substance(Figure 3.0). In one respirometer place 20 non germinating seeds. In the other you will prep the 20 germinating seeds from the plastic bag. Remove the paper towels and seeds and untangle them (Figure 2.0). Check to see if they all germinated (Figure 2.1). In order to fit in the syringe much of the living tissue will be removed, keep the growth closest to the see intact and attached (Figure 2.2).

Pointing the tube down in a sink incase of leaks as you place the plunger back into the syringe. Match the plungers so that the two respirometers have equal volume. The respirometers are then placed into the 16 ounce cup of room temperature water; capillary tubes facing upwards (Figure 3.2). After 5 minutes use a pipet to add red manometer fluid to the top of both capillary tubes. This creates a closed system to observe the seed respiration. Mark and record the movement of the red fluid every minute for 10 minutes (Figure 3.3). After the data is fully recorded properly dispose and clean lab materials.

Data and Pictures

Screen Shot 2016-04-20 at 6.36.23 PM

Screen Shot 2016-04-20 at 6.36.34 PM

Screen Shot 2016-04-20 at 6.36.47 PM

Screen Shot 2016-04-20 at 6.37.20 PM

Discussion

The nature of this experiment is to track the rate of respiration and oxygen consumption between germinating and nongerminating seeds. This can be determined using a respirometer, and tracking the movement of red manometer fluid down the capillary tube. The rate in which the fluid moves down the tube correlates with the rate of respiration in the seeds.

In this experimental trial, the germinating seeds consumed substantially more oxygen than the non-germinating seeds during the time period. There was an immediate response to the dropping of the red manometer fluid in the germinating seed as is shown by the steep slope of the graph. This is likely due to high activity in the germinating seeds as they had began to sprout. The higher level of respiration caused the red manometer fluid to rapidly go down the tube. The results of the non germinating seeds were more typical as the red fluid gradually went down the tube. This reflects the lower level of respiration taking place in the non-germinating seeds.

Conclusion

As we expected, with support from our pre-lab research, the results of our experiment show that germinating seeds consume more oxygen than non-germinating seeds This trial was conducted using barley seeds, and therefore our conclusion can only be credible in that scenario. Our seeds surpassed the germination period quicker than expected, and therefore we had to cut the seedlings prior to inserting them into the respirometer. Another error in our experiment was that the respirometers were not fully submerged in water. The purpose of the water was to regulate temperature in the experiment, because of temperature’s severe effect on respiration. Repeating the experiment with other types of seeds, dead seeds, or with different water temperatures would test other variables in cellular respiration.

Resources

https://s3.amazonaws.com/echo_files/20140121/_1390325189_Cellular%20Respiration%20Lab.pdf