Oxygen Consumption Observed to Localize

Glycolysis and Cellular Respiration in Flesh

Flies Sarcophaga bullata
Kayla Guillory
March 29, 2016
Biology 155-General Biology-Lab Section 3T
Lab Partners: Bailey Trosclair and Alexis Collins

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Abstract
This experiment was performed to determine which part of flesh fly thoraxes consume
oxygen in order to distinguish Glycolysis and Cellular Respiration. A dye, Methylene Blue, was
added to each tube to localize the presence of oxygen. We used centrifugation and
homogenization methods to separate the key constituents from the thorax where cytoplasm and
active mitochondria remained. Amongst seven test tubes, we distinctively added some of the
factors (glucose, succinate, pellet, supernatant, whole homogenate) inside the tubes and placed
them in water to observe bleaching. A tube would bleach when respiration occurred and
contained pellet, succinate, and/or whole homogenate. We examined the tubes that did not have
mitochondria (pellet), which indicated no sign of bleaching. Our results show that when
mitochondria was absent, oxygen was also absent and respiration did not occur. Without the site
of respiration, mitochondria, Cellular Respiration cannot occur even with its fueling agent
succinate. The tubes that had no reaction and remained blue, concluded to only Glycolysis taking
place. Glycolysis is the first of the metabolic pathways and produces the products for Cellular
Respiration to proceed. Although oxygen consumption is important to take note of, it is also
significant to notice what is going on at a subcellular level. That of which, the fly thorax
undergoes a series of enzymatically reactions that tie in with each process. A glucose molecule
only degrades partially, then gets completely utilized as it goes through the second process. From
the two metabolic pathways we can conclude that when oxygen is present more reactions can
take place and yield a greater amount of energy opposed to a process that does not require
oxygen. Overall, Glycolysis and Cellular Respiration can only occur when the proper elements
are present.

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Introduction
Glycolysis and Cellular Respiration are two major fundamental cycles carried out by
many organisms including; humans, flies, crickets, lizardsetc. Specially, eukaryote organisms
establish these key processes and provide the components for which we can study more
carefully. A scientist, Sidney Weinhouse (1972), studied high aerobic glycolysis in tumor cells.
He touched on a famous figure in cancer research Otto Warburg experiment (1930), that of slices
of tumor cells would produce an abundant amount of lactic acid from glucose when incubated in
the absence of oxygen (Weinhouse 1972). In contrast, when the tissue cells were incubated in the
presence of oxygen, glycolysis no longer appeared. Many studies later and advancements found
that Glycolysis occurs in the cytoplasm and does not require oxygen which we will test
ourselves.
Glucose enters in Glycolysis and essentially produces two molecules of pyruvate that
generates ATP, energy. Once the two pyruvate molecules are provided, a series of oxidation
reactions perform called Cellular Respiration (Losos et al., 2008). Now, what might this lactic
acid be found by Warburg? Lactic acid is a by-product produced when anaerobic respiration
occurs and the muscle tissues release energy. Like stated above, the two pyruvate molecules
produced from the splitting of glucose enter into Cellular Respiration, requires oxygen. From
Warburgs study, lactic acid appears and preforms a reverse process in order to maintain its
ultimate goal, energy. Cellular Respiration can be found in the mitochondria undergoing a series
of enzymatically reactions and oxidation-reduction reactions that yields a sufficient amount of
energy. However, it is also important to observe the intracellular constitutes and how they work
on a subcellular magnitude. In order to utilize the two systems and notice if oxygen is consumed,
we must obtain the site where Glycolysis and respiration occur. In this experiment flesh flies,

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Sarcophaga bullata, thoraxes were homogenized to do so. Flesh flies are exceptionally aerobic
and contain a lot of active mitochondria which is where respiration occurs. The flies thorax
consists of cytoplasm and mitochondria and therefore was the key object in this experiment. A
dye, Methylene Blue, indicates oxygen consumption and bleaches if so (Schultz 24). Methylene
Blue was used in the experiment as the buffer mix. The purpose of this experiment is to identify
which part of the fly thorax homogenate undergoes Glycolysis and which part undergoes Cellular
Respiration. The homogenate contains cell fragments and the constituents for the experiment. In
this case, cytoplasm and mitochondria. Centrifugation will further separate the components into
the two major processes and allow us to observe which part of the fly thorax carries out
Glycolysis and respiration. A series of tubes and buffer mix was used to help indicate if oxygen
was present or not in each tube. The tubes that contain mitochondria or the whole homogenate
should have bleached via Cellular Respiration. Moreover, tubes that do not contain any
mitochondria or whole homogenate should not bleach indicating the process of Glycolysis.

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Materials and Methods

To prepare the homogenate, the Flesh Flies have to be immobilized by isolating them in a
closed plastic tube on ice (Schultz 25). This allowed us to easily work with the flies and cut off
the thoraxes. The flies then were distributed amongst the lab groups and with a blade, we quickly
had to cut off the wings, abdomens, legs, and heads to obtain just the thorax itself. During this
process it was essential to work fast to avoid the flies from warming up and then becoming
mobilized again making it unable to work with them anymore. Once all the thoraxes are
separated, the homogenate can be created. This process should be performed by a lab tech to
insure consistency with the experiment. At the end, about 30 milliliters of homogenate was
made and 15 ml was reserved on ice with the remaining 15 ml brought to be centrifuged. This
process was also done by a lab tech. Centrifuging allows the homogenate to separate into two
components; pellet and supernatant. The cytoplasm was soluble and the mitochondria was
insoluble which during centrifugation, the insoluble substance sunk to the bottom, pellet, and the
soluble substance remained on top, supernatant. The supernatant was collected by carefully
pouring it into a clean tube and separating it from the pellet. At the end of creating the
homogenate, pellet, and supernatant, each of the tubes should be labeled and kept on ice for later
use. Next, each lab group gathered seven test tubes, seven pipettes, a pan, and thermostat to
carefully maintain the water temperature at 35 degrees Celsius. The test tubes were then labeled
and water was added to the pan. As shown in figure 1, buffer mix was added to all tubes.
Additionally, mannitol was added to all tubes except 4 to monitor its effects on bleaching, if any.
Then, using a different pipette we added glucose, fueling agent for Glycolysis, to tubes 1-4.
Exact measurements can be found in figure 1. Next succinate, fueling agent for respiration, was
added to tubes 5 and 6. We added H, P, and S last because the reaction starts immediately after

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adding them. Figure 1 shows the indicated amounts for each tube. Each tube was thoroughly and
rapidly mixed after adding the H, S, and/or P to the tubes to insure complete mixing. Once all the
tubes were done and placed on the test tube rack, we placed the rack into the pan with water and
recorded the initial time. It is also crucial to not agitate or move the rack while it is in the pan of
water. To maintain the water temperature around 35 degrees Celsius, we had to remove or add
hot water to the pan without causing disturbance to the tubes. During the whole experiment my
partners and I monitored the tubes to watch for bleaching. A time was recorded after a tube
bleached.

Figure 1 Volumes shown in milliliters of each ingredient that belongs in the tubes.

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Results
In science, many trials are conducted to insure precise data and results. Each of the tubes
conducted in this experiment contained different elements in order to observe whether oxygen
was being consumed or not. When mitochondria were present, the tubed bleached (fig. 2).
Because different factors were added to each tube, the time it took for bleaching to occur varied.
Glycolysis and respiration each have a substrate in order to fuel its process like mentioned
before. Based on the results, there was a significant increase for the reaction to take place when
its substrate was present (fig. 2). For the tubes that did not show any bleaching results failed to
have oxygen present therefore undergoing Glycolysis.

Figure 2: Trial 1 The time it took for each tube to bleach if

bleaching did occur.

Figure 4 Trial 3, the time it took for each tube to

bleach if bleaching did occur.

Discussion

Figure 3 Trial 2, time it took for each tube to bleach if

bleaching did occur.

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Glycolysis and Cellular Respiration in flesh flies thorax provides evidence to closely
observe which process requires oxygen and which does not. From our results, Methylene Blue
bleached in the presence of oxygen. We observed that tube 5, which had the lowest bleach time,
contained succinate and pellet. Since succinate and pellet contain the mitochondria and fuel for
respiration, oxygen was not only present in the tube, but its substrate quickly reacted for Cellular
Respiration to proceed. The tubes with not all the necessary components to move forth in
respiration did not bleach, as expected. However, there were some unexpected results shown in
tube 7. Figure 2 shows that tube 7 took the longest time to bleach and only whole homogenate
was present. Compared to bleached tube 1 which had whole homogenate and glucose, tube 7
should not have bleached because there was not any fuel to undergo Glycolysis. One explanation
of this observation occurring is the glucose reserves from the fly thorax homogenate provided the
substrate for the reaction to take place. The bleaching took longer than the other tubes because
the substrate for the reaction was not initially added from our procedure, therefore the reserves
eventually caused the reaction. Glucose being absent in tube 7 played a key factor in slow
reaction rate because it fuels Glycolysis which is analogous with succinate fueling respiration.
The results we obtained revealed that the pellet, succinate, and whole homogenate were capable
of bleaching the tube but without mitochondria, the reaction could not take place. The results
showed us that not only whole homogenate or succinate can be present in order for the tube to
bleach, but also glucose and pellet. Weinhouse (1972) proposed that the high aerobic glycolysis
is the result of a defect in respiration, that cancer results when the cell responds to an irreversible
injury to its respiratory mechanism (Weinhouse, 1972). When one process is effected, the other
has to adjust and can lead to serious malfunctions. However, from our experiment we were able
to isolate the sites of the metabolic pathways and examine why oxygen was present or not.

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Oxygen consumption examined through bleaching breaks down the two major process in order
to grasps the significance of how important it is to contain the right elements for the pathways to
proceed.

Works Cited

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Losos, Jonathan B., Kenneth A. Mason, Susan R. Singer, Peter H. Raven, and George B.
Johnson. Biology. 10th ed. Boston: McGraw-Hill Higher Education, 2008. Print.
Schultz, D. L. 2006. Biology 155 General Biology I Laboratory Supplement. 78 pp.
Weinhouse, Sidney. "Glycolysis, respiration, and anomalous gene expression in experimental
hepatomas: GHA Clowes memorial lecture." Cancer research 32.10 (1972): 2007-2016.