This experiment is performed to demonstrate which part of the fly thorax cell homogenate and carries out glycolysis and which part carries out respiration. Flies mainly have three body parts namely the head, thorax and abdomen. The thorax is the part holding the wings and legs. The lab experiment was conducted basing the general knowledge on flies and their body parts. More importantly, it was based on good knowledge of living cells of the flies which is useful in discussing the transportation of glycolysis and carrying out of respiration in the body of a fly. The lab results were then obtained by collecting the flies whose body parts were used to test the assumption that the mitochondrion cells are found in the thorax. Color observations were recorded to determine the usage of oxygen and therefore giving required results. It was observed that glucose was used fast in the test tubes used for the experiment. It was therefore concluded that respiration takes place in the thorax of flies. This experiment is significant in finding out the location of cells used for respiration in flies.

Introduction

Cell Fractionation

There are two major pieces of evidence that give us the details and the process on intercellular booth; (1) evidence obtained from biological and physical separation of intercellular constituents actual (2) inferences obtained through observation by an aid of a microscope from microscopic observation (Keeton, n.d.). The experiment aims at provision of evidence for localization of the cellular respiration of glycolysis and that of mitochondria as cytoplasm soluble; hence, verify the various function of particular cell part. This experiment furthermore aims at a separation of different cell organelles to obtain mitochondria using sophisticated lab research to exploit on the procedure to execute these results. I chose a system that yields a high harvest of mitochondria that are keys our procedure yields an abundant harvest of mitochondria.

In the experiment, I employed two methods on my research: Centrifugation and homogenization. Homogenization produces a solution is which consist of a suspension which is insoluble and soluble cell constituents. The suspension contains intracellular as reference results which have mitochondria organelles (Raven & Johnson, n.d.). Suspension and soluble cell components were separated using centrifugal. The material was spun in a centrifuge machine on its axis where centrifugal force caused the heavy stuff (suspension) directed the axis outwards. The suspension moved and collected at the end of the centrifuge tube.

My choice of experimental material is the need to obtain a high number of respiratory and glycolytic and separability of mitochondria from the soluble hence my selection of insect flight muscle. As for my hypotheses, the null hypothesis (H0) is that there is an increase of glycolysis and respiration enzymes in cells, whereas the alternate hypothesis (H1) is that there is no significant respiration process relation glycolysis and respiration in insect wing.

Methods

Part A: Preparation of homogenate

The instructor assigned one team of two students to prepare homogenizer. The team was to obtain a clean homogenizer and chilled it for 5-10 minutes before use in an ice bath. The instructor distributed 60 flies among the other teams as the one team was obtaining the homogenizer. The flies were immobilized by keeping them in a closed plastic tube on ice because cold causes anesthesia in insects. The wings, legs, heads, and abdomens were cut off quickly by razor blades from every fly. The cut was done quickly to avoid warming. The thorax of each fly was then saved. The entire class did the cutting simultaneously as fast as possible. Each team was to cut each thorax in half to facilitate grinding tissue. The thoraces were then put into chilled glass homogenizer tube as it sat on ice (Biology 155 Laboratory Supplement, n.d.).

15.0 ml of ice-cold homogenizing medium was added to the homogenizer tube i.e. 0.32M mannitol containing 0.02M phosphate buffer, pH 7.4. The team that was designated to prepare homogenizer made the homogenate for the entire class. The homogenizer was then run up and down into the mix of medium and thoraces until the mixture became thick (like a milkshake). During the process, the homogenizing tube was kept on the ice. A different pair of students, assigned before, prepared a filtering device which consisted of a 5inch diameter circle of cheesecloth, two layers thick, wetted with homogenizing medium (but not dripping), placed in a short-stemmed glass funnel. The center of the cheesecloth was pushed down as far as the beginning of the stem. The stem, in turn, was fitted into a 50ml graduated cylinder. It helped to put the cylinder in a beaker of ice.

The homogenate was then transferred to the cheesecloth where it began to filter through. The funnel was to be slightly shaken if the filtration did not start immediately. For the case where the shaking could not still work, it was squeezed through by clean hand. An additional 10ml of the ice-cold medium was added to the homogenizing vessel and the homogenizer run to suspend any residual tissue debris. The medium was then transferred to the cheesecloth in the funnel and was used to wash down the material trapped on the cheesecloth. The washing of the homogenizer and the cheesecloth was repeated with an additional 5.0ml of ice-cold medium. The cheesecloth bag was finally squeezed into the funnel with clean hands. The filtration provided 30ml of the homogenate in the cylinder while the materials hold back in the cheesecloth were mostly pieces of thoracic integument and large muscle fibers.

Thoroughly filtered homogenate was then mixed (the mixing is crucial), and the total volume was recorded to the nearest milliliter. 15ml of the homogenate was then transferred to a clean tube which was marked H (for the whole homogenate). The tube was kept on the ice every time. The remaining 15ml homogenate was transferred to a clean centrifuge tube placed in a beaker of crushed ice. A balance tube was prepared by putting 15ml distilled water into a new centrifuge tube like the one used before. Both tubes were then placed in the refrigerated centrifuge, on opposite sides of the rotor and centrifugation was done at 5000rpm for 20 minutes (Biology 155 Laboratory Supplement, n.d.).

Another crucial step was to stop the centrifuge immediately, and the tube containing the homogenate was retrieved, carefully holding it at the same angle at which it laid in the centrifuge. All the supernatant was poured into a clean (rinsed with distilled water and shaken dry) 25ml graduated cylinder. The pellet (whitish in color) was not poured to achieve a "clean" separation. The volume of the supernatant was restored to 15ml with the homogenizing medium, shaken well to mix the contents. The contents were then transferred to a clean tube, marked S, and kept on ice. The volume of S was exactly equal remaining 15ml homogenate that had been moved to clean centrifuge before.

The ice-cold homogenizing medium was then added to the pellet. The amount of medium added was made to be just enough to make the final volume of re-suspended pellet exactly equal to the original volume centrifuged (15.0ml) (This step was also crucial). The tube was the stoppered and shaken to resuspend the pellet thoroughly. When the pellet was re-suspended, it was labeled P. The three labeled tubes for un-centrifuged homogenate (H), re-suspended pellet (P) and supernatant (S) were then place on ice. The pellet contains nuclei, glycogen (polysaccharide) granules, mitochondria, and bits of the muscle's contractile apparatus. The supernatant contains most soluble muscle constituents, including glycolytic enzymes and some membranous material (reticulum) which is too small to centrifuge out at the speeds used. Finally, 1ml of H, 1ml of S, and 1ml of P was then obtained in the labeled tubes and kept in a beaker of crushed ice

Part B: Biochemical analysis of fractionated homogenate

Each team of two students obtained the following materials in the quantities indicated. They were kept in clean, dry, labeled test tubes in a rack at room temperature and not on the ice.

Substance	Concentration	Quantity
Mannitol buffer*	0.32M	3ml
Buffer-cofactor-dye mixture**		5ml
Glucose	0.015M	3ml
Succinate	0.2M	3ml

Table 1

* Mannitol buffer = homogenizing medium

** Potassium phosphate buffer, pH 7.4 (0.2M); ATP (0.0125M); MgCl2 (0.005M); Methylene blue (0.5mg/ml).

1ml plastic pipettes for these solutions and three pipettes for dealing with tubes H, S and P were obtained. Each pipette was marked (to avoid confusion) and left in the tube. Seven clean, small glass test tubes (identical in size and numbered 1-7 using a grease pencil) were used as reaction vessels. A pan containing water adjusted to 35 degrees Celsius and about two inches deep was also used (Biology 155 Laboratory Supplement, n.d.).

The table below shows what was added to each of the seven reaction tubes:

Ingredient/Reaction Tube Number	1	2	3	4	5	6	7
Mannitol	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Buffer mix	0.45	0.45	0.45	0.45	0.45	0.45	0.45
Glucose	0.20	0.20	0.20	0.20	0	0	0
Succinate	0	0	0	0	0.20	0	0
Whole Homogenate (H)	0	0	0.25	0.25	0	0	0.25
Pellet (P)	0	0.25	0	0.25	0.25	0	0
Supernatant	0	0	0.25	0.25	0	0.25	0

Table 2

The rack of reaction tubes was placed in the pan of water at 35 degrees Celsius and time were recorded. The temperature was made constant at 35 degrees Celsius. The exact time at which individual tubes lost blue color was recorded.

Results and Analysis

	Results
Trial 1
Tube #	1	2	3	4	5	6	7
Did bleaching occur	Yes	No	No	Yes	Yes	No	Yes
Time took for bleaching (min)	18	None	none	16	8	none	25

Trial 2
Tube #	1	2	3	4	5	6	7
Did bleaching occur	Yes	No	No	Yes	Yes	No	Yes
Time took for bleaching.	19	None	none	17	11	none	28

Trial 3
Tube #	1	2	3	4	5	6	7
Did bleaching occur	Yes	No	No	Yes	Yes	No	Yes
Time took for bleaching.	16	None	none	17	10	none	27

Table 3.Table showing Trials on test tubes

The ingredients used include the following:

Homogenized solution of mannitol at a concentration of 0.320M 3.0ml, Succinate solution of 0.20M 3.0ml, 5.0 ml homogenized cofactor of dye mixture, and 3.0ml of 0.015 M glucose.

Fig 1.1 Test Trial 1

Fig1.2 Trial 2

Fig 1.3 Trial 3

From the results, there is no color change in tests tube numbers two, three, and six

In test tube four, the time taken is seventeen minutes for trial 1 and two while trial one took sixteen minutes for the same number of test tube.

In all the test tubes, number seven (7) to the longest time in all the trials with trial 2 being the highest at twenty eight.

Discussion and conclusion

Consider the reagents table below;

Concentration of Ingredients
Ingredients/ Test tube number	1	2	3	4	5	6	7
Mannitol	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Buffer mix	0.45	0.45	0.45	0.45	0.45	0.45	0.45
Glucose	0.2	0.2	0.2	0.2	0	0	0
Succinate	0	0	0	0	0.2	0	0
Whole Homogenate	0	0	0.25	0.25	0	0	0.25
pellet (P)	0	0.25	0	0.25	0.25	0	0
Supernatant	0	0	0.25	0.25	0	0.25	0

Figure 2. Table showing concentration of fractionated homogenate used

The water was kept at a level of the that makes the test tubes slightly be it and maintained at a constant temperature of 35.0 Celsius and reason for this was to allow the reagents to be active, hence the reason for adding hot water to the water bath

As noted earlier, the indicator of reaction in the tube is dye color. The color of methylene changed from blue to colorless and was seen from test tubes 1, 4, 5, and 7.

Test tube 1 and test tube 7 has the same components except that test tube one has glucose and seven has whole Homogenate, The absence of glucose in test tube seven resulted to test tube seven taking longer time than any test tube in a color change for all the trials. This is a great analogy that we obtained meaning that glucose was used fast in the test tubes as the cells use it to produce energy through glycolysis and respiration resulting in changing of color (Baker & Allen, n.d.). Also, glucose is the initial substrate of respiration its process and glycolysis. Our discussion also sets that we fail to reject our hypothesis, Ho, that there is an increase of glycolysis and respiration enzymes in cells at presence of cells containing mitochondria. Flight muscle contains a great number of mitochondria, hence, cells from the centrifugal process yielded our desired results (Loewy & Siekevitz, n.d.)

References

Baker, & Allen,. The Study of Biology (3rd ed.). 195-232.

Biology 155 Laboratory Supplement.

Keeton,. Biological Sciences (3rd ed., pp. 137-138,163-180).

Loewy, & Siekevitz,. Cell structure and Function (2nd ed., pp. 310-314).

Raven, & Johnson,. Biology (4th ed., pp. 193-205.).