Lab report Due Friday March 20, 2016!!!

This lab report is about flies.
Format 12pt. font Times New Roman or Arial
Double Space (Except Abstract)
Page Numbers but not on title page

Title Page
Must have a Scientific Name

Purpose of this Lab
To demonstrate which part of the fly thorax cell homogenate and carries out glycosis and which part carries out respiration

You can relate to other source just have to cite it.

I posted the methods we used for the experiment but it have to be reword.

You can look up information about the fly and include it in the report.

I included the graphs and some results. All graphs must be label.

Student’s Name:_______________________________ Date:_______________
Lab Report Evaluation Matrix
Good Average Poor Not Present
Title Page (5pts)
Descriptive but not wordy (5pts) _______ ________ _________ _________

Abstract (20pts)
250 words or less (2pts) _______ ________ _________ _________
Problem investigated
and hypothesis (3pts) _______ ________ _________ _________
Methods (3pts) _______ ________ _________ _________
Major Results (4pts) _______ ________ _________ _________
Conclusions (4pts) _______ ________ _________ _________
Significance of
Findings (4pts) _______ ________ _________ _________

Introduction (20pts)
Sufficient Background info
on organism (2pts) _______ ________ _________ _________
Background info on System (6pts) _______ ________ _________ _________
Citations (3pts) _______ ________ _________ _________
Clearly state
goals of project (4pts) _______ ________ _________ _________
Hypothesis and predictions (5pts) _______ ________ _________ _________

Materials and Methods (15pts)
Clearly state methods used
so someone else can repeat
the experiment (5pts) _______ ________ _________ _________
Purpose of procedures
(Briefly explain why you are doing each step) (4pts)
_______ ________ _________ ________
No mundane details and no
pronouns (3pts) _______ ________ _________ ________
Passive voice and past tense (3pts) _______ ________ _________ _________

Good Average Poor Not Present
Results (20pts)
Tables and/or figures present (4pts) _______ ________ _________ _________
Figure or table has proper labels
(axis labels with units and error bars) (4pts)
_______ ________ _________ _________

Figures and tables
summary headings (4pts) _______ ________ _________ _________
No interpretation
of the results (2pts) _______ ________ _________ _________
Reference figures in text (2pts) _______ ________ _________ _________
Briefly summarize main points of
figures and/or tables in text (4pts) _______ ________ _________ _________

Discussion (20pts)

Interpretation of results (why) (8pts) _______ ________ _________ _________
Explanation of the significance
of the results (4pts) _______ ________ _________ _________
State if your hypothesis
was rejected or accepted (4pts) _______ ________ _________ _________
Citations (1pts) _______ ________ _________ _________
Summarize conclusion in a
brief ending statement (3pts) _______ ________ _________ _________

Literature Cited (5pts)
Listed alphabetically (1pts) _______ ________ _________ _________
Every citation in text is
present in literature cited (1pts) _______ ________ _________ _________
Correct format of citations (2pts) _______ ________ _________ _________

Scientific Journal Present (1pt) _______ ________ _________ _________

Biology 155 Lab Report
Title Page 5 points

Abstract 20 points
The abstract should be a concise summary of all aspects of your report: background, system purpose hypothesis, prediction, methods, results, conclusion, and relevance. Should be one paragraph on how to perform lab results. Separate Page summarize
Problem investigated
Method
Hypothesis
Major results
Conclusion

Introduction 20 points
1. give sufficient background for the reader to understand the problem being investigated
2. describe the problem or concept being investigated
3. describe the organism or system being used being investigated
4. describe the purpose of the investigation
5. state any hypotheses or prediction that will be tested

Methods 15 points
Methods should be described what was done and how it was done in narrative style. Should be in 3rd person past tense

Results 20 points
Result should thoroughly summarize you data. There should be text that should describe your data that you obtained and you interpretation of that data. Include tables or figures. Tables should have descriptive titles and all columns should be labeled. Figures should have descriptive captions. Graphs must have all axes labeled. Any simple direct conclusion you can make about your data can be stated in the results. Save broad conclusion or those that require logical development for your discussion.

Discussion 20 points
The discussion should be developing the major conclusion of your paper. It should relate your results to the concept or problem that was presented in the introduction. It should state whether hypotheses developed on the introduction or rejected are supported or can be rejected. It should relate your results to other people’s work and to the world beyond your experiment.

Literature Cited 5 points
All literature used to prepare the paper should be cited in the paper wherever it is relevant.

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

Synopsis — This experiment utilizes the method of differential centrifugation, together with
enzyme assays, to separate mitochondria from a homogenate and establish where the key processes
of respiration and glycolosis are localized in eukaryotic cells.

Objectives –
• Learn the basis and use of differential centrifugation for cell fractionation.
• Learn the use of indicator dyes (in this case methylene blue) in chemical experiments.
• Deduce the subcellular location of glycolysis and respiration from the results of the experiment.

INTRODUCTION
Cell Fractionation
Two major types of evidence have provided our present concept of the compartmentation of
intracellular function: (1) inferences from microscopic observation, and (2) evidence from actual
physical separation and biochemical analysis of intracellular constituents. This experiment will
provide evidence for the localization of cellular respiration in mitochondria, and that of glycolysis
in the soluble portion of the cytoplasm. The purpose of the experiment is not merely to verify that
a given function resides in a particular cell part, but rather to introduce you to an extraordinarily
important method of studying living matter and to lead you into some of the mental processes
required to infer how things work at the subcellular level.
Note that in this experiment we will be concerned with the separation of cell organelles.
Our separation will not be complete; that is to say, we will not obtain pure mitochondria, but will
exploit a rapid procedure which lends itself to easy execution while illustrating more complex
procedures used in the research laboratory. Our procedure yields a rich harvest of mitochondria
which are functional as judged by the most sensitive biochemical criterion available: the ability to
carry out ATP synthesis coupled to electron transport.
We will actually use two methods: homogenization and centrifugation. You are already
familiar with homogenization from the experiment on the enzyme action. Remember that
homogenization produces a solution of soluble cell constituents and a suspension of insoluble
constituents. The latter include intracellular organelles such as mitochondria. These may be
separated from the other organelles and soluble material by means of centrifugation. In a
centrifuge, material is spun on an axis of rotation and thereby subjected to a force (centrifugal
force) directed outward from the rotational axis. This force causes the suspended cell organelles to
move away from the axis of rotation down the length of the centrifuge tube. If the centrifuge runs
fast and long enough, the organelles will eventually become sedimented at the bottom of the tube
(pellet).
The rate of sedimentation varies for different kinds of suspended particles. At a given speed
of rotation, heavier and larger particles move faster than lighter, smaller ones. With elaborate
centrifugation procedures, nuclei, which are relatively large and dense, may be separated from
“microsomes” (bits and pieces of endoplasmic reticulum with any attached ribosomes).
Our choice of experimental material derives from the need for high glycolytic and
respiratory metabolism and the easiest possible separability of mitochondria from the soluble
fraction. We therefore selected insect flight muscle, specifically that of flesh flies (Sarcophaga).
As in many other flies, these have extraordinary contractile performance, causing wingbeat
frequencies of many hundreds to over a thousand cycles per second. This is energized by
glycolysis and respiration with glucose as a fuel.
Biology 155 Laboratory Supplement: 23
The most striking features of flight muscle are (1) the enlarged size of the muscle fibrils
whose contractions are responsible for the performance mentioned and (2) the equally striking giant
mitochondria. These features lend flight muscle to our present purposes for two reasons: (1) the
activity of glycolytic and respiratory enzymes is very high, permitting easy detection; and (2)
owing to the abundance and large size of the mitochondria, they can be isolated in substantial
quantity at relatively low centrifugal forces.
Recommended Reading
Baker & Allen: The Study of Biology, 3rd ed., pp. 195-232.
Keeton: Biological Science, 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.
Biology 155 Laboratory Supplement: 24

PROTOCOL
Special Preparations Before Coming To Lab
Carefully study the logic of the experiment (see table) in order to understand why each tube
is necessary and why each component is used. Know the function of the substrates glucose and
succinate in cellular metabolism.
Assay for Glycolysis and Respiration
To understand the assay method, it is crucial to know the meaning of the terms
glycolysis and respiration. Glycolysis is best defined as the conversion of glucose into two
molecules of pyruvic acid (pyruvate). This conversion does not consume oxygen, and
accomplishes only a partial chemical degradation of a glucose molecule, thus affording the cell
only a partial utilization of glucose’s potential as a metabolic fuel. In contrast to glycolysis,
cell respiration consumes oxygen, and pyruvic acid is completely oxidized to carbon dioxide
and water. As you should expect, both glycolysis and cell respiration are accomplished
enzymatically; the former is carried out by the glycolytic enzyme system, and the latter by the
enzymes of the Krebs cycle and electron-transport chain. NOTE: The usage of the term
respiration in some texts embraces both glycolysis and the conversion of pyruvate to carbon
dioxide and water, which requires water. The usage here conforms to that of the scientists
who established this field of biochemistry and is more convenient for a variety of reasons.
As a consequence of the points stated in the preceding paragraph, a homogenate which
is glycolyzing but not respiring will not use oxygen. If oxygen is utilized, its concentration in
solution will fall. We will use the dye, Methylene Blue to indicate when the oxygen is used
up as a result of respiration. When the concentration of dissolved oxygen is very low, the dye
becomes colorless. This happens as a result of chemical reduction of the dye. Reducing
equivalents from metabolic intermediates in the Krebs cycle are passed to NAD (or in the case
of succinate, FAD) and from these nucleotides on via the electron-transport chain. The dye
intercepts this passage and gets reduced. The dye color disappears when it is reduced.
Therefore, if we supply glucose to a homogenate in the presence of Methylene Blue
and the dye becomes colorless after a period of time, we have evidence for both glycolysis and
respiration. If instead of glucose we supply a substrate in the Krebs cycle derived from
pyruvate (we will use succinate for this purpose) and the dye bleaches, we have evidence for
respiration but not glycolysis. If we supply glucose to a homogenate capable only of
glycolysis, the dye would not be reduced and any inference as to the occurrence of glycolysis
would have to be based on other data. Be sure you understand the reasoning behind all these
points.
The strategy of the experiment is designed to reveal which part of the muscle
homogenate carries out glycolysis and which part carries out respiration. The procedure
breaks down into four sections: (1) preparation of the homogenate; (2) fractionation of the
homogenate by centrifugation; (3) biochemical analysis of the enzymatic capabilities of the
fractions obtained; and (4) microscope observation of the fractions (optional). Students should
work in pairs. Because the preparation of the homogenate is a relatively complicated
procedure, a flow sheet has been prepared for you (see Figure 1). Read the instructions very
carefully.
Biology 155 Laboratory Supplement: 25

Materials:
• 1 stoppered shell vial or small Erlenmeyer flask for flies
• 70 flesh flies, Sarcophaga bullata (ice-cold to anesthetize them)
• 1 wire test-tube rack (small mesh)
• a few paper towels to cut the flies on
• 1 razor blade
• 1 enamel pan
• 1 thermometer
• 1 grease pencil
• 1 piece of plastic wrap
• 1 large plastic beaker filled with crushed ice
• 7 large glass test tubes to hold reagents, homogenate, and centrifuged fractions
• 7 glass reaction tubes (SMALL size, about 3 inches tall)
• 7 plastic pipettes, 1 ml and controls

Procedure: This is the methods that should be included in to lab report PUT INTO YOU OWN WORDS
Part A- Preparation of homogenate
1. The instructor will assign one team of two students to prepare the homogenizer. This team
should obtain a clean homogenizer. Chill it for 5-10 minutes before use in an ice bath.
2. Meanwhile, the instructor will distribute 60 flies among the remaining teams. If the flies are
kept in a closed plastic tube on ice, they will be immobilized. Cold causes anesthesia in insects.
3. Using the razor blades, QUICKLY (to avoid warming) cut off the wings, legs, heads and
abdomens. Save the thorax of each fly. The thorax is the segment of the body where the wings
and legs were attached. The entire class should do this at the same time, as rapidly as possible.
Finally, each team should cut each thorax in half (to facilitate grinding tissue). The thoraces
should be put into the chilled glass homogenizer tube as it sits in ice.
4. Add 15.0 ml of ice-cold homogenizing medium to the homogenizer tube (0.32M mannitol
containing 0.02M phosphate buffer, pH 7.4).
5. The team designated in Step 1 above should now make the homogenate for the entire class.
Run the homogenizer up and down into the mix of medium and thoraces until the mixture
becomes thick (like a milk-shake). During this process keep the homogenizing tube on ice.
6. A different pair of students, assigned in advance, should prepare a filtering device. This
consists of a 5 inch 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 should be pushed down as far as the beginning of the stem. The stem, in turn,
should be fitted into a 50 ml graduated cylinder. It helps to put the cylinder in a beaker of ice.
7. Transfer the homogenate to the cheesecloth. It should start to filter through. If it does not,
shake the funnel slightly to start filtration. If it still does not, squeeze it through by hand,
making sure your hands are clean before doing so.
8. Add an additional 10.0 ml of ice-cold medium to the homogenizing vessel and run the
homogenizer to to suspend any residual tissue debris. Transfer the 10.0 ml to the cheesecloth in
the funnel, using this 10 ml to wash down the material trapped on the cheesecloth. Repeat the
washing of the homogenizer and the cheesecloth with an additional 5.0 ml of ice-cold medium.
Finally, with clean hands, squeeze out the cheesecloth bag into the funnel. This filtration
should provide about 30 ml of homogenate in the cylinder. Most of the material held back in
the cheesecloth includes pieces of thoracic integument and large muscle fibers.
Biology 155 Laboratory Supplement: 26
9. Mix the filtered homogenate thoroughly. This is crucial. Measure its total volume, and record
this value to the nearest mililiter. Transfer 15.0 ml of the homogenate to a clean tube marked H
(for whole homogenate). Keep this tube on ice at all times.
10. Transfer the remaining 15 ml homogenate to a clean centrifuge tube and place the tube in a
beaker of crushed ice.
11. Prepare a balance tube by putting 15 ml distilled water into a new centrifuge tube like the one
used in the previous step.
12. Place both tubes in the refrigerated centrifuge, on opposite sides of the rotor. Centrifuge at
5000 rpm for 20 minutes.
13. Another crucial step: immediately after the centrifuge stops, retrieve the tube containing the
homogenate, carefully holding it at the same angle at which it lay in the centrifuge. Pour all the
supernatant into a clean (rinsed with distilled water and shaken dry) 25 ml graduated cylinder.
Do not pour out any of the pellet (the pellet is whitish in color); the point is to achieve a
“clean” separation. Restore the volume of the supernatant to 15 ml with the homogenizing
medium; shake well to mix contents. Transfer to a clean tube, mark it S, and keep it on ice.
The volume of S must exactly equal that in step 10 above.
14. Add ice-cold homogenizing medium to the pellet. The amount of medium added should be just
enough to make the final volume of resuspended pellet exactly equal to the original volume
centrifuged (15.0 ml). This is crucial. Stopper the tube and shake it to resuspend the pellet
thoroughly. When the pellet is resuspend, label it P.
15. You should now have three labled tubes on ice: uncentrifuged homogenate (H), resuspended
pellet (P), and supernatant (S). 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.
16. Each individual team of students should now obtain 1 ml of H, l ml of S, and 1 ml of P in
labeled tubes. Keep them in a beaker of crushed ice.
Biology 155 Laboratory Supplement: 27

FLOW CHART
Cut 100 flies, ON ICE
Distribute 15 flies/team for preparation of thoraces
Students return halved thoraces (wings, legs, head, and abdomen removed)
Grind Pool thoraces in homogenizer, ON ICE
Add 15.0 ml homogenizing buffer
Homogenize for 2 minutes, rheostat set at 50
Filter Through cheesecloth, into a 50 ml graduated cylinder
Wash & Rinse homogenizer with 10 ml homogenizing buffer; pour through
cheescloth
Repeat Rinse with another 5 ml of homogenizing buffer; squeeze out
cheesecloth
Divide Pour off 15 ml of homogenate into a labeled tube
Centrifuge The remaining 15.0 ml homogenate for 20 minutes at 5000 rpm.
Separate Supernatant (S): Restore to 15.0 ml with homogenizing buffer; mix well
from Pellet (P):Restore to 15.0 ml with homogenizing buffer; mix well
Distribute 1 ml of H/team, 1 ml of P/team and 1 ml of S/team

Part B- Biochemical analysis of fractionated homogenate
1. Each team of two students should obtain the following materials in the quantities indicated.
Keep them in clean, dry, labeled test tubes in a rack at room temperature, not on ice.
Substance Concentration Quantity
Mannitol buffer* 0.32M 3ml
Buffer-cofactor-dye mixture ** 5ml
Glucose 0.015M 3ml
Succinate 0.2M 3ml
* Mannitol buffer = homogenizing medium
** Potassium phosphate buffer, pH 7.4 (0.2M); ATP (0.0125M); MgCl2(0.005M); Methylene blue (0.5
mg/ml).
2. Obtain 1 ml plastic pipettes for these solutions, and three pipettes for dealing with tubes H, S,
and P. Each pipette should be marked to avoid cross-contamination of solutions and should be
left in the tube rather than placed on the table top.
3. Obtain seven clean, small glass test tubes to be used as reaction vessels in the next step. Be
sure these test tubes are identical in size. Number the tubes clearly 1-7, using a grease pencil.
4. Obtain a pan containing water adjusted to 35°C, about two inches deep.
Biology 155 Laboratory Supplement: 28
5. The following table shows what to add to each of the seven reaction tubes. You will work
faster if you add the first ingredient (mannitol) to all the required tubes, then the buffer mix, and
so on down the line. Do not add homogenate, pellet, or supernatant until last, and only when
you are really ready for the reactions to start. The exact time of adding H, P, and S should be
recorded carefully. Be sure tubes H, P, and S are thoroughly agitated before pipetting from
them) their contents may settle). All volumes shown in the following table are in milliliters.
Ingredient Reaction Tube Number
1 2 3 4 5 6 7
Mannitol 0.25 0.25 0.25 – 0.25 0.25 0.45
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 – – –
Succinate – – – – 0.20 0.20 –
Whole Homogenate (H) 0.25 – – – – – 0.25
Pellet (P) – 0.25 – 0.25 0.25 – –
Supernatant (S) – – 0.25 0.25 – 0.25 –
Immediately after adding H, P, or S, each tube should be rapidly and thoroughly mixed. Gentle
sloshing is no good. The best index of complete mixing is uniformly distributed dye color
(blue) in every tube. Immediately go on to step 6.
6. Place the rack of reaction tubes in the pan of water at 35 °C, noting the exact time. Do not
agitate the tubes at all from this point on. The water must be deep enough to come to a level
above the reaction mixture in the seven tubes. During the course of the experiment it will be
necessary to add hot water to maintain the temperature at 35 °C. When you do this, add it away
from the tube rack and mix the water in the pan thoroughly without any disturbance, even
slight, to the position of the tubes.
7. As noted earlier, the indicator of reaction in the tube is dye color. When oxygen is exhausted,
methylene blue turns from bright blue to colorless. The tube then takes on whatever color its
other contents have, in this case homogenate color. Note the exact time at which individual
tubes lose blue color and record this. The time is not necessarily the same for all tubes and
some may “never” bleach, i.e., in more than one hour. Note: the bleaching of a tube depends
on metabolism using oxygen faster than it can diffuse back in from the air. When a tube
bleaches, you will still see a bluish upper margin at the surface in contact with air. This can be
disregarded for present purposes. It may take 10 minutes or longer before tubes bleach.
8. (Optional for the curious: when a tube has bleached, slight agitation will re-aerate it and turn it
blue again. This can be demonstrated easily and the tube will bleach all over again. Why does
it bleach? Why is the time different from step 7?
9. Tube 7 deserves special attention. Among cell biologists, this is loosely termed a “no-substrate
control tube”. This tube was set up to test a specific question, namely: does the action in tube 1
depend on the addition of glucose? What happened to the dye in Tube 7, if anything? What
conclusions does this permit you to draw? Is there any way in which the results which this tube
provides can still be consistent with the idea that glucose is the initial substrate for glycolysis
and respiration in vitro and in vivo?
Biology 155 Laboratory Supplement: 29