This lab report is about flies.

 

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 3^rd 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