Cell Fractionation Lab Report

Amanda PopekAnn Karras

Ara JoSaharYaghoobi

VolkmarGaussmannJanuary 14, 2011

Biology 211 Section A

I. CELL FRACTIONATION

Tubes 1, 2 and 3 after centrifugation.

Tube 1 is separated into three layers • The white solid layer contains the most dense cell components including the nucleus and

heavier membranes.• The dark green solid layer contains less heavy cell components.• The light green liquid layer is the supernatant containing cell particles which were not

separated out during centrifugation including chloroplasts.

Tube 2 contains the remaining residue after the removal of the supernatant. This residue contains denser organelles that were separated after the first centrifugation.

Tube 3 contains the supernatant removed from tube 2 after the second centrifugation. This tube contains layers similar to those found in tube 1, in smaller proportions.

Supernatant containing lightest cell particles

Solid layer of less heavy cell components

Solid residue containing the most dense cell components and the nucleus

Solid layer of less heavy cell components

Solid residue containing the most dense cell components and the nucleusSolid residue

separated from supernatant after first centrifugation

Slide A: Wet mount of the original cheesecloth residue after adding iodine.

Chloroplasts which are un-pigmented after staining.Amyloplasts which have been dyed purple

Prior to adding the iodine several green chloroplasts could be seen as well as larger round amyloplasts.

Slide B: White residue from Exercise I, tube 2 after the addition of Iodine

Amyloplasts dyed purple

Amyloplasts which have been dyed purple

Chloroplasts which are un-pigmented after staining

Amyloplasts dyed purple

Leucoplasts

Slide C: Green residue from Exercise I, tube 3 prior to the addition of iodine.

Amyloplasts

After the addition of iodine the majority of the slide turned purple indicating the presence of amyloplasts.

Slide D: Supernatant from Exercise I, tube 3 stained with Janus green stain.

Mitochondria particles

Amyloplasts

Mitochondria particles

Tubes 1-6 after the addition of Methylene Blue in tubes 1, 3 and 5 and Tetrazolium in tubes 2, 4 and 6.

Tubes 1-6 after sitting in a hot water bath overnight.

II. MITOCHONDRIA TEST

PURPOSE

The purpose of this experiment is to first illustrate the biological technique of isolating and separating the organelles in cells, and second, identifying mitochondria using different metabolic tests.

The two metabolic test indicators that the group used to identify mitochondria were Methylene Blue and Tetrazolium Chloride. Six test tubes were labeled 1 through 6. Test tubes 1, 3 and 5 contained 4 drops of Methylene Blue indicator while test tubes 2, 4 and 6 contained 3mL of Tetrazolium. Test tubes 1 and 2 were the control for each indicator and the three Methylene Blue indicator test tubes were topped with 2mL of salad oil. Methylene Blue indicator was used

to determine if oxygen was being used up by the mitochondria during aerobic cell respiration. If the test tube remains blue, then there is oxygen present in the solution, therefore mitochondria are not present. If the indicator turns colorless (clear) after being left overnight, then oxygen is no longer present, therefore used up by the mitochondria. The salad oil is used to make sure oxygen from the atmosphere does not enter the solutions interfering with the testing and adding more oxygen. The Tetrazolium Chloride indicator was used to determine whether oxidation-reduction reactions were occurring in the respiratory enzymes of the mitochondria. When mitochondria are present, the normally colorless oxidized form of Tetrazolium that was added will turn a bright red/pink overnight, meaning reduction has taken place.

HYPOTHESIS

During the blending process, the cell walls and cell membranes are broken allowing the free floating organelles that are contained in the cytoplasm to be released.  Based upon the understanding that the mitochondria contain both an inner and outer membrane and that their relative densities are higher than most other organelles we believe we will find them in the residue.  We believe that these membranes are the primary sites for aerobic cell respiration. Therefore, we expect to receive positive test results in test tubes 3 and 4.

PROCEDURE: 

Tube 1 contained the sucrose phosphate buffer with several (4) drops of Methylene Blue. Tube 2 contained the sucrose phosphate buffer with 3mL of Tetrazolium. Tube 3 had the residue from the original centrifuge tube 1. It was centrifuged three times. The bulk of the residue had accumulated after the first centrifugation step. To this, several (4) drops of Methylene Blue were added. Tube 4 had the same solid material as #3, with added Tetrazolium.

The majority of tubes 3 and 4 were relatively heavy pieces that were captured by the first centrifugation step. Tube 5 had the supernatant from the original centrifuge tube 1, after it was centrifuged for a second time. To this, several (4) drops of Methylene Blue were added. Tube 6 had the same material as #5, with added Tetrazolium. The majority of tubes 5 and 6 were tiny pieces. They must have been tiny because they were still floating in supernatant after being centrifuged twice.

In summary, the process was to generate solid deposits that were centrifuged three times, and to generate supernatant consisting of very light-weight cell pieces that were still in suspension after two differential centrifugation processes. 

RESULTS

Mitochondria Test Initial Observations

Tube #

Contents Observations

1 Methylene Blue/Control Blue color

2 Tetrazolium/control Clear

3 Methylene Blue/Residue Dark opaque green

4 Tetrazolium/Residue Lime green

5 Methylene Blue/Supernatant Opaque green

6 Tetrazolium/Supernatant Lighter lime green

Mitochondria Test Observations After Hot Water Bath

Tube #

Contents Observations Presence of Mitochondria?

1 Methylene Blue/ Control There was an obvious separation of layers. The bottom layer was blue colored from the

methylene blue and the top layer was made up of the salad oil we used.

NA

2 Tetrazolium/ Control The whole solution was completely clear. NA

3 Methylene Blue/ Residue

Separation into two layers. The bottom layer was a transparent lime green color with some residue at the bottom. The top layer consisted of the salad

oil we used. The top layer was also transparent with some blue color from the methylene blue.

Yes

4 Tetrazolium/ Residue Orange/brown color. At the bottom of the test tube there was residue. Upon further examination

the residue had red specks in it.

Yes

5 Methylene Blue/ Supernatant

Separation into two layers. Top layer has apparent white residue. Between the two layers, there is a

thin layer of methylene blue, and the bottom layer ranges in color from light yellow towards the top, and more neon yellow towards the bottom, with

residue at the bottom.

Yes

6 Tetrazolium/Supernatant Dark opaque crimson color. Yes

ANALYSIS AND CONCLUSION:

It was known that the primary sites of aerobic cell respiration were located in the mitochondria. The laboratory experiment attempted to isolate the mitochondria and identify the correct organelles in specific spaces in the test tubes. Because the supernatant in tubes 5 and 6 changed color according to the expected reactions of the Methylene Blue and Tetrazolium Chloride indicators, we could clearly identify that the primary sites were somewhere in these tubes.

The group had initially predicted that the heavy inner and outer membranes of the mitochondria had to be the place of aerobic cell respiration, therefore ending up in the bottom residue of the test tube after 3 centrifuges. However, our hypothesis was not correct. The wet mount of the supernatant, under the microscope, showed what looked like collections of threads. The primary sites of aerobic cell respiration in the mitochondria were not destroyed during the preparatory blending process (the part that was done by laboratory staff). These primary sites must be very light-weight, which is why they were still floating in the supernatant after two centrifugation steps. Thus, these primary sites could not be heavy membranes. They must be light-weight cristae.

While we did receive positive results in both tubes 5 and 6, we also observed changes in tubes 3 and 4. We concluded that this was due to many factors, but mainly human and centrifuge error. We were using a pipet in order to extract the supernatant from the centrifuge tubes. Unfortunately we were not able to accurately obtain it all and more than likely picked up some residue in the process. The centrifuges themselves were old and did not have speed settings for our use. This would affect the preciseness by which we could separate the cell components.

In conclusion, the light-weight cristae were floating in the supernatant of tubes 5 and 6, and these cristae are the primary sites of aerobic cell respiration by the mitochondria. We know this because of the color change when Methylene Blue and Tetrazolium Chloride indicators were added. This lab also helped the group improve on working as a group in laboratory as well as understand the concepts learned in lecture.