110008107 Marilena Spanou BS11004: Laboratory and Research Skills 1B

INVESTIGATION FOR THE SELECTIVE UTAKE OF SOLUTED BY YEAST

Statement of Hypothesis:
The addition of different hydroxide solutions at varying pH levels, affects the uptake of solutes by Yeast. The Null Hypothesis is that, there is no relationship between the addition of hydroxide solutions of different pH levels and solute uptake by yeast. Any relationship is due to human error or chance.

Results:
Colour changes: (Week 1) Suspension with neutral red solution Residue Filtrate Pale Red Pale Yellow Before After Pale Red Pale Orange Standard yeast suspension Residue Filtrate Pale Yellow Pale Yellow Before After Pale Yellow Pale Yellow

Filtration Water Bath

Table 1: Colour change of the suspension when filtered and when put in the water bath

Hydroxide Solution

Colour at the Beginning

Controlled Volume of Hydroxide Added

Colour Change

Volume of Hydroxide Added for Colour Change to Occur 48 drops 10 drops 3 drops 10 drops 20 drops 48 drops

Mg(OH)2 Ca(OH)2 NH4OH KOH NaOH

Pale Red Pale Red Pale Red Pale Red Pale Red 48 drops

Paler Red Dark Yellow Yellow - Orange

Pale Yellow Orange - Red No colour H2O Pale Red change Table 2: colour change of the suspension when the hydroxides were added

110008107 Marilena Spanou BS11004: Laboratory and Research Skills 1B

pH Measurements: (Week 2) pH of Hydroxide Hydroxide Solution solution Ca(OH)2 Mg(OH)2 KOH NH4OH NaOH H2O 12.22 10.12 12.36 10.85 11.92 7.03 pH of yeast suspension before adding -OH 8.29 8.29 8.29 8.31 8.37 8.38 Appearance of cells under the microscope Yellow Orange Yellow Orange Pinkish Pinkish

pH after filtration 8.93 8.58 8.96 8.98 8.62 8.50

Table 3: Appearance of Yeast cells under the microscope and pH of solutions. Discussion and Conclusion: The internal environment of yeast cells is slightly acidic, with pH between 5.5 and 6.0. When Neutral Red Solution was added to the Yeast Suspension, the yeast cells turned red. This was due to the diffusion of the dye into the cells, and because Neutral Red Solution is red in acidic conditions and turns yellow in a pH of about 8.0. The 1% Sodium Bicarbonate solution added to the suspension has a pH of 8.5, but it is not absorbed by the cells, hence the cells did not change colour. When the test tubes were put in a water bath, the permeability of the cell membrane increased, mainly because the proteins found on the cell membrane were denatured [3], causing the Bicarbonate solution to diffuse into the cells. As a result the colour of the dye changed from red to yellow. Additionally, when the yeast suspension containing the dye and the standard yeast solution were filtered, the colour of the filtrates was the same. This was an indication that the Neutral Red Solution diffused into the cells. If it was not absorbed by the cells it would appear in the filtrate. As shown in table 3 in the Results section, when the Hydroxide solutions were added into the yeast suspensions, some changed colour whereas others did not. This was due to the fact that some of the solutions diffused into the cells. Calcium Hydroxide and Potassium Hydroxide caused the colour of the dye that was already in the cells to change from red to yellow, which confirms that they diffused into the cells. As the table shows, these two solutions have the highest pH, which caused the membrane of the cells to become more permeable, whereas the other bases which have a lower pH did not cause any change to the membrane of the cells, thus not resulting in any colour change. Also, water was added to one of the suspensions and was used as a control, in order to compare our results.

110008107 Marilena Spanou BS11004: Laboratory and Research Skills 1B

After the end of the experiment, some conclusions were drawn. The uptake of solutes by yeast is affected by the changes of pH of the external environment. As justified by the results, if the pH of the external environment is too alkaline the permeability of the cell membrane increases, causing more solutes to diffuse into the cells. All in all, the results that our group obtained support our hypothesis that the addition of different hydroxides with varying pH, affect the solute uptake by yeast cells. The hypothesis is mainly supported by Tables 2 and 3 in the Results section, since the ones that showed a distinct colour change, i.e. from pale red to yellow, are the cells which absorbed the hydroxide solution, while the ones that had minor or no colour changes are the ones that did not absorb the hydroxide solutions. Appendix:
Week 1: 1. In a 450mL beaker, add 5g of Dry Yeast (20 g/L) and 250mL of Deionised Water and 100mL of 2% Sodium Bicarbonate 2. Mix evenly 3. Extract 50mL of the Standard Yeast Suspension into a conical flask 4. Add 2mL of Neutral Red Solution and record the colour immediately 5. Leave to stand for 5 minutes and observe any colour changes 6. Filter 10mL of the Yeast Suspension which contains the Neutral Red Solution. 7. Pour 10mL of the Standard Yeast Suspension in a test tube and 10mL of the Yeast Suspension which contains the Neutral Red Solution. 8. Place the two test tubes in a water bath and let there to stand for 5 minutes. Observe any colour changes 9. Label the test tubes with the Hydroxide Solutions to be used 10. In each test tube add 10mL of the Standard Yeast Suspension. Then add 0.5mL of Neutral Red Solution and record the colour of the suspension 11. Add the Hydroxide solution drop by drop 12. Place the test tubes in a water bath and let stand for 5 minutes and record any colour changes. Week 2: 1. In a 450mL beaker add 5g of Dry Yeast (20g/L) with 250mL of Deionised Water and 100mL of 2 % Sodium Bicarbonate Solution 2. Mix evenly for 5 minutes 3. Extract 50mL of the Standard Yeast Suspension into a separate conical flask 4. Add 2mL of Neutral Red Solution and record the colour immediately 5. Leave to stand for 5 minutes and observe any colour changes 6. Filter 10mL of the Yeast Suspension that contains the Neutral Red Solution and 10mL of the Standard Yeast Suspension 7. Label the test tubes with the Hydroxide Solutions to be used.

110008107 Marilena Spanou BS11004: Laboratory and Research Skills 1B 8. In each test tube add 10mL of the Standard Yeast Suspension. Then add 0.5mL of Neutral Red Solution and record the colour change 9. Using a pH meter, measure the pH of the Yeast Suspension and the pH of the Hydroxide Solution 10. Add the Hydroxide solutions drop by drop 11. Observe any colour changes and record the number of drops required for this colour change to occur 12. Leave the Suspension with the Hydroxide to stand for 5 minutes 13. Filter the suspensions and take samples of the yeast cells 14. Observe the yeast cells under the microscope, to see their colour. [1],[2]

Figure 1: Results for the addition of Hydroxide solutions

Figure 2: Results when the test tubes were put in the water bath and after filtration

110008107 Marilena Spanou BS11004: Laboratory and Research Skills 1B

Figure 3: Notes taken for the addition of Hydroxide solutions

References:
[1]

http://www.flinnsci.com/Documents/demoPDFs/Biology/bioActivities/BF10157.pdf

[2]

https://my.dundee.ac.uk/bbcswebdav/courses/BS11004_CLS_D65_201112/BS11004%20Modu le%20Handbook%202011-12final.pdf (pages 30-33)

[3]

http://www.ehow.com/about_5516866_effect-temperature-cell-membranes.html