An investigation to find the lowest temperature that kills all the yeast cells in a suspension of bakers yeast.

Plan

Risk assessment

Pilot study

Method

1. Firstly set up a test tube rack with eight test tubes in.

2. Add 0.5cm³ of 10% yeast suspension to each test tube using a graduated pipette for a high degree of accuracy.

3. Set up a Bunsen burner on a heat proof mat under a tripod and gauze.

4. Half fill a 250 cm³ beaker with water and place on the gauze above the tripod.

5. Place a thermometer in the distilled water and with controlled use of the Bunsen burner achieve the starting temperature of 35ºC.

6. Add 0.5cm³ of 10% glucose solution to the first test tube using a different graduated pipette. The yeast can use the glucose as a respiratory substrate.

7. Place the 1st test tube of yeast and glucose solution in the beaker.

8. Using a stop clock, leave the test tube two minutes to react.

9. Continually maintain the desired temperature.

10. After two minutes use a pipette to add three drops of Methylene blue to the yeast glucose solution.

11. Leave one minute for the Methylene blue to react.

12. Then remove the test tube from the beaker.

13. Shake the test tube and then use another pipette to remove a few drops of the solution and place it on a slide and cover with a slip.

14.  Carefully place the slide under an electronic light microscope, focus it and examine the slide.

15. Make a note of whether or not the majority of the yeast is dead remembering that colourless is alive and blue is dead.

16. Repeat the stages 6 to 15 for temperatures of 40 ºC, 45 ºC, 50 ºC,

     55 ºC, 60 ºC, 65 ºC and 70 ºC.

Results

Conclusion

Based on these results, to the nearest 5 ºC, it appears the respiratory enzymes denature at 50 ºC resulting in the death of the fungi.

Evaluation

This was strictly a preliminary experiment as there were many aspects which make it simply a guideline for a further experiment. Firstly there was only one test tube for each temperature meaning it was not an average reading which is far more accurate. The other flaw was that just saying whether or not the yeast was dead or alive seemed unsatisfactory. By means of a counting chamber, it would be possible to discover how much yeast survived in a particular area. However as a preliminary experiment it served its purpose as it gives an approximate suggestion to where yeast enzymes are denatured, which means a more suitable range of temperatures can be used.

Variables – Dependent and Independent

My independent variable is the single variable I will adjust in order to study the effect it has on the experiment is the temperature of the water bath the test tubes of yeast and glucose and submerged in.

The dependent variable is the percentage of yeast alive at the end of the experiment, which is by my prediction directly proportional to the temperature.

Constant Variables

• Concentration of yeast – Changing how much yeast is present in 0.5cm³of solution each time would give a useless value for the percentage alive.
• Concentration of glucose – Increasing or decreasing the amount of glucose per 0.5cm³ would change the rate of reactivity for reactions inside the cell.
• Volume of yeast – This should be kept the same for each temperature so the same number of yeast cells is affected each time
• Size of beaker – To ensure that the depth of water is constant and thus the surface area of yeast exposed to the water – the size of beaker must be constant.
• Test tube size – Similarly to the beaker it is important that the same surface area of yeast is exposed to the water.
• Time of test tubes in water bath – So that all yeast cells are exposed to the same environment

Equipment and materials list with justifications for use

• Heat proof mat – To prevent heat damage to table surface
• Tripod – To mount the gauze and beaker on
• Gauze – To mount the beaker on
• Beaker (250cm³) – To act as a container for the water to act as a water bath
• Bunsen burner – to heat the water bath to various temperatures
• Electric light microscope – To study the slides of yeast cells and determine the percentage of yeast alive.
• 18 test tubes – To contain the yeast/glucose mixture
• 2 Test tube racks – To safely hold test tubes on the surface
• 15 slides and cover slips – To make slides out of the yeast samples
• Two Graduated pipettes – To measure the volumes of yeast and glucose to a very precise degree of accuracy
• A normal pipette – to add a small volume of Methylene blue solution
• A 250cm³ conical flask of 10% yeast solution – The aim of the experiment is to see how the organism yeast behaves at different temperatures, so yeast itself is required; the conical flask is to hold the solution.
• A 250cm³ conical flask of 10% glucose solution – To act as the respiratory substrate for the enzymes in yeast; the conical flask is to hold the solution.
• A steady supply of cold tap water – To use in the water bath
• Goggles – Prevents chemical damage to eyes
• Lab coat – Prevents chemical damage to clothes and body
• Gloves – Prevents chemical damage to hands
• Matches/lighter – To ignite the Bunsen burner
• Thermometer – to measure the temperature of the water bath and ensure it is at the desired temperature
• Stop clock – To measure the time the yeast/glucose mixture is immersed in the water bath.
• A vial of 1% Methylene blue solution – To act as an indicator to whether or not the yeast is alive or dead.

Precision and accuracy

The equipment used was very accurate. The graduated pipette has a minute systematic error of 0.01cm³ which makes the device a valued piece of precision glassware for measuring the volumes of both yeast and glucose solution. The thermometer is also very accurate with an error as small as 0.5ºC making it reliable for an experiment where temperature is the key variable. To keep results precise a simple average of three results is taken e.g. if at 50ºC the first result suggests 60% of yeast survives, the second shows 40% survives and the third shows 30% survives the average would be that 43% of yeast survives (2 significant figures).

Final method

1. Use a graduated pipette to fill 18 test tubes with 0.5cm³ of 10% yeast solution and place them in two test tube racks.

2. Set up a Bunsen burner on a heat proof mat under a tripod and gauze.

3. Half fill a 250 cm³ beaker with water and place on the gauze above the tripod.

4. Place a thermometer in the distilled water and with controlled use of the Bunsen burner achieve the starting temperature of 35ºC.

5. Add 0.5cm³ of 10% glucose solution using a new, clean pipette to the first three test tubes. The yeast can use the glucose as a respiratory substrate.

6. Place the 1st three test tubes of yeast and glucose solution in the beaker.

7. Using a stop clock, leave the test tube two minutes to react.

8. Continually maintain the desired temperature.

9. After two minutes use a pipette to add a few drops of Methylene blue to the yeast glucose solution.

10. Leave one minute for the Methylene blue to react.

11. Then remove the test tubes from the beaker.

12. Shake the test tubes and then use another pipette to remove a few drops of the solution and place it on three slides and cover with slips.

13.  Carefully place the slides under an electric microscope, focus it and examine the slides.

14.  Using a counting chamber of 1cm² measure how many yeast cells are alive in this area.

15.  Take the mean for how many cells remain alive in this area.

16.  Repeat stages 5 to 16 for 40ºC, 45 ºC, 50 ºC, 55 ºC and 60 ºC.

Intended treatment of results

Once the experiment is completed I will process the results into a table.

-A table to show the percentages of yeast cells alive at various temperatures-

The first average where 100% of the yeast cells are killed gives the lowest temperature where all yeast dies. By studying the range and uncertainty it can give an approximation to how accurate the results are and account for any significant anomalies. This means that if a significant anomaly is detected it can be disregarded or re –run to account for the random error.

References

Evaluation of references

Although wikipedia has a bad reputation for reliable information the two pages I used had their information verified by other sources such as the biology handbook. Some sources had limited use, for example  had detailed and useful pictures and diagrams but the texts and descriptions were limited in there use.