An investigation to find the lowest temperature that kills all the yeast cells

The aim of this investigation is to determine at what exact temperature all the yeast cells in a 10% suspension die with no interference from other factors which may affect the results. As shown in my appendix I am looking for when the decline part of the mixtures metabolic state is absolutely 0. (ref. appendix 1)

Bakers yeast is a species of yeast formally known as Saccharomyces Cerevisiae. It is eukaryotic and its key metabolic process under normal conditions used in producing cellular energy required is aerobic respiration.

When a yeast cell respires aerobically alike human tissue cells sugar (glucose/sucrose) and oxygen are used and produce along with energy (ATP) the waste products of water and carbon dioxide.

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(1)

C6H12O6 + 6O2 6CO2 + 6H2O

A main use for yeast is its ability to produce alcohol (ethanol) from sugar during anaerobic respiration. Anaerobic respiration is commonly known as fermentation and occurs only when there is no oxygen present. When Yeast cells respire anaerobically they produce energy (ATP), alcohol and carbon dioxide.(2)

C6H12O6 2C2H5OH + 2CO2

Microbes such as yeast have optimal temperatures within which they work most efficiently, the reason for this is that the enzymes within the yeast cell are the direct reason that yeast has its particular qualities and the biochemical reactions they carry out are most active at the optimal temperature. If the temperature goes too far above the enzymes optimal temperature they will become denatured and the cell will be inactive.(3)

Methylene blue is a redox indicator and is therefore blue in its oxidised state (when aqueous).

Methylene blue can detect whether or not my yeast cells are living, it does this as all it is detecting is the presence or absence of metabolic activity. It will indicate blue if there is no metabolic activity (yeast cells are dead) and the mixture would remain the same colour as the yeast and sugar mixture was before if the cells are still metabolising therefore alive.

For this investigation to be fair I must keep certain factors the same i.e. have control variables. The control variables in this investigation will be:

* Time - each sample would undergo heating of its particular temperature for the same amount of time. I would regulate this by using a stop clock and cease observations after 10 minutes (reason for 10 minutes is that I wouldn't do it for too long as the indicator could reoxidise which would cause confusion and likely to produce inaccuracy in my results.) Also time between heating ceases and reading is taken on colorimeter as at some temperatures the yeast enzymes may start to go through renaturation and therefore give inaccurate readings for that particular temperature.

* Volumes - I would keep volumes of sugar solution, methylene blue solution and amount of yeast suspension the same as different volumes will take different times to reach temperature and also if one sample has more sugar it would respire faster and more overall this is due to there being more sugar substrate to convert due to this I would keep all of these volumes the same using a 25cm3 measuring cylinder to measure the amounts I am putting into the beakers.

* Concentrations - the reason I will keep the concentrations the same is similar to that of the volume i.e. if there is a higher concentration of sugar in a certain mixture then it is likely that this mixture would respire at a faster rate producing an anomalous result regulate with the measuring cylinders and only using 10% suspensions of yeast and sugar.

* I would also stir the different mixtures for the same amount of time and at the intensity giving fair results as they will all be mixed to the same degree. The way I would ensure this is by using an electromagnetic stirrer constantly throughout each test.

As indicated earlier in this plan my independent variable I will be changing is the temperature. I already know that yeasts optimum temperature/environment is around 30 - 37oC (similar to bacteria and enzymes in the human body) I will start my test temperatures at 40oC and go up to 90oC in 10oC intervals (this shows a good trend of how the yeast cells die well as shown in my preliminary work ref. appendix part 2) and then go up 1oC between the 2 temperatures which the activity changes from present to non present.

The dependent variable which will be directly affected by the temperature is weather or not the yeast cells are alive. I will determine if the yeast sample is alive using methylene blue indicator. To do this I must have something to compare the colour of the mixture after it is left for 1 minute, I shall therefore set a control sample which I shall heat up to 100oC which is more than overkill to the yeast cells and will definitely kill all of them (and turn the mixture blue) with this sample I shall take a colorimeter density reading and with this and I can then take samples of each temperature samples and compare them to that of the control using the colorimeter.

I think that the cells will be completely killed at around 70 - 80oC the reason for this is that as I said earlier yeast cells generally work optimally around 35oC and after optimal level (exponential phase) it reaches a deceleration phase which usually is quite gradual and I predict that it would take around 30 - 40oC above the optimal temperature to reach the decline phase which is a quick drop in number of living cells and also my preliminary work (ref. appendix part 2) shows that this is the region of temperature where all of the yeast cells were killed.

Apparatus

10% suspension of yeast

10% solution of glucose

1.0% methylene blue indicator

16 50cm3 beakers

2 25cm3 measuring cylinders

Stop clock

6 digital thermometers

Water bath

Bunsen burner

Colorimeter

Method

1. Firstly I will preheat the 6 water baths to their corresponding temperatures i.e. 40, 50,60,70,80 and 90oC ready for the main part of the investigation.

2. I shall then make my control sample when I heat the mixture to 100oC. to do this I must first make the solution, using the measuring cylinders I shall measure out 10cm3 of 10% yeast suspension and 10cm3 glucose solution and mix them together in one of the beakers and start the electromagnetic stirrer.

3. Using the Bunsen burner I shall heat the solution up to 100oC and keep it at this temperature for 10 minutes ensuring all of the yeast cells are killed and then leave to settle for 1 minute with the stirrer still on.

4. I shall then add 5 drops of methylene 1.0% solution and wait for that to stir in completely.

5. Then I will take a sample of the mixture in a cuvette and take a density reading using the colorimeter then put the sample to the side in case it is needed for reference/comparison later.

6. I shall then heat firstly a 10cm3:10cm3 glucose: yeast mixture up to 40oC and then place in the 40oC water bath and leave to maintain that heat for 10 minutes adding the second repeat 2 minutes later and the third a further 2 minutes later. So the last of the mixtures to be taken out of the water bath will come out when the stop clock reads 16 minutes.

7. I shall do step 6. for each of the 6 different temperatures and take samples of each with 5 drops of methylene blue solution mixed in and record colorimeter density readings for each exactly 1 minute after taken out of the heat.

8. Once this is done I will be able to look at my result table and see clearly between which two temperatures all the yeast cells get killed.

9. Once the two temperatures are found I will take the half way temperature (75oC if it lies between 70 and 80oC) showing if it's in the higher half or lower half of this temperature bracket.

10. I shall then repeat this two more times to make sure the first was not an anomaly.

11. Once I have found if it is above or below the 5oC marker I will then work in the direction of proposed fully killed yeast cells in 1oC intervals until I get 2 consecutive temperatures with the same densities as the control sample. Still doing 2 repeats of each and using the colorimeter to record and compare densities.

Safety and precautions

All solutions I use in this investigation are relatively non hazardous however when using methylene blue take precautions such as:

* Avoid contact with eyes as may cause mechanical irritation, wash out immediately if it does happen making sure water gets under eyelids.

* Contact with skin (wear latex gloves) as may colour the skin a bluish colour if it does come into contact with skin wash immediately with lots of soap

* If inhaled may cause shortness of breath therefore if inhaled stop and go where there is fresh air

* If ingested a burning sensation of the mouth will be noted immediately and it may cause nausea, vomiting, diarrhoea and gastritis. If ingested induce vomiting immediately as directed my medical personnel.(4)

I shall also take caution when using the water bath as water would be extremely hot and avoid contact of water with electrical components also take caution whilst using Bunsen burner as naked flame is potentially dangerous - will not leave unattended.

I shall also handle the boiling tubes and thermometers with care as they are made from glass and if cracked are sharp and dangerous.

Results tables

Temperature (oC)

100 (control)

40

50

60

70

80

90

Colorimeter density reading

I shall repeat my test 3 times for each temperature, these repeats of the test from 40oC up to 90oC show weather my results are anomalies if no anomalies seem present an average can be worked out by adding the first, second and third density reading for the particular temperature together and then dividing by 3.(if it seems that there is an anomaly in my readings I shall repeat another time and use the 3 that correlate most.

Presume the control density was 83, there will be 2 temperatures within this table which the first will be less than this value and the later would be the same. Lets say 70oC and 80oC are these temperatures 70oC having a density of 77 and 80oC having 83. The lowest temperature at which all the yeast cells are killed lies between these two points and I shall then produce a graph showing the temperatures between 70oC and 80oC and their densities I shall then do 3 tests at each of these temperatures starting with 75oC (this meaning less time wasted as you can tell if it is above or below the middle value) which will give me the exact temperature at which all the cells in a 10% suspension of bakers yeast are killed correct to 1oC.

Temperature (oC)

71

72

73

74

75

76

77

78

79

Colorimeter density reading

Bibliography

(1) http://faculty.uca.edu/~march/bio1/aer_resp/aerresp_yeast_f03.htm

(2) http://en.wikipedia.org and OCR planning literature

(3) http://www.madsci.org

(4) http://www.jtbaker.com/msds/englishhtml/m4381.htm

(5) bio factsheets January 1999 number 33.

Appendix

1. Below is a graph showing the 4 stages that my yeast enzymes will undergo.(5)

It consists of 4 main parts A - the lag phase, B - log/exponential phase, C - deceleration phase and the main one which concerns my investigation D - decline phase. The aim of this investigation is to determine at what temperature does this line stop by meeting the x-axis/temperature-axis.

2. Preliminary investigation

I carried out a simple preliminary investigation to determine at roughly what temperature my yeast enzymes will all die and stop producing CO2 gas and therefore give me an indication of the temperature range for the actual investigation. The results are as follows.

Temperature oC

Observations

40

Lots of bubbling, yeast coloured mixture.

50

Less but still lots of bubbling and still yeast coloured

60

Less bubbling and starting to go very pale blue

70

Very little bubbling and deeper blue

80

No bubbling and very blue

90

Same as 80

From this preliminary work I know that this is a good range of temperatures to use as it shows the trend of how the yeast enzymes are respiring well and between 50 and 80oC get denatured and therefore kill the yeast enzymes I will still use 90oC in my actual investigation as this shows that it is definitely below 80oC as they have the same results.