Investigation of the effect of different carbohydrate substrates on yeast growth

sucrose will have no effect on the yeast growth.

Variables

Independent; glucose, maltose and sucrose (the carbohydrates)

Dependent; the growth of the yeast cells (numerical growth/ change in population) is what will be measured and the yeast cell will be counted by a method called:

“The Haemocytometer technique” using a light microscope and a counter to count the yeast, use of a microscope will be required due to cell size being no bigger than ≈10μm.

The experiment will be made more reliable by taking two counting’s and taking an average of the two counting’s.

Fixed;

1. Temperature will be kept constant at 25 °C due to that this is the optimum temperature for the yeast cells to grow.
2. PH will be kept at 5.5, this will be done with the help of a buffer solution, due to this being the optimum ph for growth.
3. The percentage of the glucose, maltose and starch to be used will be kept at a constant.
4. The yeast will be kept in an environment that will be as sterile as possible, by means of a piece of cotton on top of the boiling tube.
5. The percentage of the yeast used will be constant and stirred to keep the distribution and concentration equal.

Control test

The change in carbohydrate is the independent variable hence not changing the carbohydrate and just adding water will be the control test due to that water is not a carbohydrate and doesn’t provide a source of ATP for growth, hence the control test wil be yeast added to distilled water. Only the carbohydrates will be the ones to affect the growth hence the solution without carbohydrates, without the control there would be no evidence of the carbohydrates being responsible for the growth.

Apparatus

1. Heamocytometry pack
2. Stopwatch
3. Distilled water
4. yeast
5. stirrir
6. ph meter
7. 100 cm³ glass beaker x2   +/- 1 cm³
8. 5 cm³ pipette +/- 0.05 cm³
9. 10 cm³ pipette +/-0.1 (1.1%)-
10. 1 cm³ pipette +/- 0.01 cm³ (1%)-
11. glucose, fructose and sucrose.
12. 6 test tubes
13. Graduated Pipette and filler.
14. buffers at 5 and 6 ph
15. marker for labelling tubes
16. balance

Safety first

1. dealing with glass careful due to the fact that it may break and cause lacerations.
2. no harsh chemicals will be dealt with but regular safety gear should be put on in case of spillage, eyewear goggles and lab coat.
3. washing hand after experiment will be advised.

Procedure

1. I will collect all the apparatus.
2. I will label the test tube 1,2,3 and c; 1 will be the glucose solution affecting the yeasts growth;2 will be the maltose solution affecting the growth of the yeast;3 will be the sucrose solution affecting the yeast growth; c being the control group with no carbohydrate just distilled water
3. I will measure 10 gram of the carbohydrates on the balance pre setting the balance to zero with filter paper on it ass I will only be using the carbohydrates. This 10 grams of the named carbohydrates will be added to 90 cm3 of water to make a 10 % solution of carbohydrate. I will stir the solution for 15 seconds.
4. due to high percentage of yeast I will perform serial dilution making the yeast percentage lower and easier to count underneath the light microscope ast next; 1ml % of yeast into 9ml of solution making it 0.1% yeast then I will pipette 1ml of the 0.1% yeast into 9 ml of  solution making the yeast 0.01% and dilte enough to use for counting.
5. the ph will be maintained by use of ph meter and ph buffer solutions at ph5.5 using the calibrated ph buffer and meter
6. I will pipette 5ml of the carbohydrate solution with 5ml of the 0.01% yeast to get 10ml total volume this will be done with test tubes 1,2,3 and test tube c will only be added to 5ml yeast and 5ml distilled water;
7. I will put rubber lids on top the test tube with the known solutions of yeast into the water bath 10 minutes apart from each other to have enough time to count to yeast. This will be done for the maintenance of the optimum 25C temperature for yeast to grow.
8. after the initial counting of the yeast, I will count the yeast over a time span of one hour for the first six hours then 24 hours after first count then 25 hours after first count then 26 hours after first count.
9. for the counting I will use the haemocytometer, I will pipette just one drop of yeast onto the slide which will be prepared by sliding glass over the haemocytometer and pipette the yeast gently on the glass till its equally distributive.
10. using the 10 times magnification I will be able to do the yeast count more clearly. Using the next technique and counting only two side if bordering the line on the grid; as next illustrates.

        

11. I will do this counting twice hence taking average of the 5 b squares and making the data more reliable by doing to counts.
12. the volume of the b square is 0.004 mm3   hence I will divide the yeast count by the volume of the b square getting the number of yeast cells per mm3; this will be used in the data analysis by plotting a graph comparing the yeast growth.
13. these procedure will be done accordingly for all the carbohydrates and the control.
14. I will collect all the data and I will analyse it by means of graphical format.

Results in format

• Taking the two counting’s from the two haemocytometer grid an average will be taken from both counting’s
• After the counting I will use the known volume of the haemocytometer to calculate the number of yeast cells per mm3 as followed:  divide the counted average by 0.004mm3= number of yeast cell per mm3
• I will put the results in tabular format:

        

The data and graph

Lag time where the yeast cells are not dividing; there is only an increase in cell mass, but not in cell number due to adapting to their environment.

Log; time  where there’s an exponential growth, the population can increase exponentially due to no limiting factors.

Stationary time where number of new cells formed is equal to the number of cells dying. Limiting factors, such as nutrient supply, have started to influence further increase in population size.

Death; time for autolysis. they have run out of supply and digest themselfs.

The statistical test

This test will be done to either accept or reject the null hypothesis. The equation below is the one that will be used to work out the value of chi squared.

O= the observed result

E=the expected result

Degree of freedom is the number of environments and subtract one of it. In this experiment 3 -1 = 2 df, 0.05 probability, if the value is less than 5.991 than one can accept with 95% confidence the null hypothesis.