C6H12O6 2 CO2 + 2 C2H5OH + 2 ATP
As can be seen from the equations, anaerobic respiration/fermentation produces only 2 ATP molecules whilst aerobic respiration produces 36/38 ATP molecules and so aerobic respiration produces much more ATP per glucose molecule than fermentation/anaerobic respiration. So aerobic respiration produces more energy for the yeast growth.
In this investigation I will be investigating the effects of different carbohydrate substrates on yeast growth. I have chosen to be using a monosaccharide (glucose), a disaccharide (maltose) and a polysaccharide (starch).
Glucose; Maltose; Starch;
Alternative hypothesis;
Starch will have the least effect on the yeast growth. (Lowest number of yeast cells)
Glucose will have the largest effect on yeast growth. (Highest number of yeast cells)
Maltose will have a slight effect on yeast growth. (Moderate number of yeast cells)
Null hypothesis;
Starch will have no effect on the yeast growth.
Glucose will have no effect on yeast growth.
Maltose will have no effect on yeast growth.
Expectations;
I will be expecting to see unequal strong foaming in the 3 test tubes (The 3 conditions, glucose, maltose and starch).due to their different effects on the yeast growth, according to the alternative hypothesis.
Variables;
Independent variable; is the different carbohydrate substrates
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Glucose; which will be used as it is the main respiratory substrate
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Maltose; As it consists of 2 glucose monomers, so it will be used to see the effect of a double glucose unit on the yeast growth.
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Starch; will be used as it is a large polymer, and so it could be difficult for the yeast to hydrolyse it, as they do not contain the enzymes for its hydrolysis.
Dependent variable; the growth of yeast cells.
This will be measured using the Haemocytometer technique. This would be carried out using a Light microscope, focusing with the x10 lens- as this provides the clearest image of the yeast cells sample. The counting of the yeast cells would be facilitated with the use of a counter, so that accurate counts are obtained. There would be a strict regulation of stirring the yeast cultures before mixing, so that the sample obtained for counting, contains an equal and adequate distribution of yeast cells, this would enable an accurate and reliable count of the yeast cells, which would then result in an accurate growth graph and so an accurate and reliable study of the yeast growth. To increase the degree of reliability, 2 samples will be pipetted for counting and the average per B square will be calculated and then average of both of the averages would be derived from the 2 samples to give one figure which will then be used to calculate the number of yeast cells per 1 mm3, which will then be plotted on a growth graph.
Fixed variables
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temperature; it would be fixed at 25°C , which is the optimum temperature for yeast growth, as yeast have an optimum temperature range from 20°C -30°C .This will be controlled by fixing the temperature of the water bath at 25°C.If the temperature was way below the chosen e.g. 19°C , then the temperature would be inadequate for the enzymes involved in the yeast’s living processes e.g. cell division. and if it was too high then this could lead to the dying out of the yeast cells , as the enzymes involved in its living processes would denature at such high temperatures, which in turn would affect the rate of growth , as the yeast cells will move on to the death stage abnormally fast, and so the graph plotted (growth graph) would not be accurate , as it would not reflect the effect of the carbohydrate on the yeast growth, but it will reflect the effect of high temperature on the yeast growth.
- pH; would be fixed at 5.5 , which is the optimum pH for yeast growth, this would be fixed using standard pH buffers of 5-6, along with a pH meter. if the pH was too acidic/alkaline , although yeast do have some degree of acid tolerance, however this is not sustained for long enough and so the yeast cells would not be able to survive in those extreme conditions , and so they could die, which would again result in only the death stage being observed and plotted and so the other phases (lag, log and stationary) would not be observed. And so an inaccurate growth graph. (if the pH and temperature were not fixed at optimum , then the yeast would grow much slower, and so the time assigned for the experiment will run out, without the yeast cells developing from the lag phase to the other phases of growth)
- The % concentration of the carbohydrates; Will be fixed at 10%,as it is a moderate concentration to use , if it was too high then this would give the solution a more negative water potential value , which will result in the osmotic movement of water to move out of the yeast cells and into the solution .This , could result in the shrivelling of the cells, making it difficult for them to be seen and counted under the microscope, and so the incorrect number will be counted, making the investigation inaccurate. Moreover the increasing sugar concentration will inhibit the process of the active transport of glucose, as all the binding sites of the carrier proteins would be occupied, so the only way to transport it is via facilitated diffusion, which is less efficient for the yeast. this would result in less glucose available for cell growth and respiration, which would limit the cell division , and so the population would reach the stationary phase at a faster rate than usual, giving an inaccurate growth graph.
- The % concentration of the yeast; will be provided at 5%, and then a serial dilution will be carried out to dilute to down to 1/100, which will then be used in the investigation. the reason for choosing the dilution , as it would make it less difficult to visibly see and count the yeast cells clearly under the microscope, which would result in inaccuracies occurring, making the whole investigation inaccurate , and so difficult to draw conclusions from. (Note. The concentrations of both the yeast and the carbohydrates were planned in a way that they are in a ratio of 1:2, so that there is twice as much carbohydrate than yeast, as the yeast divide exponentially, so the concentration was made so, so that the carbohydrate supplies the doubling population at least through the early log phase.
- the stirring of the yeast culture; will be standardised for a set amount of time (10 seconds).this ensures that under each condition , the carbohydrate and the yeast are surrounding each other, with the carbohydrate being fully dissolved . If this was not standardised then the yeast growth in the conditions stirred the most (fully dissolved) would be higher, making it biased.
- The conditions will be fixed to be sterile and at anaerobic respiration; sterilising the yeast environment, is very important, especially as its growth is studied, if a bacterial contamination occurred, then the bacteria will compete for the resources, affecting both the growth rate and the final population size, and so acting as a major confounding variable. This would be done, by placing a rubber lid on the test tubes. Doing so will also control another confounding variable, which is the fact that yeasts are facultive anaerobes, and so placing a lid, limits the oxygen supply, and so the respiration would be standardised at anaerobic respiration. Different respiration (anaerobic/aerobic) produce different amounts of ATP energy and in turn would have different effects on the rate of yeast growth. Aerobic respiration will provide more ATP energy for growth and so the yeast may grow faster, but if was left aerobic then there would be a great and quite discrete risk of bacterial contamination.
- The time the yeast culture (yeast and carbohydrate) remains in the water bath; will be controlled to be 1hour and 45 minutes. since yeast cells would require time to adapt to the conditions before beginning to divide, (characteristic of the lag phase).As the experiment has to be done in 1 day, 1hr 45min is the longest possible time for each condition (including the control test).If the culture remains in the water bath for less time, then this would not be enough to observe all the growth stages of the yeast population and so this would prevent all the phases to be plotted on the growth graph , resulting in an inaccurate graph.
- Time intervals between removing and counting a sample; is going to be 15minutes, as it is a reasonable time to observe a difference in the growth of the yeast population. If it was taken every 5 minutes then this would be inadequate for a difference to be seen. Moreover as I will be counting two samples, then 15 minutes is adequate to isolate and count the samples. If the sample was taken earlier i.e. whilst the population is still in the lag phase, this means that there would not be yet a change in numbers, as during the lag phase yeast only change in mass, but not in number. So, it would be pointless to count whilst it is still in the lag phase. If the subculture was taken during that phase, this would increase the length of the lag phase of the subculture, as it will disrupt the time for the yeast cells to adapt to their conditions. This would lead to wasting the time allocated for each investigation (1hr 45 min), and so would not allow the other phases from being fully observed.
Control test
The control test will involve adding yeast and distilled water to a test tube, and placing it under the same conditions as with the other substrates (so the same temperature and pH etc…) .This will be done in order to investigate if it was only the nutrients (glucose, maltose and starch) are the ones affecting the yeast growth, And it is to ensure that it is only the independent variable affecting the dependant variable. If there was not a control, then it would note be certain to state that it was the carbohydrate substrates affecting the yeast growth.
Apparatus list
- Test tubes x6 (n%) -% error
- Powdered glucose, maltose and starch.
- 5% yeast stock.
- Distilled water
- Glass stirrer
- Stop clock
- Balance
- Pipette filler
- Test tube rack x2
- Black marker
- Weighing bottles
- Rubber test tube lids x4
- Heamocytometry set
- pH meter
- Standard buffer solutions at pH 5 and 6.
- Graduated transfer (plastic) pipette x2
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100 cm³ glass beaker x2 +/- 1 cm³ (1%)-1/100x100
- 5 cm³ pipette +/- 0.05 cm³ (1%)- 0.05/5x100
- 10 cm³ pipette +/-0.1 (1.1%)- 0.1/9x100
- 1 cm³ pipette +/- 0.01 cm³ (1%)- 0.01/1x100
Safety precautions
This experiment is a low risk one, as it does not contain any dangerous chemicals. However, still lab coats and goggles should be worn. There is a risk of getting cuts with using glassware, plus a risk of breaking it. Care should be taken when sliding the coverslip onto the haemocytometer grid, as it is delicate and so could break easily. All spillages should be avoided, and wiped as soon as they occur. I will never look into the mouth of a tube, or beaker while stirring or heating or adding reagents. Any accident, breakage, faulty equipment and any other hazards would be immediately reported. Hands will be washed before leaving the laboratory.
Procedure
- I will collect all the apparatus.
- I will label the test tubes A-C .Test tube A will be for the glucose investigation, B for the maltose investigation and C for the starch investigation.
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I will carry out the investigation by investigating the effect of each carbohydrate on the yeast growth, one by one; this is to prevent confusion and distraction. I will start with the glucose. I will weigh out 10g of powdered glucose, using a weighing bottle, I will first place the weighing bottle and set the scale to 0.00g, and then I will start weighing the glucose. Then I will fill a beaker up to the 100 cm³ mark (allow the meniscus to be just above the mark).And add the glucose to the water and stir with the glass stirrer for 10 seconds (time with a stop clock).
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(Serial dilution)Another beaker would be filled with distilled water, and labelled “distilled water” – to prevent confusion. I will pipette 9 cm³ from the water, using the 10 cm³ pipette, and place it in 2 other test tubes, one labelled 1/10 and the other 1/100. 1cm³ of the yeast stock will be pipetted using the 1cm³ pipette , and placed in the test tube labelled 1/10, and stirred (10 seconds). 1cm³ of it will be pipetted and placed into the 1/100 test tube, and stirred (10 seconds). A sample will be removed for counting, to get an initial figure of the number of yeast cells available (this would be used as the time 0 point, as removing a sample during the beginning of the investigation would disrupt the lag phase of the culture, this is to see how much they would increase by, throughout the investigation.
- 5 cm³ will be pipetted and placed into the test tube A. Now this test tube would contain both glucose and the yeast, and would have a total volume of 10 ml.
- to fix the pH at 5.5; the pH meter will be calibrated by placing the electrode in the pH 5 buffer until the readings would appear in the meter, and then the electrode would immediately be rinsed with distilled water. The electrode will also be placed in the pH 6 buffer until the readings appeared on the meter. The pH meter would now be calibrated and ready to be used in the experiment.
- It would then be placed in tube A, and the reading would be shown, the pH 5 and 6 buffers would be added by very small and careful increments (using a plastic graduated transfer pipette, until the reading on the pH meter reaches 5.5.
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Now that tube A is ready, a rubber lid is placed on its top, and then it is placed in a test tube rack, in the water bath which would be regulated at 25°C.And ,the stop clock will be started. And the first sample will be taken in 15 minutes.
- By the time it gets to 15 minutes, a haemocytometer slide would be prepared, and focused under the microscope (at x10-best to get a clear image of yeast cells) any higher and the haemocytometer slide may break.
- At exactly 15 minutes, 2 samples will be pipetted from tube A, using the 2 graduated plastic pipettes. After stirring the solution (10 seconds), to allow a sample with an adequate distribution of yeast cells.
- One sample will first be counted using the haemocytometry technique, by placing the slide cover, by gently pressing towards as sliding it on the top of the central platform around the grid. A “rainbow” pattern would be seen, then a small amount of the sample will be added at the corners , counting should take place only at the 4 corner and the middle B squares on the main type C square (with the thick border around it).
- the number of yeast cells at each of the 5 allocated B squares will be counted, with
the aid of a counter-to prevent confusion and to help to count the yeast cells faster) And the numbers will be recorded on the recordings grids, for example;
Sample 1 Sample 2
- The average number of yeast cells in the allocated B squares will be calculated;
Total number in the 5 Bsquares/5 this will be done for both of the samples. Then both the average numbers of each of the samples will be divided by 2, to get the mean of the 2 samples.
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Using this, the population density per 1 mm³ will be calculated as the following; the sides of each B square is 1/5= 0.22 mm. The area of each B square is 0.04 mm².the volume of each B square is 0.04x0.1= 0.004 mm³.to work out the number of yeast cells per 1mm³= the average no. of B squarex1 / 0.004. As I have planned to dilute the yeast stock to 1/100 and so I will have to multiply it by 100 to get the actual number of yeast cells per 1mm³.The final figure would then be recorded on a recording table. This will later be plotted on a growth graph.
- The collecting, counting and the calculation of the sample, would be repeated for each time interval (every 15 minutes) up to 105 minutes.
- The whole same procedure will be repeated for the other remaining carbohydrates (Maltose and Starch).
- When the investigation has been done with all 3 carbohydrates, I will now carry out the control test. I will label a test tube with “control” to avoid confusion with the other test tubes.
- I will pipette 5cm³ of distilled water into the test tube, and then I will pipette 5cm³ of the yeast stock (1/100) and place in the test tube. This will be then placed in the water bath for 1hr 45 min, with the count being taken every 15 minutes.
Sample Results tables/graphs/diagrams;
As mentioned before when counting the yeast cells on the 5 type B squares, the numbers of each will be recorded on a figure as such;
For the Glucose investigation; (every 15 minutes; )
Sample 1 Sample 2
(n)= the number of yeast cells in this square)
For the maltose investigation; (every 15 minutes; )
For the Starch investigation (every 15 minutes; )
Sample 1 Sample 2
For the control experiment (every 15 minutes; )
Results tables
For the glucose investigation;
For the maltose investigation;
For the starch investigation;
Control experiment
From the data on the recordings tables on the 3 investigations (In addition to the control test) of the different carbohydrate substrates (glucose, Maltose and Starch), a growth graph for each will be drawn;
Lag Log Stationary Death
The phases refer to the following;
Lag refers to a time where the yeast cells are not dividing; there is only an increase in cell mass, but not in cell number. This occurs as the yeast are still adapting to their environment.
Log; refers to a period where there’s an exponential growth, the population can increase exponentially as there are no limiting factors.
Stationary refers to; where the population remains the same as the 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. The yeast also produces changes in its environment, its metabolic products, such as ethanol may make it toxic and so unsuitable for further growth.
Death; the individuals are unable to compete successfully, so they die and undergo autolysis. This is due to all the energy sources running out.
The statistical test which I will be using to accept/reject my null hypothesis;
Is the Chi-squared test;
The degree of freedom (df);
Total so it will be the number of carbohydrates used-1=
Number
Of categories -- 1 3-1= 2
The degree of freedom is 2
Using the table of probabilities with the (df) of 2 and the standard 0.05 level of probability. the tabulated value is 5.991,If the numbers derived from the chi-square calculations was less than the 5.991 number then at a 95% of confidence , it could be stated that the events( the growth of the yeast cells under each condition) was only due to chance alone. In this case the null hypothesis will be accepted.
And, if the number was higher, then the events that have taken place are not due to chance alone, and so highlights the significance of the cause and effect. In this case the null hypothesis will be rejected.
References;
http://www.wooster.edu/biology/wmorgan/bio306/secure/A1.pdf
http://www.pc.vccs.edu/biology-labmanual/lab6cellresp/cellrespiration.htm
Yeast, mould and plant protoplast, edited by J.R. Villanueva, Garcia- Acha
S.Garson and F.Uruburu pages 168 and 182.
Yeast a practical approach, edited by I Campbell & J H Duffus. Pages 1 and 2.
http://en.wikipedia.org/wiki/Yeast
Bio- fact sheet, practical techniques in Microbiology II: Measuring the growth of micro organisms.
Revise A2 biology by Graham read and ray Skwierczynski, pages 8 and 9 on Respiration.
Revise AS biology by Graham read and ray Skwierczynski, pages 2 and 4 on carbohydrates (Monosaccharides, disaccharides and polysaccharides)
Karen Arthur’s notes on Respiration and on serial dilutions.
Steve Wain’s notes on calculating Chi-squared and probabilities.