Yeast use oxygen to build membranes components that are essential to replication. These cell-wall building blocks (unsaturated fatty acids and sterols) can sustain yeast growth even when present at very low levels.
In anaerobic respiration, when free oxygen is not present, hydrogen cannot be disposed of by combination with oxygen. The electron transfer chain therefore stops working and no further ATP is formed by oxidative phosphorylation. If a cell is to gain even the two ATP molecules for each glucose yielded by glycolysis, it is essential to pass on the hydogens from the reduced NAD that are also made in glycolysis. This pathway is involved in anaerobic respiration of yeast cells. The hydrogen from reduced NAD is passed to ethanal (CH3CHO). This releases the NAD and allows glycolysis to continue:
GLUCOSE
ADP
2H REDUCED NAD
NAD
ATP
2H
PYRUVATE ETHANAL ETHANOL
First pyruvate is decarboxylated to ethanal; then ethanal is reduced to ethanol (C2H5OH) by the enzyme alcohol dehydrogenase. The conversion of glucose to ethanol is referred to as alcoholic fermentation3.
VARIABLES
The independent variable in this investigation is temperature. The yeast cells shall be given five different environments of varying temperatures ranging from 20oC to 60oC.
The dependent variable in this investigation is the number of yeast cells counted using a haemocytometer after being exposed to different environmental temperatures.
The control variables in this experiment, which shall be kept constant to ensure a fair test throughout the investigation, are:
∗ Concentration of yeast suspension used. From preliminary experiments it was evident that a yeast concentration, which was too strong created viewing problems when attempting to count yeast cells on the haemocytometer. A concentration of 0.1moldm-3 shall be used throughout the investigation to reduce these problems.
∗ Volume of yeast suspension. 1cm3 of yeast cells shall be used for each temperature. This ensures a fair test throughout the investigation, as an initial larger volume of yeast solution would inevitably contain more yeast cells.
∗ Volume of nutrients supplied to yeast. 50cm3 of cider shall be added to each 1cm3 of yeast solution. If more cider were given to some yeast solutions than others were then cell division would inevitably be more rapid. This would clearly affect the reliability of the results as it would not show that temperature alone has an overall effect on the growth of yeast cells.
∗ A cork bung shall be fastened onto every conical flask once the solutions have fully mixed. This is to prevent the yeast cells being supplied with any additional oxygen. Although yeast cells can respire anaerobically these reactions merely 'buy time'. They allow continued production of at least some ATP even though oxygen is not available as the hydrogen acceptor. However, since the products of anaerobic respiration are toxic, cell growth cannot continue indefinitely until the nutrients in the yeast suspension begin to run out. The yeast cells begin to slow down their rate of growth and division, as they compete for an ever-decreasing quantity of nutrients. Moreover, the toxic products of anaerobic respiration begin to accumulate and may also inhibit their metabolism and slow down their rate of growth and division.
∗ Time. The suspensions of yeast shall be kept in the different environments for 24 hours. This ensures that each yeast solution is given the same length of time in each environment. Subsequently this increases the reliability of the results obtained by maintaining a fair test throughout. Giving some yeast solutions more time than others would clearly allow more time for cell division and growth to occur.
NUMBER AND RANGE OF MEASUREMENTS
I intend on using five different temperatures of water bath ranging from 20oC to 60oC at 10oC intervals. A sample of yeast suspension shall be extracted every 2 hours for 24 hours. This shall allow a population/time graph to be plotted for each temperature. I.e. 5 graphs
For each of the five environments the number of yeast cells counted in the populations shall be repeated three times. This shall provide three results at each temperature, which inevitably increases the reliability of the results obtained.
PRELIMINARY WORK
As I planned this investigation I had two possible methods to measure the population of yeast when in different environmental temperatures.
- Using a microscope and a special slide known as a haemocytometer slide. It is designed so that a known volume of sample covers a ruled grid. A representative sample of cells can thus be counted and estimates can be made of the number in the total sample. A haemocytometer has an etched grid on it. It consists of a 1mm² square known as an A square which is divided into 25 B squares which have an area of 0.04mm². These B squares are each divided into 16 C squares which have an area of 0.0025mm².
- Using a colorimeter. A colorimeter is a machine that is used to see how much light can pass through a liquid. It shows how much light is being transmitted through a sample of liquid. As the number of cells gets higher, less light will be transmitted through the sample. Special thin walled test tubes are used in the colorimeter so that they do not affect the amount of light passing through the sample.
I have used colorimeters for many previous experiments i.e. investigating the affect of temperature on the release of pigment from beetroot cells. I found that they were particularly unreliable providing at times incoherent results. In the above investigation I believed that increasing the temperature would increase the amount of pigment released from the beetroot, due to the breakdown of the phospholipid bilayer. Less light being transmitted through the sample as the temperature increased would portray this. However, my results had no real correlation:
Although, these results may have been due to experimental error, it was still particularly time consuming and inconvenient to use the colorimeter i.e. had to be repeatedly zero the dial before the next reading etc.
From a recent topic on growth I used a haemocytometer to view and count yeast cells at three different concentrations of yeast solution. I had never before used such apparatus and thoroughly enjoyed the experience. I therefore chose to use a haemocytometer to count the number of yeast cell rather than the colorimeter mainly due to my motivation of extending my abilities as a biologist in using new apparatus along with my already competent skills with a microscope.
SAFETY
APPARATUS
∙ Microscope. Simple to use and provides an accurate method of focusing on yeast cells using a haemocytometer.
∙ Haemocytometer. Simple to use and allows a representative sample of cells, which can thus be counted and estimates can be made of the number in the total sample. Method of use:
1. Clean haemocytometer and cover slip using 70% ethanol.
2. Place coverslip squarely on top of haemocytometer, lightly moistening polished surface of the slide before pressing the coverslip into position.
3. Gently redistribute cells throughout medium and take up a small sample of cells into the syringe.
4. Load haemocytometer so that the fluid entirely covers the polished surface of each chamber. Take care not to overload the counting chambers. Should overloading take place, excess fluid may be removed carefully from the groove using filter paper.
5. Using the x10 objective of the microscope, locate the upper left primary squares of each grid (10 primary squares). It may be necessary to move to a higher magnification to increase the resolution of the yeast cells i.e. ability to distinguish between two different cells5. Use a counter to count the cells in 10 primary square using the following conventions:
→The middle of the triple lines separating each primary square is the boundary. Cells that touch the upper or left boundaries are included, those that touch the bottom or right boundaries are excluded (Fig.2).
→If greater than 10% of particles are cell clusters, attempt to disperse the original cell suspension further and start again. Alternatively, assign all clusters containing more than five cells a value of five.
→If there are too many cells present to realistically count, perform a dilution using a buffer or dye.
→For accuracy and reliability, counts must be carried out in the same manner each time.
→When the haemocytometer is loaded properly, the volume of cell suspension that will occupy one primary square is 0.1 mm3 (1.0 mm2 x 0.1 mm) or 1.0 x10-4 ml.
→In practice, cell counts of 10 primary squares give the number of cells within 1.0 mm3 (10 x 0.1 mm3) or 1 x 10-3 ml. Total cell concentration in the original suspension (in cells/ml) is then:
Cells/ml = total count x 1000 x dilution factor
∙ 15cm3 Yeast at concentration 0.1moldm-3. This is a dilution of the suspension of baker's yeast. Dilute using distilled water. If provided as a 1moldm-3 solution add 10cm3 distilled water to 1cm3 of 1moldm-3 baker's yeast to make a 0.1moldm-3 solution. 15cm3 Yeast is a sufficient volume to repeat all five different temperatures three times to achieve reliable results.
∙ 750cm3 cider, which is a sufficient volume to repeat all five different temperatures three times to achieve reliable results.
∙ Five Cork bungs to cover the conical flasks whilst in the varying environments to prevent further intake of oxygen by the yeast, ensuring that they continue to respire anaerobically throughout the investigation.
∙ 1cm3 syringe. This is a highly accurate piece of apparatus to measure precise volumes of liquids. They have a very low percentage error, which increases the accuracy of the results.
∙ 50cm3 measuring cylinder. Required for measuring out 50cm3 of cider to be supplied to yeast cells as a nutrient source.
∙ 10cm3 measuring cylinder to measure 10cm3 distilled water for dilutions of baker's yeast at concentration 0.1moldm-3.
∙ Five 250cm3 conical flasks, which shall be placed into each water bath of different temperatures.
∙ Five electronic water baths. Temperatures ranging from 20oC to 60oC at 10oC intervals. Very accurate piece of apparatus allows the required temperature to be precisely maintained throughout a chosen time period.
METHOD
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Measure out 50cm3 of cider into a 250cm3 conical flask using 50cm3 measuring cylinder.
-
Use a 1cm3 pipette to carefully measure out a volume of 1cm3 0.1 moldm-3 yeast solution and add to the cider.
- Mix the solution and then push a cork bung securely into the conical flask.
-
Put this conical flask into a water bath set to 20oC for 24 hours. It may be necessary to monitor the temperature of the water bath using a thermometer. This shall improve the reliability of the results, ensuring that the yeast cells are actually in the required environment for the investigation.
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Every 2 hours remove a sample volume of 1cm3 of yeast suspension using a 1cm3 syringe. (12 samples of yeast suspension for each temperature.)
- Turn on the microscope and prepare the haemocytometer:
(a) Clean haemocytometer and cover slip using 70% ethanol.
(b) Place coverslip squarely on top of haemocytometer, lightly moistening polished surface of the slide before pressing the coverslip into position.
(c) Gently redistribute cells throughout medium and take up a small sample of cells into the syringe.
(d) Load haemocytometer so that the fluid entirely covers the polished surface of each chamber. Take care not to overload the counting chambers. Should overloading take place, excess fluid may be removed carefully from the groove using filter paper.
(e) Using the x10 objective of the microscope, locate the upper left primary squares of each grid (10 primary squares). It may be necessary to increase the magnification. This increases ability to distinguish between two different cells, whilst at a lower magnification these two cells would be viewed as just one cell. Use a counter to count the cells in 10 primary squares. Cells that are within or that touch, the left or top boundary are counted, while those that touch or are outside the lower or right hand boundary are not counted.
[Refer to fig. 2 for assistance.]
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Repeat steps 1-6 using four different temperatures of water bath: 30oC, 40oC, 50oC and 60oC.
- Repeat each temperature three times to increase the reliability of the results obtained.
- Record all results in an appropriate table
- Calculate the total number of yeast cells present using the following equation:
Cells/ml = total count x 1000 x dilution factor
REFERENCES
1Populations and interactions Biology 2 endorsed by OCR p29
2Enzymes Biology 1 endorsed by OCR p47
3Energy and respiration Biology 2 endorsed by OCR p12
4
5Cell structure Biology 1 endorsed by OCR p7
6Growth and development endorsed by OCR p17