Measuring the population growth over time using turbidity
Equipment
Equipment
- Water bath
- For creating an optimum temperature for the yeast to grow at
- Sterile Pipettes
- Used for transferring broth into a test tube
- Colorimeter and Cuvette
- For measuring the absorbance of light of your diluted yeast
- Nutrient Broth
- The sterile nutrient medium suited for growth
- Bunsen Burner
- By flaming the neck of a flask you can prevent bacteria getting inside, keeping something sterile over time is imperative
- Conical Flask
- Contained the nutrient broth and yeast, it was necessary to be large enough that we could take small samples multiple times
- Distilled Water
- Water that was aseptic was used as any contaminants would have competed for nutrients with yeast meaning altered values
- Dried Yeast
- We used yeast in our experiment so we had to add dried yeast to our broth
- Test tubes
- Test tubes were used to measure out our 1:9 cm3 solutions
- Cotton wool
- Used as a bung for our conical flasks to ensure they remained sterile
Variables
- Independent Variable
- The time at which we took measurements of our the absorbance of light
- Dependant Variable
- The absorbance of light of our yeast broth solution
- Control Variables
- Concentration of solutions, when we recorded our results we used a 1:9 cm3 solution, if we did not use the same concentration every time our results would vary widely
- Cuvette used, different cuvettes are made of different thickness of plastic meaning different base absorbances, to make sure this is not an issue we have to use the same one every time
- Put 0.025g of dried yeast into 100cm3, then after shaking to ensure even distribution of yeast take 1cm3, using a pipette, and put into 99cm3 of sterile broth. This gives a a very low concentration of 0.0025 g/l. It was necessary to use such a low value as the mixture would have gone cloudy very quickly meaning very little analysis if we had a higher concentration
- Place in yeast nutrient broth into a water bath, this was set to 30 degrees which was a close to optimum temperature for yeast growth
- To measure the absorbance of the yeast solution take 1cm3 of the broth and add to 9cm3 of distilled water in a test tube. Then use a reference cuvette which is just 1:9 ratio of sterile broth (with no yeast) and water. After referencing it use the same cuvette and fill it up with the 1:9 cm3 solution. Then measure the absorbance by pressing “T”. Repeat this 3 times in one session and average out the result
- Repeat step 3 three times a day at as regular an interval as possible, for our group this was first thing in the morning (08:00), lunchtime (14:00) and last thing at the end of school (18:00)
- Ensure to record all results and record the results in a graph
Results
You can see from this table our absorbency results, due to being averaged across three recordings are large decimals. To simply this we can plot the results on a graph. This also allows to see trends and view anomalies.
These as a graph over time show a clear trend. The graph starts of with a low gradient for the first 18 hours and then the gradient increases dramatically and the absorbance initially peaks it then for the next 14 hours does not reach near the same value. After the second distinct peak the graph begins to fall until we stopped the readings which was after 74 hours.
Analysis of results
Before interpreting our results it is important to remember that the absorbance of light directly correlates to population of cells. Our results have a clear trend to them which I think mirrors that of the intended shape of number of cells over time. Because cell growth is exponential the growth is initially very slow causing what is known as a “lag phase” this can be seen up until 18 hours into the experiment. This is because the gradient is quite low relative to the rest of the graph. The sharp increase of the gradient after 18 hours up until 30 hours in known as the “log phase” in which most of the growth of the population occurs very quickly. Up until this point our results have mirrored the intended graph however our results fall and then rise. I believe our results here are incorrect as if we were to remove our results at 42 hours, 50 hours and 54 hours our graph would have a flat or very low gradient. This is indicative of the stationary phase in which the population has reached carrying capacity (K). This means that the population has reached the point where, for whatever reason (e.g. nutrients or space) the medium cannot support any more yeast cells. At the end of the stationary phase, or 66 hours, the population begins to fall. This is known as the death phase. This is most likely due to the nutrients, which was in the broth being exhausted by the large number of cells, or when the cells respire and reproduce they leave behind toxic material which over time accumulates and can eventually kill the population.
Analysis of results
Before interpreting our results it is important to remember that the absorbance of light directly correlates to population of cells. Our results have a clear trend to them which I think mirrors that of the intended shape of number of cells over time. Because cell growth is exponential the growth is initially very slow causing what is known as a “lag phase” this can be seen up until 18 hours into the experiment. This is because the gradient is quite low relative to the rest of the graph. The sharp increase of the gradient after 18 hours up until 30 hours in known as the “log phase” in which most of the growth of the population occurs very quickly. Up until this point our results have mirrored the intended graph however our results fall and then rise. I believe our results here are incorrect as if we were to remove our results at 42 hours, 50 hours and 54 hours our graph would have a flat or very low gradient. This is indicative of the stationary phase in which the population has reached carrying capacity (K). This means that the population has reached the point where, for whatever reason (e.g. nutrients or space) the medium cannot support any more yeast cells. At the end of the stationary phase, or 66 hours, the population begins to fall. This is known as the death phase. This is most likely due to the nutrients, which was in the broth being exhausted by the large number of cells, or when the cells respire and reproduce they leave behind toxic material which over time accumulates and can eventually kill the population.
Evaluation
I believe our experiment was quite successful, overall I felt our results were very close to what was intended meaning that we did our experiment aseptically and followed the instructions well. I feel our only problem was that our stationary phase didn’t quite match to what it should have been. However as it was not a single result that off but three which had a general correlation between each other I feel that the issue was most likely the reference solution which we calibrated the colorimeter with. If it were not made with precise measurements with exact measurements it would affect readings taken uniformly. IF the reference solution had slightly more broth in that would lower the readings we would have taken. Although there is no way to prove this theory is correct, I believe it provides a logical answer to why our results went down when they should have not.
I believe our experiment was quite successful, overall I felt our results were very close to what was intended meaning that we did our experiment aseptically and followed the instructions well. I feel our only problem was that our stationary phase didn’t quite match to what it should have been. However as it was not a single result that off but three which had a general correlation between each other I feel that the issue was most likely the reference solution which we calibrated the colorimeter with. If it were not made with precise measurements with exact measurements it would affect readings taken uniformly. IF the reference solution had slightly more broth in that would lower the readings we would have taken. Although there is no way to prove this theory is correct, I believe it provides a logical answer to why our results went down when they should have not.