lab 1 - yeast growth PDF

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lab 1...

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Introduction The objective of this experiment was to determine and compare the exponential growth rate of an unicellular organism, yeast(Saccharomyces Cervisiae), in two different medias, nutrient broth and Sabouraud’s broth. Yeast is often used in biological studies because it can be easily cultured at an economic price( Investigation of the Best Saccharomyces cerevisiae Growth Condition). Yeast can reproduce through two approaches, the asexual way of budding and the sexual way of mating. The most common way is the budding, which the chromosomes are duplicated in a mother cell and then one copy of them is distributed to the new daughter cell. The daughter cell forms as a bud on the mother cell. (yeast textbook 175page 7.1) Budding can occur in both diploid and haploid yeast cells. For haploid cells, the budding is exhibited in an axial pattern, where the mother cell buds adjacent to the daughter cell. On the contrary, diploid cells bud in a bipolar pattern. The bipolar pattern is slightly more complicated. The mother cell can bud on either its ends, whereas the daughter cell bud away from its mother cell.(yeast textbook 175page 7.1.1.1) Budding has been enormously studied by Ira Herskowitz and other collaborators. Based on their research, budding is not a random process, but rather a controlled process. The bud site is actually genetically controlled by a morphogenetic

pathway.(Ira 文章 文章）Under starving conditions, yeasts will undergo the mating to reproduce spores sexually.(yeast textbook 175page 7.1)

Similar to other microorganisms, the growth of a yeast population has 4 distinct phases. As a small amount of yeast is placed in an appropriate media, it will experience the lag phase. The growth rate at lag phase is negligibly slow because the newly arrived cells need to modify themselves to benefit from the new environment so they can grow exponentially later.(Predictive modelling of the microbial lag phase: a review) After the lag phase, the cells enter the exponential phase, where the population rapidly grows at a constant rate. The rate is constant because the cells are in a steady state. The exponential phase ends as the stationary begins. The rapid growth finally stops mainly due to three reasons, the exhaustion of nutrients, the accumulation of harmful metabolic wastes and the change in ion equilibrium.(THE GROWTH OF BACTERIAL CULTURES) The population of yeast remains stable at the stationary phase, and then decreases as it moves to the death phase. There are some factors which will affect the growth of yeast. Yeasts are able to live in either the aerobic environment or the anaerobic environment. The aerobic environment is obviously more favourable since the aerobic cellular respiration is way more efficient than

the anaerobic respiration.(Investigation

of

the Best

Saccharomyces cerevisiae Growth Condition) As for temperature, the growth rate of yeast is at maximum when the temperature is between 30°C and 35°C. (GROWTHOFSACCHAROMYCESCEREVISIAEANDSACCHAROMYCESU VARUMINATEMPERATUREGRADIENTINCUBATOR). In addition, the pH plays a role in affecting the growth of yeast as well. The most comfortable environment for yeast is when the pH equals to 4, so yeasts like acidic environment.

(Investigation of the Best Saccharomyces cerevisiae Growth Condition) The medias, Saboraud’s broth and nutrient broth, are different in terms of composition. Saboraud’s broth is composed by distilled water, dextrose and polypeptone or neopeptone with a pH of 5.8.(BAM Media M133: Sabouraud's Dextrose Broth and Agar). Nutrient broth contains beef extract, peptone and distilled water with a pH of 6.8±2.(BAM Media M114: Nutrient Broth) In the case that all other conditions are held equal except pH and nutrients composition for the two medias. It is reasonable to hypothesize that the yeast in the Saboraud’s broth will grow more than the yeast in the nutrient broth since Saboraud’s broth contains sugars(dextrose) and has a lower pH, which best satisfies the favourable condition for the yeast. Thus, the exponential growth rate of the sample in Saboraud’s broth should be higher than that of the nutrient broth. Material and Method After being provided with all the essential information, the one-week long experiment was initiated on October 18. In the beginning, a Bunsen burner was ignited for the sake of establishing a sterile environment, which is extremely crucial for this experiment, and all the following works would be done near the fire. Two pipette tips were then placed above the fire for a moment to be sterilized. 1 mL of yeast suspension was pipetted by a sterilized pipette and added to an Erlenmeyer flask containing nutrient broth(aka NB) and another same 1 mL of yeast suspension was added the flask containing Sabouraud’s broth(aka SB). The two flasks were stirred so the yeasts were evenly spread. Finally, the two flasks were tightly sealed by two foils

respectively and placed in an environment with a temperature of 25°C to let the yeasts reproduce. Observing is certainly an indispensable step in this experiment. In order to observe the phenomena and record the related data, sampling the culture is the first thing that must be done. 1 mL of yeast culture from each of the two flasks(NB and SB) was added into a test tube by a sterilized pipette. Afterward, two drops of methylene blue dye were added to each of the two test tubes, and a few drops from each of them were pipetted to the two sides of a clean haemocytometer. The haemocytometer was then covered by a glass slide and placed on a microscope with a magnification of 40X. At this point, it was time to count the living and dead cells on the 16-squared haemocitometer. The blue ones are the death cells and the transparent ones are the living cells. The observing and recording part was repeated on the day 1,2,3,4,5,6. One thing noticeable was that on the day 2,4,5 and 6, the sample culture was diluted by distilled water as diluting was necessary for the proper progress of the experiment. The water added for day 2 was 1mL, and the water added for day 4,5 and 6 was 2 mL. After all the procedures mentioned above were completed, calculation fulfilled the last step of the method part. There are two formulas associated with this experiment. The first one is for calculating the cell density, cell density = (number of cells/number of squares) x 4000 x 1000 x dilution factor; the second one is for obtaining a k value which stands for the rate of exponential growth, k = (logN t logN0)/(log2 x t). For the cell density formula, the content inside the parentheses are

pretty straightforward, the 4000 x 1000 means the sum of squares times the conversion from mm3 to mL, and the dilution factor is simply the water added plus the yeast suspension added divided by the yeast suspension added. For the k value formula, Nt

represents the density of a specific day after the experiment

began(ranging from day 1 to day 6), and N 0 represents the density of day 0. The t in the denominator is the time measured in hours. (CITATION NEEDED, FROM PPT?) Result

Cell density in NB 70000000

64800000

60000000

Cell density cells/mL

49600000 50000000 40000000 31200000 30000000 21450000 16200000 20000000 7800000 10000000 0 Day 0

14400000 8700000

6600000

Day 1

Day 2

Day 3

8800000

15200000 9600000

Day 4

Day 5

Time/Days Alive

Dead

Graph I - Cell density in NB over the week

8000000 7200000

Day 6

Cell density in SB 120000000 101700000

Cell density cells/mL

100000000 80000000

68000000

60000000

49200000 41800000

40000000

15000000 12900000 20000000

26950000

24800000

17100000

24000000 20800000 8000000

2400000 0 Day 0

1600000 Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Time/Days Alive

Dead

Graph II - Cell density in SB over the week After collecting all the raw data, cell density was derived by using the cell density formula mentioned above. Graph I and II illustrate the change in cell density in NB and NB over the week. For both of them, the densities of living cells on day 3 and day 4 are predominantly higher than others. In NB, the living cells’ density starts with 7800000, fluctuates a little bit, finally drops to 6600000 on day 2, then it is followed by a dramatic soar that the density reaches the maximum of 64800000 on day 4. After that, the density intensively declines to 15200000 and reaches 8000000 on day 6. For the dead cells’ density, one thing interesting is that the trend of dead cells’ density and the trend of living cells’ density are roughly opposing to each other. The dead cells’ density starts with 16200000 and ends with 7200000. One thing noticeable is that on day 3, the dead cells’ density reaches is maximum at 31200000. The living cells’ density and dead cells’ density in SB pretty much have the same

trend. The density of

K value for NB 5 3.8

43.7

3.2

K value in percentage(%)

3 2 1 0 Day 1 -1

1.3 0.87 0.25

-0.52 Day 2

Day 3

Day 4 -0.92

-0.08

0.03

Day 5

-0.81 Day 6

-2 -3 -3.7 -4 -5

Time/Days Alive

Dead

K value for SB 6 4

K value in percentage(%)

1.7 2 0 Day 1

3.7 1.8

4.2 2.5

2.4

0.75

0.75

0.4 Day 2

Day 3

Day 4

-2 -4 -6 -8 -10-11 -12

Time/Days Alive

Dead

Day 5

-0.48 Day 6

-2.2

Reference https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308499/ 开头引用

https://www.sciencedirect.com/science/article/pii/009286749190015Q Ira 文章

https://www.sciencedirect.com/science/article/pii/S0168160504000698 Lag phase

https://onlinelibrary.wiley.com/doi/epdf/10.1002/j.2050-0416.1977.tb06813.x Ph

https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm063580.ht m SB

https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064169.ht m

NB...