BIOLOGY 313: CELL BIOLOGYSPRING 2007

Lab 5: Yeast Cell Growth Curve1

A. INTRODUCTION

A significant portion of biotechnology requires the ability to maximize the growth of desired types of cell. This week’s lab uses the growth of yeast cells to illustrate some basic concepts of cell growth measurement and specific growth rate calculations. The final outcome of the lab is to determine the concentration of glucose that should be used in yeast growth medium to result in maximum profit for a biotechnology company.

The Cell Biology class has been hired by a bioprocessing firm that would like to supply yeast (Saccharomyces cerevisiae) to the brewing industry. The firm knows the general formulation for the growth media. However, because glucose is expensive and known to be growth-limiting for yeast, they have asked the class to tell them the optimal glucose concentration to use in the growth media. They would like to maximize their profit, so the class will need to determine how much yeast can be produced in a year using different amounts of glucose, and calculate the profit based on yeast production and the cost of glucose. To accomplish this goal, each team will calculate the yeast growth rate at one concentration of glucose and post their result. Each team will then use the data from the entire class to determine the glucose concentration that will produce maximum profit.

In order to construct a yeast cell growth curve, cell counts must be obtained from a culture of growing yeast every hour for up to 24 hours. This presents some problems for completing this study in a single three hour lab period!! Fortunately, your team will be provided with data for yeast growing at a single glucose concentration, so you will not have to collect these data points. However, the data will be supplied as absorbance, not as cell number!! Your team’s job during the lab period will be to construct a standard curve of absorbance as a function of yeast cell concentration. You will then be able to use this standard curve to determine the cell concentration at each of the absorbences given to you, and use this information to calculate the profit from growing yeast cells at your team’s glucose concentration.

In constructing the standard curve in today’s lab, your team will collect three data points for each concentration of yeast. Comparison of the reproducibility of the protein standard curves from different teams with the reproducibility of the yeast cell standard curves from different teams will provide an opportunity to observe the effect of replicate sampling on the reproducibility of the data.

B. OBJECTIVES

Detemine the largest annual profit that can be attained by growing yeast cells for sales to microbrewers, and the initial glucose concentration in which yeast cells should be grown to produce this profit.

C. METHODS

hemacytometerbright field microscopepastuer pipettes

10 ml pipettes & pipettertest tubes & rack5 dilutions of yeast cell culture

spectrophotometer (visible range) & cuvettes

absorbance of yeast grown in a specific concentration of glucose.

  1. Collect three separate 4 ml samples from one of the 5 dilutions of the yeast cell cultures, placing each sample into a different test tube.
  2. Immediately measure the absorbance at 600nm of each of the separately collected samples. Use a "blank" of growth medium in which no yeast have been grown to adjust the spectrophotometer to zero absorbance at 600nm, thoroughly mix the sample and then measure its absorbance. Repeat for the other two samples, and record this information in each “A600” box for Sample #1, #2 and #3 in the first row of Table I.
  3. Load both sides of the hemacytometer from the one of the samples and count 100 to 150 cells on each side. Repeat the cell count for the other two samples. Record this information in each “Cell count” box for Sample #1, #2 and #3 in the first row of Table I.
  4. Calculate the cell concentration for each of your three replicate samples. Show a sample calculation and record this information in each “Cells/ml” box in the first row of Table I.
  5. Calculate the mean and standard deviation of A600, and of Cells/ml, for the three replicate samples you obtained in Steps 2 and 4. Enter these values in the last two columns of row 1 of Table I.
  6. Repeat steps 1 through 5 for each of the other 4 yeast cell dilutions in exactly the same manner as steps 1 through 5 were conducted the first time, but entering your data and calculation in rows 2 through 5 of Table I.
  7. Post your team’s calculation from step 5 on the blackboard before you leave the lab.
  8. Use the mean“Cells/ml” and the mean “A600” from each of your team’s five samples to construct a standard curve.
  9. Use your team’s standard curve to determine the cell concentration at each absorbance of your team’s growing yeast culture. These absorbances will be given to your team. Enter these determinations in the first and second rows of Table II.
  10. Convert each yeast cell concentration in row 2 of Table II to mass/L. Show a sample calculation and enter the mass/L in the third row of Table II.
  11. Construct the growth curve in the glucose concentration given to your team for yeast cells(yeast cell mass/L as a function of time) on both linear and semilog graph paper.
  12. Determine the slope of the straight line portion of the semilog plot. This slope is equal to the specific growth rate, m = mass increase per liter/hr. Post this specific growth rate and the glucose concentration for this growth rate on the bulletin board inside the lab door.
  13. Record the specific growth rate and glucose concentration posted for all teams in the first two rows of Table III.

D. ANALYSIS AND CONCLUSIONS

  1. Your instructor will accumulate the data from all teams and post a plot of all team’s standard curves on the Cell Biology class folder. You are responsible for comparing the team to team reproducibility of these standard curves.
  1. Use the information posted by all nine teams to construct a plot of specific growth rate as a function of glucose concentration.
  1. Harvesting of the yeast occurs when the cell concentration reaches 100 g/l. Determine the time it takes for the yeast to reach this level at each glucose concentration using the equation

X = X0em t. In this equation,

  1. X = Cell concentration when the cells have been growing for time = t
  2. X0 = starting cell concentrationAssume an initial yeast cell concentration of 0.0049 g/l.
  3. em = log of the specific growth rate
  4. m = specific growth rate
  5. t = the time that the cells have been growing

Record this information in row 3 of Table III.

  1. In step 3 you calculated the run times (time it takes to reach 100 g/l of yeast) for each glucose concentration. Now calculate the number of runs in a year for each glucose concentration and enter this information in row 4 of Table III.
  1. Glucose costs $1.00 per kilogram while yeast cells sell for $5.00 per kilogram. This allows one to calculate the profit generated by one run. Using this information, determine the profit per liter per run at each specified initial glucose concentration. Enter this information in row 5 of Table III.
  1. Nowuse the results of steps 4 and 5 to calculate the annual profit for each glucose concentration and enter this information in row 6 of Table III.
  1. Construct a plot of profit per year per liter versus initial glucose concentration. Use this plot to determine the initial glucose concentration that produces the largest annual profit.
  1. According to your team’s calculations, the largest annual profit is , and this profit is

obtained with an initial glucose concentration of .

  1. Enter this information in the blanks provided on the last page of this lab guide.

Table I: Standard Curve Data

(methods steps 1 through 6)

Sample #1 / Sample #2 / Sample #3 / Avg & Std Dev
Dilution / A600 / Cell count / Cells/ml / A600 / Cell count / Cells/ml / A600 / Cell count / Cells/ml / A600 / Cells/ml
1
2
3
4
5

Table II: Conversion of Absorbance to Cell Concentration

(Methods steps 9 through 12)

A600
Cells/ml
Mass/L
(= g/L)
Hours of Growth

Table III. Specific Growth Rate and Profit/Year as a function of Glucose Concentration

(Methods step 13 and Analysis steps 2through 6)

[Glucose]
Specific Growth Rate
Run Time
Runs/Yr
Profit per Liter per run
Profit per Liter per Year

According to your team’s calculations, the largest annual profit is , and this profit is

obtained with an initial glucose concentration of .

Lab5S2007BI313YeastGrowth.DOC11/16/20181