Yeast Plate Count Lab
Making a Serial Dilution

Robert Koch, a German physician, observed bacteria growing on an old boiled potato and realized that each colony he saw came from a single cell that had previously landed on the potato. We will be inoculating 4% glucose medium with an unknown number of yeast cells, and like Koch’s conclusion that each bacterial colony on the potato came from a single, live bacterium, we will assume that each yeast colony we find growing on our agar plates came from a single, live yeast (a colony-forming unit or CFU). We will use this information to calculate the average number of yeast cells in a packet of yeast. There are too many yeast cells in a packet to count them all, so we must dilute them, but if we keep track of how much they were diluted, we can use the number of colonies growing on our plates to calculate the number of cells in a yeast packet. Because the yeast must be very dilute to get countable results, we need to spend time discussing the math involved in figuring out the number of cells per packet.

For example, think about how you make Campbell’s Chicken-Noodle soup. What two things do you mix together, and what do you get as a result? The soup concentrate would be an aliquot from the big batch made at the soup factory, and the water you mix with it would be the diluent. Answer the following soup-making questions, then push the “Am I Right?” button to see if you were right.

1. In a dilution, the dilution factor is equal to the final volume divided by the initial volume of solution, or DF = V_f ÷ V_i. How many times as dilute is the soup than the soup concentrate?
   times as dilute
2. The concentration factor is equal to the initial volume divided by the final volume of solution (the inverse of the dilution factor), or CF = V_i ÷ V_f. How many times as concentrated is the soup than the soup concentrate?
   times as concentrated
   Suppose that the diluted soup makes 2 bowls of soup, thus the volume of 1 can = the volume of 1 bowlful. If each bowl of soup contains 50 noodles, we could express its concentration as 50 noodles/bowl.
   
3. How many noodles were there in the soup concentrate?
   noodles
4. What was the concentration of the soup concentrate in units of noodles/bowl?
   noodles/bowl
   Now, suppose you have a really big family and want to stretch the can of soup, so for one can of soup concentrate you add 99 cans of water, thus your final volume = 100 cans.
   
5. Expressed in scientific notation (powers of ten), how many times as dilute is the soup than the soup concentrate?
   10 as dilute
6. Expressed in scientific notation, how many times as concentrated is the soup than the soup concentrate?
   10 as concentrated
7. If each bowl of soup contains 1 noodle, how many noodles were there in the soup concentrate?
   noodles
8. What was the concentration of the soup concentrate in units of noodles/bowl?
   noodles/bowl

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   First, a packet of yeast will be suspended in 100 mL of water. Why is it not necessary to use sterile water for this step?
   
9. If one packet of yeast was put into 100 mL of water, what percentage of the yeast cells from the original packet are now in the 100 mL of suspension?
   %
   

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   Out of that 100 mL, each person will use a 0.10 mL aliquot. A 0.10 mL pipet will be used to obtain this aliquot.
   
10. Expressed in scientific notation, how many of the original yeast cells will be in your pipetful of suspension?
    10
    

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    This 0.10 mL aliquot will be placed into 9.9 mL of sterile water and mixed thoroughly.
    
11. What will the total volume in the first test tube be?
    mL
12. What, in scientific notation, will the concentration factor be for the first test tube as compared to the pipetful of original suspension?
    
    10
    

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    Because this is a serial dilution, an aliquot will be removed from the first test tube and mixed with 9.9 mL of water in the second test tube. Using sterile technique, 0.10 mL of the suspension from the first test tube will be added to the second test tube.
    
13. Expressed in scientific notation, how many of the original yeast cells will be in this pipetful of suspension?
    10
14. What will the total volume in the second test tube be?
    mL
15. What, in scientific notation, will the concentration factor be for the second test tube as compared to the first test tube?
    10
16. What, in scientific notation, will the concentration factor be for the second test tube as compared to the original suspension?
    10
    

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    In this serial dilution, using sterile technique, 0.10 mL from the second test tube will now be put into 9.9 mL of water in the third test tube.
    
17. Expressed in scientific notation, how many of the original yeast cells will be in this pipetful of suspension?
    10
18. What will the total volume in the third test tube be?
    mL
19. What, in scientific notation, will the concentration factor be for the third test tube as compared to the second test tube?
    10
20. What, in scientific notation, will the concentration factor be for the third test tube as compared to the original suspension?
    10
    

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    Once again, 0.10 mL will be obtained from the third test tube. However, this suspension will now be used to inoculate a plate of your sterile medium.
    
21. Expressed in scientific notation, how many of the original yeast cells will be in this pipetful of suspension?
    10
22. Expressed in scientific notation, how many of the original yeast cells will be on your agar plate?
    10
    

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    Here’s a summary of all the steps. You should also inoculate an agar plate with 0.20 mL of suspension from your third test tube.
    
23. Expressed in scientific notation, as compared to the number of CFU on your plate, how many more yeast cells are in the original packet?
    10
24. Expressed in scientific notation, how many of the original yeast cells will be on your 0.20 mL agar plate?
    
    (Hint: use the format 1x10e5 to indicate superscripts.)
25. Expressed in scientific notation, as compared to the number of CFU on your 0.20 mL plate, how many more yeast cells are in the original packet?
    
    (Hint: use the format 1x10e5 to indicate superscripts.)