Question

A student in Biology 328 performed a serial dilution series on his dirt sample to determine the starting concentration of bacterial cells. He performed 5 dilutions by adding 1 mL of the previous culture to the next culture (sterile water) and plated the last dilution as follows: He plated the bacteria by pipetting 1 mL and 0.1 mL (Plates A and B respectively) and spreading the culture evenly over a R2A plate. Please answer the questions on the following page regarding this dilution series. 5 mLs (1 gram dirt) Questions: 1. What is the dilution factor for each dilution? (Added amount/total amount = dilution factor) 2. What is the TOTAL dilution factor for Plate A? What makes the total dilution factor for Plate B different from Plate A? (Total dilution factor = individual dilution factors multiplied by each other) 3. After the plates were incubated at 30°C for 24 hrs, 273 colonies formed on plate A and 30 colonies formed on plate B. What is the concentration, in c.f.u./mL, of the original diluted dirt sample? 4. As shown, the student has 5 mLs of the original culture. How many c.f.u/g dirt are there in the sample? 5. Say the student changed dilution 5; adding 1 mL of the previous culture to 4 mLs of sterile PBS. A. How does this change the dilution factor of dilution 5? B. How does this change the TOTAL dilution factor? C. What would be the new original dirt sample concentration, in c.f.u./g dirt, if Plate A and Plate B yielded the same amount of colonies as indicated in question 3?

Answer 1

The dilution facor for each dilution is given as;

Dilution 1: 1mL of culture + 4 mL of sterile water = 5 times diluted

Dilution 2: 1mL of dilution 1 + 4 mL of sterile water = 25 times diluted

Dilution 3: 1mL of dilution 2 + 4 mL of sterile water = 125 times diluted

Dilution 4: 1 mL of dilution 3 +4 mL of sterile water = 625 times dilution

Dilution 5: 1 mL of dilution 4 + 4 mL of sterile water = 3225 times diluted

Answer 2: Total dilution factor for plate A = 3225

Answer 3: Plating out 0.1 mL of diluted sample on plate B does not change the dilution factor.

Answer 4: The concentration in cfu/mL of the original sample is 273 x 3225 = 8.8 x 10⁵ cell/mL

Answer 5: The cfu/g of the original sample will be = 8.8 x 10⁵ x 5 = 4.4 x 10⁵

Answer 6: Changing sterile water with PBS will not change the dilution factor.