Jennifer Boyd (Mathematics)Lab Exercise 4
Counting bacteria and estimating generation time
Overview
Using serial dilutions and the plate count method, students will calculate the density of bacteria in a stock culture. After a period of incubation at either a high or low temperature, students will again calculate bacterial densities. Using these data students will determine generation time for their bacteria.
Objectives
After completion of this exercise, students should demonstrate a mastery of the techniques involved in estimating bacterial densities using the plate count method. These include aseptic technique, serial dilutions, inoculating culture plates, and counting colonies. Students should also demonstrate an understanding of the mathematics required, which include using scientific notation and exponents, and solving logarithmic equations.
Materials
E. coli culture in broth
sterile 1ml pipettes
culture tubes with 9.0 ml of sterile water
culture plates with TSA
glass spreader ("hockey stick")
90% alcohol
Methods
Serial dilution.
Label water-filled culture tubes 1-6. Using aseptic technique, transfer 1.0 ml from culture to tube 1. Using a new pipette, transfer 1.0 ml from tube 1 to tube 2. Using the same pipette, transfer 0.1 ml to the agar surface of a sterile Petri dish. Using the glass "hockey stick", spread the liquid over the surface. Sterilize "hockey stick" using the method demonstrated by your instructor. Label the bottom of the dish 1:10. Using a new pipette, transfer 1.0 ml from tube 2 to tube 3. Using the same pipette, transfer 0.1 ml to the agar surface of another sterile Petri dish. Using the glass "hockey stick," spread the liquid over the surface. Sterilize "hockey stick" and label the bottom of the dish 1:100. Continue this procedure until tube 6 has received 1.0 ml from tube 5. Using a sterile pipette, transfer 0.1 ml from 6 to a sterile plate, and label the plate with the proper dilution.
Place the 6 TSA plates and the remaining stock broth culture in the incubator at 37oC. Note: Half the class will incubate their broth cultures at 25oC.
After the broth culture has been incubated for 4 hours, remove the culture from incubation and repeat the above serial dilution procedure. Be sure to label these TSA plates in a way that distinguishes them from the previous set.
Analysis
At the next lab period remove all TSA plates from the incubator. For each set of six, identify the plate with 30-300 colonies and count the colonies. Record your results in table 4.2 below.
Questions
1. Complete the following tables.
Table 4.1
Dilution # |
ratio of culture to water |
dilution expressed as a fraction |
dilution expressed in decimals |
dilution expressed in exponents |
1 |
1:10 (1ml added to 9 ml) |
1/10 |
0.1 |
10-1 |
2 |
||||
3 |
||||
4 |
||||
5 |
||||
6 |
Table 4.2
no. colonies |
volume |
cfu/0.1ml |
dilution factor |
Bacterial Density |
|
Initial |
|||||
Final |
2. Why is it important to use a new pipette for each dilution? _____________________
3. Using the equations
determine the generation time (G)
for your bacteria in minutes. _________________
4. How does your estimate of generation time compare to published values for E. coli?
5. What effect did incubation temperature have on generation time? ________________
6. For each dilution/plate count, compare the number of colonies on plate 6 with the number on the plate you counted. Based on your plate counts, what would you predict should be the number of colonies on plate 6?
7. Use the following equation to determine the percent error that might be associated with counting the plate with the lowest number of colonies.
8. Why are colonies counted on plates with 30 - 300 colonies? ___________________
| Objectives Lab 4 Word Problems Assesment Timeline Future Projects |