4.1.2: Serial Dilutions and Viable Plate Counts Activity

Activity Preparation

1. Melt the agar deep tubes by placing them in boiling water or by autoclaving. After they are melted place them in the hot water bath at 45C to prevent them from solidifying.
2. Label your sterile water blank tubes # 1 - 7.
3. Label your petri dishes as follows: 1A, 1B, 2A, 2B, 3A, 3B

The following diagram shows the steps you will carry out to complete the serial dilutions of your stock sample and the transfer of samples to petri dishes.

Serial Dilutions

1. Obtain the E. coli stock culture and flick the tube until the bacteria are evenly suspended throughout the media.
2. Use a sterile pipette and pipette pump to aseptically obtain 1 ml of the sample and transfer it to the sterile blank tube #1.
3. Mix tube #1 to evenly distribute the sample throughout the solution.
4. Aseptically obtain 1 ml of sample from tube #1 using a fresh sterile pipette and transfer it to sterile blank tube #2.
5. Mix tube #2 to evenly distribute the sample throughout the solution.
6. Aseptically obtain 1 ml of sample from tube #2 using a fresh sterile pipette and transfer it to sterile blank tube #3.
7. Mix tube #3 to evenly distribute the sample throughout the solution.
8. Aseptically obtain 1 ml of sample from tube #3 using a fresh sterile pipette and transfer it to sterile blank tube #4.
9. Mix tube #4 to evenly distribute the sample throughout the solution.
10. Aseptically obtain 1 ml of sample from tube #4 using a fresh sterile pipette and transfer it to sterile blank tube #5.
11. Mix tube #5 to evenly distribute the sample throughout the solution.
12. Aseptically obtain 1 ml of sample from tube #5 using a fresh sterile pipette and transfer it to sterile blank tube #6.
13. Mix tube #6 to evenly distribute the sample throughout the solution.
14. Aseptically obtain 1 ml of sample from tube #6 using a fresh sterile pipette and transfer it to sterile blank tube #7.
15. Mix tube #7 to evenly distribute the sample throughout the solution.

Inoculation of Petri Dishes

You will use the serial dilution tubes #4, #5, #6, and #7 to inoculate the plates that were labeled to begin this activity.

1. Mix tube #4 to ensure the bacteria are evenly distributed throughout the solution. Use a fresh pipette to aseptically transfer 0.1 ml of the sample to petri dish 1A.
2. Mix tube #5 to ensure the bacteria are evenly distributed throughout the sample. Then using a fresh pipette transfer 1 ml of sample to petri dish 1B.
3. Using the same pipette obtain 0.1 ml of sample from tube #5 and transfer to petri dish 2A.
4. Mix tube #6 to ensure the bacteria are evenly distributed throughout the sample. Then using a fresh pipette transfer 1 ml of sample to petri dish 2B.
5. Using the same pipette obtain 0.1 ml of sample from tube #6 and transfer to petri dish 3A.
6. Mix tube #7 to ensure the bacteria are evenly distributed throughout the solution. Use a fresh pipette to aseptically transfer 1 ml of the sample to petri dish 1A.
7. Obtain one 45°C molten agar tube, wipe moisture from the outside of the tube.
8. Using sterile technique pour the molten agar into petri dish 1A. Close the lid to the petri dish and gently rotate the plate to move the agar through the sample until it has been evenly distributed throughout the dish.
9. Repeat steps 7 & 8 for petri dishes 1B, 2A, 2B, 3A, and 3B and then allow the agar to solidify within the dishes.
10. After the agar has solidified, invert the petri dishes and place them in an incubator at 37°C for 24 hours.

Counting Colonies

After incubation obtain all of your petri plates Examine them and select those plates with 30 - 300 colonies for counting. If a plate has less than 30 colonies use a marker to write TFTC on the plate. TFTC stands for “too few to count.” If a plate has greater than 300 colonies use a marker to write TNTC on the plate. TNTC stands for “too numerous to count.”

If the plate has TNTC or TFTC you will also record that as the “# bacterial colonies in your results table.

1. Count the number of colonies on each of the plates yielding 30 - 300 colonies. If one is available you can use a Quebec colony counter (digital colony counter). Your instructor will show you how to utilize the device. If not, a handheld counter can be utilized.

2. Calculate the number of bacteria in the original sample. Do this by multiplying the #of colonies by the dilution factor. Record your data in the provided table.

Example: You count 190 colonies on a plate that was diluted 1:1,000,000 (10-7). The dilution factor is the reciprocal of the dilution. So, the dilution factor would be 107. Answers will be denoted using scientific notation

190 x 107 = 1.9 x 109

Results: