"Experiment 2" - Growing and Diluting

Goal: Attempt to get reasonable trends in bacterial growth by plating at different OD600 and at different concentrations.

Method:

1. Grow cells until they reach an OD600 of 0.10, 0.15, 0.20, 0.25, 0.30
2. Take 1 mL of solution at each OD and dilute 6, 7, 8 and 9 times (each dilution corresponds to a 10-fold decrease in concentration)
3. Plate bacteria, count colonies the next day

Results:

 

Figure 1: Shows that generally the higher the OD, the higher the colony count. As the dilutions get stronger, the trend becomes less obvious

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Figure 2: Shows that generally the stronger the dilution, the lower the colony count. This graph also shows that the effect is stronger for higher OD's

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I personally take these results very lightly. The overall trend is what should be expected for the most part. Whether or not the details (exact slope, colony count, etc.) match what should be expected is a different story. In this batch, I got a large amount of contamination (which wasn't obvious until 2 days after) which decreases the accuracy and value of the results.

What I learned not to do next time:

• don't start dumping flasks until AFTER you take a sample of the cells in it (hence the missing OD 0.3 data)
• set up dilutions beforehand to avoid manic pipetting
• start the spectrophotometer early in the day because it takes a long time
• either don't take OD readings as often or add sufficient amount of liquid to avoid consuming it all before reaching your desired level
• Most importantly... do not add too much liquid to the plate when plating bacteria! I realized after a few plates that adding 0.5 mL of solution to the agar plates was far too much (which I believe is responsible for the contamination. I decided to continue using 0.5 mL for the rest of the plates to keep the data consistent, but never again!