Dear to whom it may concern,
I have recently isolated several bacterial strains from soil samples. To obtain these strains, I treated the soil samples in a specific bacterial culture broth to encourage the growth of selective bacteria capable of surviving in that environment. Afterward, I streaked these liquid cultures onto agar plates to isolate pure cultures (single colonies). These single colonies were then incubated in LB broth, and bacterial stock suspensions were prepared using 20% glycerol.
Once I obtained the bacterial strains, I examined their growth curves by measuring the optical densities of each strain at 600 nm over time. To ensure that the data was repeatable and reliable, I prepared five replicate samples of each bacterial strain in a single experiment, which I repeated three times. However, I noticed that the growth curves of each bacterial strain displayed different behaviors upon repeating the experiment, despite using the same conditions (medium and temperature).
In my own opinion, I doubt that the single colonies from the agar plates that I collected were not pure enough. However, I am not sure of what I am thinking, would you mind suggesting some potential reasons and practical solutions to overcome this problem to me?
Thank you so much,
Best regards.
It's possible to have a "mixed colony", even after streaking out to single colonies. Re-streak out to single colonies a few more times for each colony of interest.
Another possibility is that you need to work on your bench techniques. Technical replicates of the same biological sample should have minimal variation. See if an experienced person in the lab can observe/train you since they will have good practical advice.
You'll get this figured out!
Quynh Khoa Pham If you describe the main difference between bacterial growth curves (such as time for lag-, log, stationary phases), it would be easier to suggest the possible reasons. So far, mixed colonies or plasmid loss are the most common reason for such difference.
Obviously, the first possibility is that it is not the same microorganism. The first thing to do is to make sure that you are working with pure cultures, and confirm the identifications with a reliable method (16S RNA, mass spectrometry...). Another issue that could have an influence is whether you have worked from frozen stocks, as the fact that they have been preserved in 20% glycerol seems to indicate. When working from frozen stocks, it is advisable to make at least an initial pass to plate, and work from the growth obtained, since working directly from the frozen stock sometimes leads to erratic results.
Good question.
Yes one colony may not be pure. Otherwise perhaps some cells show phenotypical adaptations while others not
If it is the same bacteria, the same culture medium, and the same incubation conditions, the reason is due to the size of the inoculum
Have you tried a control experiment where you prepare single starter culture and then split it into 3 or 4 growth cultures for growth curves? They should be identical and that will be a good control for some methodological issue (or instrument issue).
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