We have looked at so many papers and are still uncertain why we have a consistent problem where the OD's of our bacterial cultures which are completely inconsistent. We are trying to express recombinant protein using E.coli - mainly BL21 (PLysS). We have tried alternative cell lines too to rule that out. Below is what happened today, a typical example of what we face on a regular basis so I will put details below. Any insight would be very much appreciated. I have run a gel and will upload when I get a chance. **Phage has been ruled out as an issue**.
-Starter culture grown overnight in LB+antibiotics, used 10 flasks (exactly the same, baffled). Have also tried glass with cotton bungs.
-Inoculated 5x 1L flask of T-Broth and 5x 1L L-Broth (appropriate media volume and antibiotics)
-Cultures incubated at 37 degrees C, 200RPM. OD's checked regularly. Induced with IPTG between OD (600nm) 0.6-0.8. Temperature reduced to 18 degrees C and cultures left over night (~21 hours). ODs of all flasks were checked before harvesting.
L-Broth ODs (600nm)
0.849
2.219
1.151
2.603 (this sample was diluted as OD was really high, got a lovely band for this one on the gel)
0.312
T-Broth ODs (600nm)
2.367
0.859
2.616
1.152
1.457
Anything with an OD
So, you are pooling the starter cultures before inoculating the 1L cultures, right?
Can you please standardized your culture by using McFarland standard. Best wishes
Alejandro there is only one started culture that is used to inoculate the other flasks containing media.
Benjamin - the measurements were taken using a spectrophotometer, unfortunately we do not use McFarland standards. Is there a benefit to doing this at all?
We are trying to understand why the ODs are so variable between our cultures that have been grown in exactly the same conditions. We have been able to rule out contamination but again, we can revisit this.
Thank you!
Have you tried measuring the OD of the starter culture, and making sure that each sample that of your starter culture has an identical OD prior to inoculating the the 1 L flasks? You can also use MacFarland's standards, but those are oftentimes up to the eye of the beholder - meaning that one standard to you may look like the OD of your culture, but to another person, they may say it looks like a different standard.
So the OD of all of the cultures is that same at the point of induction? Are you preparing media in each flask separately, or making all of the media at once and aliquoting it into each flask? I'm getting at that the media might be slightly different between flasks causing different growth rates.
Also, I (and some colleagues) have seen cultures grow differently depending on where they are in the incubator. No explanation for that, however... Be sure that when you are measuring ODs that your culture is truly suspended. The cells can start to sediment and change the OD value depending on where you took your sample from in the culture. Good luck!
Morning all! Yes IPTG is optimised :)
We don't make the media ourselves, we have a department that do it for us. I will check the OD of the started cultures in flasks, thank you for that Angie! They media is the same through all of the flasks but there can be very obvious visual differences in colour between them. We have been working to try and solve this and one solution would be making it ourselves. OD of all cultures are the same (give or take but all 0.6**) at induction. Thank you Austin, I had wondered about this too - the incubators we have are quite dates and we have had a few issues with flasks falling out and smashing etc, (not great when the shaker keeps going to grind all the glass in!) but we have ordered new ones. Do you think there is a temperature or o2 variance?
Thank you all again!!
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