Technical question: Why use a bacterial starter culture? - (Dec/01/2011 )

Ok, I have a question for someone who really understands the details of bacterial growth.

When growing hundred-milliliter-quantities of bacteria in amp culture (i.e. for a maxiprep) I'm wondering if someone can tell me why it's a good idea to use a starter culture rather than inoculating directly from a plate.

I have a vague understanding that we want the bacteria to "hit the ground running" in the larger culture volume - i.e. we want them to be in log phase as soon as they're added to the larger culture. I understand that when bacteria are transferred from a colony on a plate to a liquid culture, they go through several hours of "lag phase" where they're not dividing. We let them make that transition in the starter culture, and then they continue their logarithmic growth in the larger culture volume.

The thing that never makes sense to me about this is: Why not just let them make the lag->log transition in the larger volume? On the surface at least, it seems that we're actually making the whole process take longer by using a two step culture. If I start my colony in 5mL, and then after 8 hours I use 500uL of that culture to inoculate 500mL of new LB, I've just reduced my total bacteria by a factor of 10. Assuming a doubling time of 45 min during the log phase, that's just added two hours to the process!

I can think of a couple of reasons why we might want to use a two-step culture:

1. It's a race between cell growth and ampicillin inactivation. If cells are are secreting beta-lactamase during the lag period, but they're not growing, they may inactivate all the ampicillin early in the culture process, giving un-transformed bacteria a chance to compete for the LB. I've even seen some people say you should spin down your bacteria before you transfer them to get rid of B-lactamase in the medium.

2. Using a starter culture might give more consistent results, because when we physically transfer a colony from a plate (I use a clean 10uL pipette tip) we might sometimes get 500,000 bacteria, and sometimes 500. Maybe that could lead to large differences in the amount of time it takes to populate a large culture volume. By growing up the starter culture first, we're starting the larger culture with way more bacteria (I'm not going to try to calculate how much more), so maybe we get a more consistent time-to-stationary-period in the larger culture.

Number 1 seems more likely than number 2, and maybe there's other reasons I can't think of. I'd love to get a handle on this.

Two related bonus questions, as long as you've read this far:

1. I sometimes autoclave LB and then keep it in the fridge without amp for a couple weeks. I add the amp just before I prep my starter culture, and then I keep the larger volume of LB+amp for the overnight culture in the fridge during the day until I'm ready to inoculate it. Am I screwing myself by not pre-warming the larger culture to 37C before I put it on the shaker? I.e., will transferring the starter colony into the cold LB put it back into lag phase?

2. I usually grow my 5mL starter culture in a 15mL flask, and my ~200mL of overnight culture in a 1L flask. I keep the caps tight in both cases, but I'm wondering if that might be inhibiting growth. Would it be better to use tin foil so there's a chance for some air exchange?

Finally, I want to say I'm one of two people doing any kind of mol-bio in my lab, and I've learned an incredible amount from all your posts on this site. Thanks for being so generous with your knowledge and experience!

jakester

Dear jakester,

both answers seem to apply to your question.
I do pre-cultures because i believe it helps to make the procedure of plasmid preps more reproducible and additionally by doing so you prevent overgrowth of your culture (because as you already suggested ...incubating too long leads to inactivation of the ampicillin and gives rise to plasmid free cells that do compromise your results). If you inoculate 500 mL with a single colony picked from a plate you have no clue how long it will take to reach a certain OD ...a lot of people do it like this ...but those are the people who complain about inconsistent yields after midi or maxi preps

Concerning your bonus questions:
1) First of all, i would not keep pre-opend LB without antibiotics in the fridge ...this can cause troubles because you do not see contaminations. I always keep plain LB that has been used already on room temperature. I would pre-warm the medium to RT before use since the cold medium won't harm the bacteria but prolongs the lag phase.

2) What do you mean by keeping the caps tight? Don't use airtight vessels for the growth of E. coli! ...they can grow anaerobically ...but failure to provide enough oxygen changes the metabolism dramatically and severly affects plasmid and protein yield!

Hope this helps a bit!
Good luck!
Best regards,
p

pdna's answer is reasonably complete but i'll add something to consider.

a starter culture will give you more comparable cultures if you need to inoculate more than one of the same.

and, you can save any remaining to inoculate from the same colony.

Thanks p & m! That's very helpful.

I used to just grab a colony with a pipette tip and throw it in the overnight culture and things worked pretty much ok (although I sometimes had inconsistent yields ). A few months ago I started having poor yields from my maxipreps that I think were due to a leaky vacuum manifold and some other issues, and I started doing starter cultures then. I've fixed the other problems I was having, but I wonder if the way I'm growing my starter cultures may actually be contributing to current problems with maxi yields. I've been growing 5mL cultures in sealed 15mL conical-bottom tubes, and then inoculating my overnight cultures from there. I wonder if the initial growth under somewhat anaerobic conditions could be affecting my yields?

if sealed means airtight ...then you are seriously running into problems ...aeration is definitly one of the most important factors.
I've done lab-scale fermentations to produce pDNA ...and anytime we had problems with the pO2 electrod and the O2 saturation was falling below 30% we recognized it in the final yields ...therefore keep your cultures properly aerated. Zymo has a nice diagramm in their manual on page 5 ...watch it here!

Best regards,
p

again, to add to pdna's answer,

unless you sealed the tube under vacuum or in a nitrogen or argon (or some other inert gas) atmosphere, you are still growing the bacteria under aerobic conditions (with limited oxygen).

however, you must ensure that you have proper agitation of the culture.