Innoculation concerned with the lag phase - (Apr/05/2011 )

normally you do a preculture, started from a single colony in ~10 mL and dilute that 1/500 up to 1/1000 fold.

Another reason that comes to my mind why one should not pour all of the 10 mL into the 50 mL flask is that by doing so you transfer all the byproducts (like acetate and other organic acids that do interefere with bacterial growth) into your "new" culture. Additionally, when you use e.g. Ampicillin for selection you transfer all the secreted ß-lactamase and this will lead to only a short periode of selection in your new culture ...and this is not really a great thing.

So, i personally think it is better to stick to the dilutions ...but as always it is a matter of taste

Regards,
p

pito on Sat Apr 9 11:19:06 2011 said:

pDNA on Sat Apr 9 00:17:41 2011 said:

it does not really matter to me either ...except you want to establish a reproducible procedure

Let's assume you want to do your "experiment" more than once ...and in a repdoducible manner ...than i would suggest to use defined values and volumes for inoculation. Then it's a great deal to know how long it will take you until you culture, inoculated with a defined starting material, take to reach a certain OD600 to e.g. induce.

Regards,
p

yeah, its the only acceptable explenation I have ever gotten about the "1ml" in stead of just using the entire tube.

Its more about "semantics" or "writing it nice" then logic.

its nicer to write down: "take 1ml of the 10ml you inoculated and put it in the 50ml" then writing "just put the entire tube (10ml) in the 50ml"

You can lose a bit of the 10ml you used to inoculate and 1ml is nicely defined quantity.

But for me: its not worth the fuss of using pipettips and all the extra work that comes with it.

The 10ml in 50ml is maybe a bad example because you indeed dont dilute a lot.

But about the by-products: they are still diluted enough + they are made anyway when you are culturing the bacteria... So I dont know if they would prevent growth a lot of have a big influence.
(+ its not that you grow your inoculum that long..)


About the Ampicillin part: yes and no.

I see your point however you work under the LAF and if even 1 (or more) not wanted colony gets in.. changes are very very small this colony will overgrow the rest .. + when you put in 10ml you allready have more of your wanted colony (so its easier for them to overgrow the other, not wanted ones).

I see your point and maybe my example of 10ml in 50ml was a bad one.. I should maybe have said 10ml in 100ml.


Anyway: I never used this "dilution" thingie where you need to take 1ml.. I just put in the entire tube.. and I never had bad results or really different ones compared to other...

@pito: what is LAF?

my brain is a bit slow today ...I've a terrible hay fever

Regards,
p

pito on Sat Apr 9 12:28:44 2011 said:

pDNA on Sat Apr 9 12:45:46 2011 said:

@pito: what is LAF?

my brain is a bit slow today ...I've a terrible hay fever

Regards,
p

You could start with as few as 1 viable cell or cfu. If you start with 10E%, it reaches max cell number sooner. If you're curous - run the experiment - spin or filter cells from medium at stationary and reinoculated a small number to determine if they'll grow in the used or spent medium..

pDNA on Sat Apr 9 12:08:44 2011 said:

normally you do a preculture, started from a single colony in ~10 mL and dilute that 1/500 up to 1/1000 fold.

Another reason that comes to my mind why one should not pour all of the 10 mL into the 50 mL flask is that by doing so you transfer all the byproducts (like acetate and other organic acids that do interefere with bacterial growth) into your "new" culture. Additionally, when you use e.g. Ampicillin for selection you transfer all the secreted ß-lactamase and this will lead to only a short periode of selection in your new culture ...and this is not really a great thing.

So, i personally think it is better to stick to the dilutions ...but as always it is a matter of taste

Regards,
p

Phil Geis on Sun Apr 10 10:59:42 2011 said:

Thank you all for participating in this topic. it were very interesting.

@ protolder .... is the 3 ud u meant ug (microgram) ? or what does ud mean ?

@pDNA ....... if the inoculum is diluted, it will/could produce purer strain. but it could slow down the cell growth rate and so, slower to get into the lag phase. does it ?

and i think the medium volume not play very important role in fast multiplying cells and entering to the lag phase. as long as the inoculum volume is increased, the growth will enter to the lag phase. eg. the inoculum ratio was 1:100 to medium volume and then, increased to 2:100 or 5:100.

and yes, culturing the cells of exponential growth will definitely help the stage moving into the lag phase. and would it be like .. they grow fast and end/die fast?

MicroGeek on Sun Apr 10 21:06:43 2011 said:

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