Innoculation concerned with the lag phase - (Apr/05/2011 )
Hi .
how to get the cells of E coli or yeasts entered faster to the lag phase growth ? if the innoculum percentage is increased altogether with the medium volume, would it be helpful to enter to the lag phase ? is the cultured medium volume important in this role or only the innoculum percentage ?
Thanks
MicroGeek on Tue Apr 5 22:00:16 2011 said:
Hi .
how to get the cells of E coli or yeasts entered faster to the lag phase growth ? if the innoculum percentage is increased altogether with the medium volume, would it be helpful to enter to the lag phase ? is the cultured medium volume important in this role or only the innoculum percentage ?
Thanks
Hola, both assumptioms are the same , the inoculum percentage is referred to the medium volume. And yes increasing inoculum means faster reach lag phase, Think in extremes. Suposse that any bacteria (E.coli) reachs lag phase at 3ud OD600nm in LB. If you seed 1l of LB with 2ooml of 3ud culture, this culture starts growing at 0.6 ud OD and needs 3-4 generatios (1h) to get newly lag phase. If the seed was only 20ml , the culture starts growing at 0.06 and needs about 6 generatios (2h ) to reach lagh phase. I hope will clear it to you. Buena suerte
Maintain the same medium of inoculum origin and growth. For some microbes, spent medium can provide growth factors such as siderophores.
Phil Geis on Wed Apr 6 10:49:13 2011 said:
Maintain the same medium of inoculum origin and growth. For some microbes, spent medium can provide growth factors such as siderophores.
Hallo,
could you explain what you mean?
Do you mean that you need to use the same medium for inoculation as for growth? By this I mean: that you dont put the inoculum in another bigger flask.
(so in stead of inoculating 10ml with a bacterium, and then transfering it after an hour to 0,5liter , you just inoculate 0,5liter at once?) Or what do you mean?
Or do you mean that when you inoculated that 10ml you simply add the 10ml in total to a new flask with 0,5liter?
protolder on Wed Apr 6 05:39:50 2011 said:
MicroGeek on Tue Apr 5 22:00:16 2011 said:
Hi .
how to get the cells of E coli or yeasts entered faster to the lag phase growth ? if the innoculum percentage is increased altogether with the medium volume, would it be helpful to enter to the lag phase ? is the cultured medium volume important in this role or only the innoculum percentage ?
Thanks
Hola, both assumptioms are the same , the inoculum percentage is referred to the medium volume. And yes increasing inoculum means faster reach lag phase, Think in extremes. Suposse that any bacteria (E.coli) reachs lag phase at 3ud OD600nm in LB. If you seed 1l of LB with 2ooml of 3ud culture, this culture starts growing at 0.6 ud OD and needs 3-4 generatios (1h) to get newly lag phase. If the seed was only 20ml , the culture starts growing at 0.06 and needs about 6 generatios (2h ) to reach lagh phase. I hope will clear it to you. Buena suerte
Hallo,
what does 3ud mean?
(ud=?)
Meant the same medium - inoculate TSB with an inoculum grown in TSB. Certainly not the only factor - but lag has been shown for some bugs to be partially associated with the inoculum modifying its environment to facilitate rapid reproduction. For example, iron is needed for cytochrome and DNA synthesis. Iron acquisition is siderophore mediated and the new medium will have none - so the inoculum has to synthesize siderophores to get iron to speed growth. If siderophores were already present (some of the spent medium) - it would shorten lag at least for that limitation.
The other comment is not really that relevant. Of course the time ultimately to reach 10E6 cells/ml is less if one starts with 10E5/ml than 100 cfu/ml. There is still a lag phase and the time for that doesn't necessary change.
Phil Geis on Thu Apr 7 11:52:35 2011 said:
Meant the same medium - inoculate TSB with an inoculum grown in TSB. Certainly not the only factor - but lag has been shown for some bugs to be partially associated with the inoculum modifying its environment to facilitate rapid reproduction. For example, iron is needed for cytochrome and DNA synthesis. Iron acquisition is siderophore mediated and the new medium will have none - so the inoculum has to synthesize siderophores to get iron to speed growth. If siderophores were already present (some of the spent medium) - it would shorten lag at least for that limitation.
The other comment is not really that relevant. Of course the time ultimately to reach 10E6 cells/ml is less if one starts with 10E5/ml than 100 cfu/ml. There is still a lag phase and the time for that doesn't necessary change.
I understand what you mean and agree, but whats your opinion on the majority of protocols that state that (I take a random example) you need to inoculate 10ml (test tube for example with some media) with a bacterium (single colony) , let it grow till it reach OD 0,6 and then take 1ml of this and put it in a larger bottle with for example 200ml to get your cells at OD 0,6 to use for transformation.
Why the 1ml, why not just pour the entire test tube in the 200ml bottle?
I never really got this and never got a straight , direct , correct answer why I should not just pour the 10ml in the bigger bottle..
(thus I often do it and there is nothing wrong with my cells...)
Me too.
Phil Geis on Thu Apr 7 11:52:35 2011 said:
Meant the same medium - inoculate TSB with an inoculum grown in TSB. Certainly not the only factor - but lag has been shown for some bugs to be partially associated with the inoculum modifying its environment to facilitate rapid reproduction. For example, iron is needed for cytochrome and DNA synthesis. Iron acquisition is siderophore mediated and the new medium will have none - so the inoculum has to synthesize siderophores to get iron to speed growth. If siderophores were already present (some of the spent medium) - it would shorten lag at least for that limitation.
The other comment is not really that relevant. Of course the time ultimately to reach 10E6 cells/ml is less if one starts with 10E5/ml than 100 cfu/ml. There is still a lag phase and the time for that doesn't necessary change.
Ok I see, but would it matter to start with more cells then? Like 10E5/ml or 100 cfu/ml? Its like pito or phage434 asked: does it matter if I simply add all most medium with cells or do I just add 1 ml ?
PS. what does ud means? Concering that OD? And how do they know that at an OD of 0,6 you are in the exponential growth phase?
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
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.