Don't stick to much to the term "overnight". Bacteria do not recognize day and night (to the best of my knowledge and at least those which are medically relevant ...).

Over night incubation is most convenient for the typical workflow in a lab which operates from 7 a.m. to 7 p.m. In the clinical lab, you incubate for some 18 hrs, before you pick colonies for doing susceptibility testing. At that time, we can assume the bacteria in that colony are somewhere in the log phase but not in the stationary phase. In the 7 a.m./7 p.m. lab, 18 hrs incubation of bacteria consequently covers "overnight".

In a 24/7 lab, 18 hrs can be at any time (e.g. a sample is inoculated at 6 p.m. and AST is therefore prepared in the afternoon, another sample is inoculated at 4 a.m. and is therefore 10 p.m.). Does it help?

Dear Silvia,

This depends on what MIC actually means.

The MIC is the lowest concentration of an antimicrobial drug that PREVENTS visible growth. This you can check after overnight incubation of your mcirobe on a plate or in liquid culture. The lowest concentration with absent grwoth is MIC.

It differs from LD50 value, a dose, which causes 50% death (or 50% inhibition of grwoth rate in log phase).

Best,

Christoph

Dear Christoph ,

thank oyou very much for your answer.  it is still not completely clear to me why you can't check it during the exponential phase..Can you explain it to me a little more?

Thanks

Sorry Christoph. I did not specify it well. What I don't understand is why you need overnight cultures when you prepare your inoculum \(when  you set your MIC up essentially,).can't I use cells in log phase to prepare the inoculum to incubate for the set-up of the MIC? and if not, why?

Silvia,

Of course you can and maybe you should check inhibition of your microorganism upon compound addition to the microorganism in the mid-log phase, but when you check depends on how your specific microbe grows etc. You can then monitor growth as a function of time. The response will depend on your microorganism, the compound etc and you are the best judge of how this compound will be working and how you can define MIC (which still should mean the lowest concentration of compound that prevents growth, but the time depends on how the organism grows without the compound and what the compound does to it).

Hi Oleg,

thanks. What I really wanted to know is why I can't use cells in log phase to prepare the inoculum to incubate for the set-up of the MIC. Why for the set-up of the MIC do I need to use overnight cultures and not cells in log phase? I hope to have been clear. Thanks

Don't stick to much to the term "overnight". Bacteria do not recognize day and night (to the best of my knowledge and at least those which are medically relevant ...).

Over night incubation is most convenient for the typical workflow in a lab which operates from 7 a.m. to 7 p.m. In the clinical lab, you incubate for some 18 hrs, before you pick colonies for doing susceptibility testing. At that time, we can assume the bacteria in that colony are somewhere in the log phase but not in the stationary phase. In the 7 a.m./7 p.m. lab, 18 hrs incubation of bacteria consequently covers "overnight".

In a 24/7 lab, 18 hrs can be at any time (e.g. a sample is inoculated at 6 p.m. and AST is therefore prepared in the afternoon, another sample is inoculated at 4 a.m. and is therefore 10 p.m.). Does it help?

Dear Caprari,

I think Oliver Nolte is correct while displaying the term "overnight" in case of microbial growth. Yes, obviously, the microorganisms have no power cells in them to recognise the day and night conditions. Off course, for our better convenience, we select the night period for their growth as we have the lab open for the entire day for some other experiments.

The MIC setup is incubated for overnight in order to maximize the chance of inhibition potential of the test substance against the test organism at various stages of growth. Perhaps, the growth of bacteria is more pronounce at exponential stage, but the bacteria should be allowed to pass all the stages of growth before the activity of the test substance become apparent, since some organisms are spore-formers, as such they cannot be captured by the effect of the test substance if the readings of MIC is determined at logarithmic phase. But, by completion of the growth cycle, all the test organisms that can withstand harsh environmental conditions and survive to the decline phase can be determined. Thanks.

I think the "overnight" period is for standardization. As other techniques like MBC uses same culturing period. 

Dear Silvia,

The overnight term is usually set as a standard procedure as most mesophiles tend to arrive to the log phase in around 12 hours (depending on the initial inoculum of course). But it will depend on the type of bacteria you are using for your study as some extremophiles may take over 24 hours to get to the log phase. 

So, you are right, you are really looking to have a log phase in your culture to do your MIC test. 

You could probably get a growth curve of your bacteria to stablish at what time it gets to log phase and if it is suitable to leave it as long as "overnight" or if it is preferable to use a shorter period of incubation.

I  hope it helps.

Good luck!

Ok,

so let's say that it is probable that the cultures after overnight incubation are still in log phase, did I understand correctly?

Yes, that´s right. They should be in log phase. But once again, it will depend on the type of bacteria you are using. 

Cultivate >>> evaluate >>> cultivate >>> evalutate . . . find out the optimal cultivation time.

Yes Sharifah, the incubation time depends on your growth curve. First find out when your bacteria gets to log phase and then you will be able to define the incubation time to do te following tests on a log phase culture. 

Yes Sharifah,

For exemple, incubation of slow growing mycobacteria is done for 7 days, if the contrôle growth is not sufficient we reincubate for additional 7 days. (MIC reading for a total of 14 days).

Why we prefer mid log phase? the answer is simple: to reach the log phase when the bacteria is in active division.

If you choose the log phase as starting material, the culture will reach rapidly the stationary phase, Cells in stationary phase produce many different types of secondary metabolites including heterocyclic compounds  also the metabolic activity is reduced and some cells can diplays other characteristics. Not the ideal to measure the effect of the drugs.