Dr. Barry Hall
Many microbiology methods begin with an overnight culture, but few discuss how to prepare a reliable overnight culture.
Typically, an overnight culture is started by inoculating a single colony into rich broth medium and shaking the culture overnight. The condition of the resulting culture can be quite variable depending on the stage the culture has reached. The culture may be in stationary phase or it may have progressed through late stationary phase and into log death phase.
In stationary phase the cells undergo numerous physiological changes including changes in cell shape, thickening and cross-linking of peptidoglycan in the cell wall, activation of the stringent response, and nucleoid condensation. Alternative transcription sigma factors are activated. In late stationary phase cells can remain metabolically active but fail to form colonies when plated https://www.frontiersin.org/articles/10.3389/fmicb.2017.02000/full.
Stationary phase represents an adaptation both to exhaustion of essential nutrients and to accumulation of waste products. Accordingly, when sub-cultured into fresh medium the lag time before assumption of exponential growth depends on both the time to reverse those physiological changes and the fraction of cells that remain able to divide. Thus, the state of stationary phase cultures is highly variable, reducing the reproducibility of experiments that are initiated from overnight cultures.
In late stationary phase cells begin to die, so colony counts will decrease although OD tends to remain the same. In log death phase cells die rapidly and OD may decrease as cells begin to lyse.
Preparing the Cultures
There are two easy ways to prepare overnight cultures that are highly reproducible and contain almost exclusively viable cells. Both methods involve limiting cell growth before stationary phase is entered.
Method 1: using rich broth media
 | Inoculate a colony into 10 ml of your favorite broth medium in a 15 ml plug-seal centrifuge tube, then tighten down the plug-seal cap and allow the culture to stand overnight at the preferred growth temperature without shaking or other method of aeration. The details are important. 10 ml of medium in a 15 ml tube allows only 5 ml of oxygen-containing head space. The plug-seal cap ensures that no additional oxygen is available to the growing culture. |
As a result, the culture becomes oxygen limited before entering stationary phase, cell viability persists for many hours, and the cells do not undergo the physiological changes associated with stationary phase. When sub-cultured or diluted into fresh medium the culture enters exponential growth within a few minutes.
The population density of such “standing overnight” culture is quite reproducible for a given medium and bacterial species.
To determine that population density:
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Method 2: using mineral salts media
Experiments that involve growth in defined mineral salts media such as M9 medium are best initiated from overnight cultures in mineral salts medium to avoid adaptation resulting from shifting from rich too minimal medium. In this case instead of limiting overnight cultures by oxygen availability, cultures are limited to early exponential phase by carbon source availability.
- Ensure that the medium contains 0.01% (w/v) glucose or other sugar, or 0.01% (v/v) glycerol and no other carbon and energy source.
- Inoculate a suitable volume of carbon-limited medium with a single colony and shake well overnight at the optimum growth temperature.
The experiment can be initiated the following morning by adding excess carbon source, 0.2% or higher carbon source. Cells will enter exponential growth within a few minutes.
For E. coli, a carbon-limited culture will be at about 1E8 cells per ml, but you should plate dilutions of an overnight culture to determine the titer for your organism and medium.
Why does this work?
The Kt for sugar transport (equivalent of Km for enzyme reactions) is in the micromolar range. As a result, by the time the cell can even notice that the sugar concentration has dropped below “excess” the sugar has been exhausted and there isn’t any carbon or energy available to undergo stationary phase adaptations. In effect, the population is in suspended animation until an energy source is restored. Cells retain full viability for many hours under those conditions.
This approach to overnight cultures is particularly important for measuring microbial growth rates. The program GrowthRates for analyzing microbial growth is available from www.bellinghamresearch.com.
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