Lag phase (WIP)

This article dives into what happens during the lag phase of a bacterial growth curve, and investigates what influences the lag phase duration.

What is the lag phase?

The lag phase is the initial period of a bacterial growth curve that occurs immediately after a population of cells is introduced into fresh growth medium. During this stage, the number of viable cells does not increase noticeably, but the bacteria are metabolically active. Cells adapt to their new environment by repairing damage, synthesizing proteins, and activating metabolic pathways that will allow them to take advantage of the available nutrients. Although growth in terms of cell division is not yet detectable, the lag phase is essential because it represents the preparatory stage for exponential growth.

Different phases of bacterial growth. Michal Komorniczak/Wikimedia Commons/CC BY-SA 3.0

Apparent lag and true lag

The apparent lag is the period until clear exponential growth can be observed in the population as a whole. In other words, it is the time when measurements show that the entire culture has transitioned into exponential growth.

The true lag, by contrast, is the point when individual cells actually resume division after inoculation. This can occur earlier than the apparent lag, because only a fraction of the cells may begin dividing at first. For example, if a culture is seeded from a cryostock where only 10% of the cells are viable, the true lag would be when those viable cells first start dividing, whereas the apparent lag would not be visible until the whole population exhibits exponential growth.

Because the true lag is extremely difficult to measure in practice, most reports of “lag phase” in microbiology, including ours, are in fact referring to the apparent lag.

Deriving the lag phase

The duration of the lag phase can be determined in different ways, most commonly by one of the two following approaches:

1. Monitoring growth directly: By following the increase in biomass or cell numbers from the moment of inoculation. However, this is often impractical because the lag phase occurs when bacterial concentrations are still very low and difficult to detect rapidly with conventional methods.

2. Tangent method: A more practical approach involves plotting a growth curve and then applying the tangent method. In this method, a straight line (tangent) is drawn along the steepest part of the exponential phase. This line is then extrapolated back to the initial concentration (N₀) , and the intercept with the initial concentration is taken as the end of the lag phase. This allows researchers to estimate the duration of the lag phase even if cell numbers are too low to measure during its course. To reliably apply the tangent method, it is essential to know the precise seeding concentration and to use an enumeration technique that is not biased by changes in cell morphology over the course of the growth curve.

The duration of the lag phase (λ) can be estimated with the tangent method by drawing a tangent to the exponential phase and identifying its intersection with the seeding concentration (N₀).

Lag phase is independent of seeding concentration

It is a common misconception in industry that the lag phase depends on the concentration of cells used to inoculate a culture. It is a misconception that stems from noise in optical density measurements, which require very high time resolution for accuracy, and are easily influenced by changes in morphology.

In reality, when seeding from a stationary-phase E. coli culture at concentrations of 10³, 10⁵, and 10⁷ cells per mL, the lag phase remains identical as shown in Figure 1. The cells in each case require the same period of adjustment to the new medium, regardless of how many were introduced at the start. Thus, the lag phase duration is not a function of seeding density.

Figure 1: A stationary-phase E. coli starter culture was quantified with BactoBox and diluted to initial concentrations of 10³, 10⁵, and 10⁷ cells/mL in three shake flasks. Growth was monitored with BactoBox. Lag time for each curve was estimated by the tangent method from the log-linear region. Estimated lag durations agreed across seeding concentrations within experimental variation, demonstrating that lag is independent of inoculum size under these conditions.

Lag phase depends on seed culture health

While seeding concentration does not matter, the physiological state of the seed culture has a large influence on lag phase length. In one experiment, thirteen shake flasks were seeded from starter cultures taken at different time points during their progression. It was observed that the lag phase lengthened as the starter culture aged. A likely explanation is that as the starter culture progresses through its growth, the cells deplete and adapt to different nutrients in the medium. When transferred to fresh LB medium, they must readjust their metabolism, resulting in a longer lag phase. This shows that culture health and history directly shape the length of the lag period.

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Cells increase in size during lag phase

Although the cell count does not rise significantly during the lag phase, bacterial cells do increase in size. This is part of the adaptation process, as the cells synthesize enzymes, ribosomes, and metabolites necessary for active division. By the time the culture transitions into exponential growth, the cells are physiologically primed and ready to divide at their maximum rate.

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