Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration

Abstract

Background: In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (tau) only up to a certain threshold cell density. Below this cell concentration (<or= 100 -1,000 CFU mL-1 ; <or= 27-270 CFU well-1) we observed an obvious increase in the tau scatter.

Results: Working with a food-borne E. coli isolate we found that tau values derived from two different microtiter platereader-based techniques (i.e., optical density with growth time {=OD[t]} fit to the sigmoidal Boltzmann equation or time to calculated 1/2-maximal OD {=tm} as a function of initial cell density {=tm[CI]}) were in excellent agreement with the same parameter acquired from total aerobic plate counting. Thus, using either Luria-Bertani (LB) or defined (MM) media at 37 degrees C, tau ranged between 17-18 (LB) or 51-54 (MM) min. Making use of such OD[t] data we collected many observations of tau as a function of manifold initial or starting cell concentrations (CI). We noticed that tau appeared to be distributed in two populations (bimodal) at low CI. When CI <or=100 CFU mL-1 (stationary phase cells in LB), we found that about 48% of the observed tau values were normally distributed around a mean (mutau1) of 18 +/- 0.68 min (+/- sigmatau1) and 52% with mutau2 = 20 +/- 2.5 min (n = 479). However, at higher starting cell densities (CI>100 CFU mL-1), the tau values were distributed unimodally (mutau = 18 +/- 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal tau distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL-1), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells.

Conclusion: These data argue that there is a dependence of growth rate on starting cell density.

Figure 1

Steady state O₂ ([O₂]: Fig 1A, open symbols), O₂ consumption rates (normalized to TAPC: Fig 1B) and E. coli cell growth (Fig 1A, closed symbols) as a function of growth time at 37°C in various media. Culture volume = 100 mL minimal defined medium (MM) or Luria-Bertani (LB) broth in a 250 mL normal or baffled Erlenmeyer flasks; 200 RPM agitation: squares = MM, normal flask; circles = LB, normal flask; triangles = LB, baffled flask; diamonds = LB, air bubbled in addition to shaking.

Figure 2

Plot of 653 observations of τ as a function of initial cell concentration (C_I; dilute stationary phase E. coli cells). Inset Figure: Frequency of occurrence of various values of τ (C_I < 100 CFU mL^-1) fit to Eq. 7.

Figure 3

Dependence of numerous t_m observations on initial bacterial cell concentration (C_I; Eq. 6) as a function of growth phase of the initial inoculum (log or stationary phase): circles = Log phase cells (τ = 16.8 ± 1.13 min); diamonds = stationary phase cells (τ = 16.8 ± 0.313 min).

Figure 4

Plot of 987 observations of τ as a function of initial cell concentration (C_I; diluted log phase E. coli cells). Inset Figure: Frequency of occurrence of various values of τ (C_I < 1000 CFU mL^-1) fit to Eq. 7.

Figure 5

Plot of 372 observations of τ as a function of initial cell concentration (C_I; LB with 75 mM EA-diluted log phase generic E. coli cells). Inset Figure: Frequency of occurrence of various values of τ (C_I = all CFU mL^-1) fit to Eq. 7.

Figure 6

Frequency of occurrence of various values of τ (all C_I; C_I > 100; C_I < 100 CFU mL^-1, from top to bottom). Left-hand side plots: stationary phase cells diluted with and grown in sterile-filtered 'conditioned' LB. Right-hand side plots: stationary phase cells diluted with and grown in LB.

Figure 7

A: Frequency of occurrence of various values of τ (all C_I; C_I > 100; C_I < 100 CFU mL^-1, from top to bottom). Left-hand side plots: mid-log phase cells diluted with and grown in LB with ~2×10⁵ CFU mL^-1 of disrupted cells LB. Right-hand side plots: mid-Log phase cells diluted with and grown in LB. B:Plot of 572 observations of τ as a function of initial cell concentration (C_I; diluted with and grown in LB with ~ 2×10⁵ CFU mL^-1 of disrupted E. coli cells LB).

Figure 8

Plot of optical density at 590 nm (open circles) and associated first derivative (ΔOD/Δt, closed circles) data associated with E. coli growth (C_I ~ 4,000 CFU mL^-1) at 37°C in Luria-Bertani broth. Inset Figure: OD and first derivative data associated with growth (C_I ~ 7,000 CFU mL^-1) at 37°C in a defined minimal medium (MM). The growth parameter, t_m, calculated using Eq. 1, is shown as at the center of symmetry about the maximum in ΔOD/Δt.

Figure 9

Typical t_m results showing its relationship (Eq. 5) with solution dilution factors (Φ) on both linear and semi-log scales. The |slope| of the line shown in the inset figure is equal to Φ (= 0.286 hrs or 17.2 min). The parameter t_m was calculated by fitting OD[t] data to Eq. 1.