Use of optical density detection times to assess the effect of acetic acid on single-cell kinetics

The growth of Listeria innocua at different acetic acid concentrations (0 to 2,000 ppm) was monitored by optical density measurements in a Bioscreen (Labsystems, Vantaa, Finland). The generated populations came from low inocula that were obtained by serial dilution. A new method to estimate both the growth rate and the lag time of single cells from the detection times (time to reach an optical density of 0.11) was developed. It assumes that the single-cell lag times follow a gamma distribution and takes into account the randomness of the inoculation level. (The initial cell number per well was assumed to follow a Poisson distribution.) In this way, relatively small numbers of replicates are sufficient to obtain a robust estimation of the distribution of single-cell lag times. The results were validated with plate count experiments. It was found that logarithms of both the growth rates and of population lag times increased linearly with the acetic acid concentration. The logarithm of the scale parameter of the gamma distribution of the single-cell lag times also increased linearly with the acetic acid concentration irrespective of the phase of the inoculum.     

Use of Optical Density Detection Times To Assess the Effect of Acetic Acid on Single-Cell Kinetics

The growth of Listeria innocua at different acetic acid concentrations (0 to 2,000 ppm) was monitored by optical density measurements in a Bioscreen (Labsystems, Vantaa, Finland). The generated populations came from low inocula that were obtained by serial dilution. A new method to estimate both the growth rate and the lag time of single cells from the detection times (time to reach an optical density of 0.11) was developed. It assumes that the single-cell lag times follow a gamma distribution and takes into account the randomness of the inoculation level. (The initial cell number per well was assumed to follow a Poisson distribution.) In this way, relatively small numbers of replicates are sufficient to obtain a robust estimation of the distribution of single-cell lag times. The results were validated with plate count experiments. It was found that logarithms of both the growth rates and of population lag times increased linearly with the acetic acid concentration. The logarithm of the scale parameter of the gamma distribution of the single-cell lag times also increased linearly with the acetic acid concentration irrespective of the phase of the inoculum.     

Single-cell and population lag times as a function of cell age

After inoculation, the times to the first divisions are longer and more widely distributed for those Escherichia coli single cells that spent more time in the stationary phase prior to inoculation. The second generation times are still longer than the typical generation times in the exponential phase, and this extended the apparent lag time of the cell population. The greater the variability of the single-cell interdivision intervals, the shorter are both the lag time and the doubling time of the population.     

Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.     

Studies on plant tissue cultures I. Relationship between inocula sizes and growth of calluses in liquid culture

1. 1. It was observed that lag of growth was longer in small inoculathan in large inocula using tobacco callus in liquid culture.
2. 2. These different growth responses between small and largeinocula were dependent on the ratio of inoculum to culture medium.
3. 3. The same result was obtained in a strain of carrot rootcallus.But the growth lag was very short in the carrot callus,whichwas subcultured for the shortest period among the 4 strainsused, even in small inocula. On the other hand, both small andlarge inocula of the strain, which were subcultured for thelongest period among the 4 strains, did not grow at all duringthe culture period; the longer the period of subculturing, thelonger the lag of growth.
4. 4. The longer lag of small inoculain tobacco callus was recoveredby gibberellin A₃ in the presenceof the acidic fraction ofcarrot root extract or vitamins suchas pyridoxine and thiamine.

(Received December 11, 1967; )     

The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain

Shorter lag phases were obtained in cultivations of Bacillus licheniformis using early-compared to late-stationary growth phase inocula and using liquid versus solid propagation medium. Flow cytometry and fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), confirmed that liquid early-stationary growth phase inoculum had a higher vitality and was more homogeneous than solid late-stationary growth phase inoculum. DNA-microarray analyses indicated that liquid early-stationary growth phase inoculum was in a more active state in terms of cell multiplication whereas solid late-stationary growth phase inoculum was induced to some spore formation potentially causing delayed growth initiation.     

Aerobic and anaerobic degradation and mineralization of 14C-chitin by water column and sediment inocula of the York River estuary, Virginia.

Potential rates of chitin degradation (Cd) and mineralization (Cm) by estuarine water and sediment bacteria were measured as a function of inoculum source, temperature, and oxygen condition. In the water column inoculum, 88 to 93% of the particulate chitin was mineralized to CO2 with no apparent lag between degradation and mineralization. No measurable dissolved pool of radiolabel was found in the water column. For the sediment inocula, 70 to 90% of the chitin was degraded while only 55 to 65% was mineralized to CO2. 14C label recoveries in the dissolved pool were 19 to 21% for sand, 17 to 24% in aerobic mud, and 12 to 21% for the anaerobic mud. This uncoupling between degradation and mineralization occurred in all sediment inocula. More than 98% of the initial 14C-chitin was recovered in the three measured fractions. The highest Cd and Cm values, 30 and 27% day-1, occurred in the water column inoculum at 25 degrees C. The lowest Cd and Cm values were found in the aerobic and anaerobic mud inocula incubated at 15 degrees C. Significant differences in Cd and Cm values among water column and sediment inocula as well as between temperature treatments were evident. An increased incubation temperature resulted in shorter lag times before the onset of chitinoclastic bacterial growth, degradation, and mineralization and resulted in apparent Q10 values of 1.1 for water and 1.3 to 2.1 for sediment inocula. It is clear that chitin degradation and mineralization occur rapidly in the estuary and that water column bacteria may be more important in this process than previously acknowledged.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

Inhibition of Listeria monocytogenes growth by the lactoperoxidase-thiocyanate-H2O2 antimicrobial system.

The lactoperoxidase-thiocyanate-H2O2 system (LP system), consisting of lactoperoxidase (0.37 U/ml), KSCN (0.3 mM), and H2O2 (0.3 mM), delayed but did not prevent growth of L. monocytogenes Scott A at 5, 10, 20, and 30 degrees C in broth and at 20 degrees C in milk. The net lag periods determined spectrophotometrically varied inversely with temperature and were shorter at 5 and 10 degrees C for cultures from shaken versus from statically grown inocula. Lag periods for cultures from shaken and statically grown inocula, respectively, were 73 and 98 h at 5 degrees C, 22 and 32 h at 10 degrees C, both 8.9 h at 20 degrees C, and both 2.8 h at 30 degrees C. After the lag periods, the maximum specific growth rates were similar for each of the three treatments (complete LP system, H2O2 alone, or control broth) at 5, 10, and 20 degrees C and were 0.06 to 0.08, 0.09 to 0.1, and 0.32 to 0.36/h, respectively. At 20 degrees C in sterile reconstituted skim milk, the LP system restricted growth of Scott A, with log CFU counts per ml at 0, 36, and 68 h being 5.7, 6.4 and 7.9 (versus 5.7, 9.8, and 11.2 for controls). Possible explanations for the decreased lag times observed for cultures from aerobically grown inocula are discussed.     

Carbon Dioxide Effects on Glucose Catabolism by Mixed Microbial Cultures

Results have shown that, with mixed culture (sewage) inocula, the lag period in aerobic catabolism of glucose can be reduced by increased CO₂ tension. Conversely, removal of CO₂ from the air supply to the growth flasks and Warburg vessels may increase the lag period.     

Early bending kinetics in response to geostimulation or unilateral IAA application in Lolium nodal segments

Abstract In Lolium multiflorum nodal segments, bending responses both to geostimulation and unilateral indole-3-acetic acid (IAA) application exhibited much variability in their lag times and speeds of early bending. Despite this variability, mean response curves to gravity and auxin stimulus were markedly similar with each having a phase of immediate, negative bending followed by phases of slow, positive bending and eventually more rapid, positive bending within 40 min of initial treatment. Comparison of lag times for response to geostimulation and unilateral IAA application, whether derived from the mean of individual replicates, or from mean curve data, showed that at least 4 min is available in this geotropic system for establishment of asymmetric auxin levels that could lead to differential growth. The hypothesis that variability in georesponse in Lolium nodal segments is linked to variable sensitivity of geosensitive tissue to auxin was tested using matching longitudinally-halved nodal segments and evidence was obtained in support of the hypothesis from lag time but not from early bending speed data. The implications of the findings for an involvement of endogenous IAA in shoot geotropism together with the necessity to understand better the complex behaviour of bending response in individual replicates are discussed.     

Individual-based modelling of bacterial cultures to study the microscopic causes of the lag phase

The lag phase has been widely studied for years in an effort to contribute to the improvement of food safety. Many analytical models have been built and tested by several authors. The use of Individual-based Modelling (IbM) allows us to probe deeper into the behaviour of individual cells; it is a bridge between theories and experiments when needed. INDividual DIScrete SIMulation (INDISIM) has been developed and coded by our group as an IbM simulator and used to study bacterial growth, including the microscopic causes of the lag phase. First of all, the evolution of cellular masses, specifically the mean mass and biomass distribution, is shown to be a determining factor in the beginning of the exponential phase. Secondly, whenever there is a need for an enzyme synthesis, its rate has a direct effect on the lag duration. The variability of the lag phase with different factors is also studied. The known decrease of the lag phase with an increase in the temperature is also observed in the simulations. An initial study of the relationship between individual and collective lag phases is presented, as a complement to the studies already published. One important result is the variability of the individual lag times and generation times. It has also been found that the mean of the individual lags is greater than the population lag. This is the first in a series of studies of the lag phase that we are carrying out. Therefore, the present work addresses a generic system by making a simple set of assumptions.     

Biofilm development in laboratory methanogenic fluidized bed reactors

Biofilm development on sand with different heterogeneous inocula was studied in laboratory-scale methanogenic fluidized bed reactors. Both the course of biofilm formation during reactor start-up and the bacterial composition of newly developed biofilms at steady-state were found to be similar irrespective of the type of inoculum applied. Biofilm formation proceeded according to a fixed pattern that could be subdivided in three consecutive phases, designated as the lag phase, biofilm production phase, and steady-state phase. Methanogenic activity and biomass content of the fluidized bed granules were found to be accurate parameters of the course of biofilm formation. More indirect parameters monitored did not give unambiguous results in all instances. The composition of the newly developed biomass as assessed on the basis of potential methanogenic activities on different substrates and of the concentration of specific methanogenic cofactors was consistent with electron microscopic observations.     

Estimating Risk from Small Inocula by Using Population Growth Parameters

Risk from an uncertain small inoculum depends on variability of single-cell lag times. However, quantifying single-cell variability is technically challenging. It is possible to estimate this variability using population growth parameters. We demonstrate this possibility using data from literature and show a Bayesian scheme for performing this task.An inoculum size effect on a bacterial population lag phase has been demonstrated in many studies of bacterial growth (2, 7, 9). These authors showed that, with smaller inoculum levels, the uncertainty in the population lag parameter increases. Pin and Baranyi (8), using a computer simulation model, demonstrated that the inoculum effect on the population lag time was not evident when more that 40 cells were used to initiate growth in their system.A bacterial population at time t, grown from an inoculum consisting of n cells, can be represented by (1) where μ is the specific growth rate for cells (we assume this is constant within the cell inoculum). We assume that the lag times of individual cells in the inoculum, L_i, are identically and independently distributed random variables. Taking the natural logarithm of the cell population in equation 1, for sufficiently large time t, and comparing the result with a biphasic model for growth gives the population lag time, λ, arising from an initial inoculum of size n as (see, e.g., reference 5) (2) In this report we will show that this model is consistent with actual observations of growth from small inocula and therefore that it can be used, in combination with easily obtainable population parameters and Bayesian inference, to estimate details of single-cell variability. This approach is in contrast to using observations from experiments initiated with small inocula, such as a bioscreen, to infer single-cell variability parameters (see, e.g., reference 5) and hence is valuable when these measurements are unavailable.Francois et al. (4) measured a comprehensive set of individual cell lag times for Listeria monocytogenes under different environmental conditions and quantified the variability of these lag times using either a gamma or Weibull distribution. We have used these variability distributions of single-cell lag phase to derive a population lag, λ, and compared modeled growth data to experimental growth data from the ComBase database (www.combase.cc). The ComBase database currently contains about 9,000 L. monocytogenes data records. We selected 32 growth curves from ComBase which closely matched the environmental conditions used by Francois et al. (4). Table ​Table11 gives the database identification numbers of the L. monocytogenes growth data used in this study, the corresponding single-cell growth parameters of Francois et al. (4), and the derived population lag for n = 10³.

TABLE 1.

Listeria monocytogenes growth data from the ComBase database with equivalent single-cell growth parameters of Francois et al. (4) and the estimated population parameters λ^a

Spectophotometric method for preparing the inocula of dematiaceous fungi.

The aim of this study was to adapt a spectrophotometric method for preparing the inocula of dematiaceous fungi used for in vitro susceptibility tests. Fifty-two isolates of 17 different species of dematiaceous fungi were used for this purpose. Homogeneous suspensions of conidia and hyphae of these isolates were obtained and adjusted for reading at 530 and 550 nm at 40% and 50% of transmittance. The suspensions were standardised to 1-5 x 10 e6 CFU/ml. Quality controls of the inocula were done by quantitative cultures on agar-Sabouraud plates. The inocula obtained by spectrophotometry showed little variability within all the isolates. This method can be useful for in vitro antifungal evaluation of dematiaceous fungi.     

Effect of Environmental and Physiological Conditions on the Phase of Adjustment of Pseudomonas fragi

The duration of the phase of adjustment of Pseudomonas fragi was affected by the physiological age and growth temperature of the inoculum, as well as by the temperature at which the growth curve was determined. Cultures in the exponential phase of growth gave shorter lags than stationary-phase and resting-phase inocula. Inocula from the latter phase gave the longest lags. Inocula grown at the temperature at which the growth curve was determined usually gave the shortest lags: the greater the difference between the incubation temperature of the inoculum and the incubation temperature of the growth curve, the longer the lag. Inocula grown at temperatures below the incubation temperature of the culture tended to produce longer lags than inocula grown at temperatures above the incubation temperature. The combined effect of physiological age and incubation temperature of the inoculum was additive. The effect of the incubation temperature of the culture upon the duration of the lag depended upon the method used to determine this phase. Lags that were measured in physical time (i.e., Lockhart's lag) decreased as the incubation temperature of the culture was increased, within the temperatures used. But Monod's lag, which measures physiological time, did not decrease as the temperature of growth increased but rather appeared to vary around some constant value dependent upon the physiological condition of the culture.     

Scalable and flexible inference framework for stochastic dynamic single-cell models

Understanding the inherited nature of how biological processes dynamically change over time and exhibit intra- and inter-individual variability, due to the different responses to environmental stimuli and when interacting with other processes, has been a major focus of systems biology. The rise of single-cell fluorescent microscopy has enabled the study of those phenomena. The analysis of single-cell data with mechanistic models offers an invaluable tool to describe dynamic cellular processes and to rationalise cell-to-cell variability within the population. However, extracting mechanistic information from single-cell data has proven difficult. This requires statistical methods to infer unknown model parameters from dynamic, multi-individual data accounting for heterogeneity caused by both intrinsic (e.g. variations in chemical reactions) and extrinsic (e.g. variability in protein concentrations) noise. Although several inference methods exist, the availability of efficient, general and accessible methods that facilitate modelling of single-cell data, remains lacking. Here we present a scalable and flexible framework for Bayesian inference in state-space mixed-effects single-cell models with stochastic dynamic. Our approach infers model parameters when intrinsic noise is modelled by either exact or approximate stochastic simulators, and when extrinsic noise is modelled by either time-varying, or time-constant parameters that vary between cells. We demonstrate the relevance of our approach by studying how cell-to-cell variation in carbon source utilisation affects heterogeneity in the budding yeast Saccharomyces cerevisiae SNF1 nutrient sensing pathway. We identify hexokinase activity as a source of extrinsic noise and deduce that sugar availability dictates cell-to-cell variability.     

Lag‐phases in alien plant invasions: separating the facts from the artefacts

Temporal trends in biological invasions are often described by a lag‐phase of little or no increase in species occurrence followed by an increase‐phase in which species occurrence rises rapidly. While several biological and environmental mechanisms may underlie lag‐phases, they may also represent statistical artefacts or temporal changes in sampling effort. To date, distinguishing the facts from these artefacts has not been possible. Here we describe a method for estimating the lag‐phase in cumulative records of species occurrence, using a piecewise regression model that explicitly differentiates the lag and increase phases. We used the von Bertalanffy, logistic, linear and exponential functions to model the increase phase, and identified the best‐fitting function using model selection techniques. We confirmed the accuracy of our method using simulated data and then estimated the length of the lag‐phase (t_lag), the maximum collection rate (r) and the projected asymptotic number of records (K) using herbarium records for 105 weed species in New Zealand, while accounting for changes in sampling effort. Nearly all the New Zealand weed species had a lag‐phase, which averaged around 20–30 years, with 4% of species having a lag‐phase greater than 40 years. In more than two thirds of the cases, the accumulation of records was best modelled with the decelerating von Bertalanffy function, despite the tendency for temporal variation in sampling effort to force cumulative herbarium records to follow the sigmoidal shape of a logistic curve. A positive correlation between r and K is consistent with the assumption that the final distribution of an alien plant species reflects its rate of spread. Seemingly rare but fast‐spreading aliens may thus become tomorrow's noxious weeds. A positive correlation between inflection year and r warns that the weeds that have only begun to spread relatively recently may spread faster than previously known invaders.     

Effect of inhibition treatment, type of inocula, and incubation temperature on batch H2 production from organic solid waste

Two types of induction treatments (heat-shock pretreatment, HSP, and acetylene, Ac), inocula (meso and thermophilic) and incubation temperatures (37 and 55 degrees C) were tested according to a full factorial design 2(3) with the aim of assessing their effects on cumulative H(2) production (P(H), mmol H(2)/mini-reactor), initial H(2) production rate (R(i,H), micromol H(2)/(g VS(i) x h)), lag time (T(lag), h), and metabolites distribution when fermenting organic solid waste with an undefined anerobic consortia in batch mini-reactors. Type of inocula did not have a significant effect on P(H), T(lag), and R(i,H) except for organic acids production: mini-reactors seeded with thermophilic inocula had the highest organic acid production. Concerning the induction treatment, it was found that on the average Ac only affected in a positive way the P(H) and T(lag). Thus, P(H) in Ac-inhibited units (6.97) was 20% larger than those in HSP-inhibited units (5.77). Also, Ac favored a shorter T(lag) for P(H) in comparison with HSP (180 vs. 366). Additionally, a positive correlation was found between H(2) and organic acid production. In contrast, solvent concentration in heat-shocked mini-reactors were slightly higher than in reactors spiked with Ac. Regarding the incubation temperature, on the average mesophilic temperature affected in a positive and very significant way P(H) (10.07 vs. 2.67) and R(i,H) (2.43 vs. 0.76) with minimum T(lag) (87 vs. 459). The positive correlation between H(2) and organic acids production was found again. Yet, incubation temperature did not seem to affect solvent production. A strong interaction was observed between induction treatment and incubation temperature. Thus, Ac-inhibited units showed higher values of P(H) and R(i,H) than that HSP-inhibited units only under thermophilic incubation. Contrary to this, HSP-inhibited units showed the highest values of P(H) and R(i,H) only under mesophilic conditions. Therefore, the superiority of an induction treatment seems to strongly depend on the incubation temperature.