Influence of stress on individual lag time distributions of Listeria monocytogenes

The effects of nine common food industry stresses on the times to the turbidity (T(d)) distribution of Listeria monocytogenes were determined. It was established that the main source of the variability of T(d) for stressed cells was the variability of individual lag times. The distributions of T(d) revealed that there was a noticeable difference in response to the stresses encountered by the L. monocytogenes cells. The applied stresses led to significant changes of the shape, the mean, and the variance of the distributions. The variance of T(d) of wells inoculated with single cells issued from a culture in the exponential growth phase was multiplied by at least 6 and up to 355 for wells inoculated with stressed cells. These results suggest stress-induced variability may be important in determining the reliability of predictive microbiological models.     

Variability in the performance of the spectrogram correlation detector for North-east Pacific blue whale calls

Spectrogram correlation has been used successfully for automatic detection of baleen whale calls. However, applying this method consistently to long time series can be challenging. To illustrate the potential challenges of the automatic detection process, recordings collected in the Southern California Bight between 2007 and 2012 were used for detection of North-east Pacific blue whale (Balaenoptera musculus) B calls. The effects of the following factors were investigated: blue whale B call frequency shift and appropriate kernel modification, seasonal variability in call abundance, analyst variability and noise. Due to intra- and inter-annual changes in the call frequency of blue whale B calls, seasonal and annual adjustments to the call detection kernel were needed. To account for seasonal variability in call production, evaluation of the detector against ground truth data was performed at multiple times during the year. Analyst variability did not affect overall long-term trends in detection, but it had an impact on the total number of detections, as well as call rate estimation. Noise, particularly from shipping, was negatively correlated with detections at hourly time scales. A detailed analysis of variability in the performance of spectrogram correlation detectors should be performed when applying this method to long-term acoustic data-sets.     

Estimation of Staphylococcus aureus Growth Parameters from Turbidity Data: Characterization of Strain Variation and Comparison of Methods

Turbidity methods offer possibilities for generating data required for addressing microorganism variability in risk modeling given that the results of these methods correspond to those of viable count methods. The objectives of this study were to identify the best approach for determining growth parameters based on turbidity data and use of a Bioscreen instrument and to characterize variability in growth parameters of 34 Staphylococcus aureus strains of different biotypes isolated from broiler carcasses. Growth parameters were estimated by fitting primary growth models to turbidity growth curves or to detection times of serially diluted cultures either directly or by using an analysis of variance (ANOVA) approach. The maximum specific growth rates in chicken broth at 17°C estimated by time to detection methods were in good agreement with viable count estimates, whereas growth models (exponential and Richards) underestimated growth rates. Time to detection methods were selected for strain characterization. The variation of growth parameters among strains was best described by either the logistic or lognormal distribution, but definitive conclusions require a larger data set. The distribution of the physiological state parameter ranged from 0.01 to 0.92 and was not significantly different from a normal distribution. Strain variability was important, and the coefficient of variation of growth parameters was up to six times larger among strains than within strains. It is suggested to apply a time to detection (ANOVA) approach using turbidity measurements for convenient and accurate estimation of growth parameters. The results emphasize the need to consider implications of strain variability for predictive modeling and risk assessment.     

Mechanisms of orthostatic intolerance following very prolonged exercise.

Nine men completed a 24-h exercise trial, with physiological testing sessions before (T1, approximately 0630), during (T2, approximately 1640; T3, approximately 0045; T4, approximately 0630), and 48-h afterwards (T5, approximately 0650). Participants cycled and ran/trekked continuously between test sessions. A 24-h sedentary control trial was undertaken in crossover order. Within testing sessions, participants lay supine and then stood for 6 min, while heart rate variability (spectral analysis of ECG), middle cerebral artery perfusion velocity (MCAv), mean arterial pressure (MAP; Finometer), and end-tidal Pco(2) (Pet(CO(2))) were measured, and venous blood was sampled for cardiac troponin I. During the exercise trial: 1) two, six, and four participants were orthostatically intolerant at T2, T3, and T4, respectively; 2) changes in heart rate variability were only observed at T2; 3) supine MAP (baseline = 81 +/- 6 mmHg) was lower (P < 0.05) by 14% at T3 and 8% at T4, whereas standing MAP (75 +/- 7 mmHg) was lower by 16% at T2, 37% at T3, and 15% at T4; 4) Pet(CO(2)) was reduced (P < 0.05) at all times while supine (-3-4 Torr) and standing (-4-5 Torr) during exercise trial; 5) standing MCAv was reduced (P < 0.05) by 23% at T3 and 30% at T4 during the exercise trial; 6) changes in MCAv with standing always correlated (P < 0.01) with changes in Pet(CO(2)) (r = 0.78-0.93), but only with changes in MAP at T1, T2, and T3 (P < 0.05; r = 0.62-0.84); and 7) only two individuals showed minor elevations in cardiac troponin I. Recovery was complete within 48 h. During prolonged exercise, postural-induced hypotension and hypocapnia exacerbate cerebral hypoperfusion and facilitate syncope.     

Estimation of Staphylococcus aureus growth parameters from turbidity data: characterization of strain variation and comparison of methods

Turbidity methods offer possibilities for generating data required for addressing microorganism variability in risk modeling given that the results of these methods correspond to those of viable count methods. The objectives of this study were to identify the best approach for determining growth parameters based on turbidity data and use of a Bioscreen instrument and to characterize variability in growth parameters of 34 Staphylococcus aureus strains of different biotypes isolated from broiler carcasses. Growth parameters were estimated by fitting primary growth models to turbidity growth curves or to detection times of serially diluted cultures either directly or by using an analysis of variance (ANOVA) approach. The maximum specific growth rates in chicken broth at 17 degrees C estimated by time to detection methods were in good agreement with viable count estimates, whereas growth models (exponential and Richards) underestimated growth rates. Time to detection methods were selected for strain characterization. The variation of growth parameters among strains was best described by either the logistic or lognormal distribution, but definitive conclusions require a larger data set. The distribution of the physiological state parameter ranged from 0.01 to 0.92 and was not significantly different from a normal distribution. Strain variability was important, and the coefficient of variation of growth parameters was up to six times larger among strains than within strains. It is suggested to apply a time to detection (ANOVA) approach using turbidity measurements for convenient and accurate estimation of growth parameters. The results emphasize the need to consider implications of strain variability for predictive modeling and risk assessment.     

Contrast discrimination and choice reaction times at near-threshold pedestals

Choice reaction times (CRTs) to contrast differences were measured and compared with contrast increment thresholds obtained from concurrently measured psychometric functions at pedestal contrasts in the vicinity of detection threshold. Contrast discrimination functions had a classical dipper shape. The main finding was that CRTs were shorter at low pedestal contrasts but longer at higher pedestal contrasts compared to detection, reflecting the behaviour of increment thresholds. Even when equalized for response accuracy, CRTs varied with pedestal contrast in a similar manner to the contrast increment thresholds. The finding that CRTs and contrast increment thresholds depended on pedestal contrast in a similar manner suggests that both share a common origin. This common origin is proposed to lie in the variability of the sensory effect which determines the variability of the information accumulation process, which in turn affects the response criterion and contrast increment thresholds. At low pedestals, a decrease in variability lowers thresholds and results in a lower response criterion, thereby accelerating reaction times. At high pedestals, increasing signal-dependent noise inflates variability and thus raises thresholds and the response criterion, which results in slower CRTs.     

Determination of d-fenfluramine, d-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of d-fenfluramine (FEN), d-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/μl), plasma (2 pmol/ μl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/μl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.     

Determination of d-fenfluramine,d-norfenfluramine and fluoxetine in plasma,brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride

A HPLC method is described for the simultaneous determination of d-fenfluramine (FEN), d-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/μl), plasma (2 pmol/ μl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/μl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.     

Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry

The rodenticide monofluoroacetate (MFA) and monochloroacetate (MCA), a chemical intermediate from several chemical syntheses, have been identified as potential agents of chemical terrorism due to their high toxicity. In preparation for response to poisonings and mass exposures, we have developed a quantification method using isotopic dilution to determine MFA and MCA in urine from 50 to 5000 ng/mL. Both analytes were extracted from urine using solid-phase extraction; extraction recoveries were 62% (MFA) and 76% (MCA). The extracts were then separated with isocratic high-performance liquid chromatography and identified using electrospray ionization tandem mass spectrometry, with detection limits of 0.9 and 7.0 ng/mL for MFA and MCA, respectively. Selectivity was established for both analytes with unique chromatographic retention times which were correlated with isotopically labeled internal standards and the use of two mass spectral transitions for each compound. The intra-day variability was less than 5% for both analytes and the inter-day variability was 7% for MFA and 6% for MCA.     

The Influence of Chemical Mutagenesis on the Properties of the Cyclosporine a High-Producer Strain <Emphasis Type="Italic">Tolypocladium inflatum</Emphasis> VKM F-3630D

The effects of treatment with the chemical mutagen N-nitroso-N-methylurea (NMU) on the survival rate, morphological variability, and the level of production of cyclosporine A (CyA) in the Tolypocladium inflatum subsporum blastosporum strain VKM F-3630D were studied. The range of exposure to the dose of the mutagen (incubation time and concentration of HMU) was determined. The lethal and sublethal doses, as well as the conditions under which the highest variability in the levels of CyA production was observed, were determined. It was shown that the most efficient method to further increase CyA production in the studied high-producer T. inflatum strain is mutagenic treatment with 0.1% HMU for 2 h. In this case, we observed a relatively high level of survival of clones (80–85%), a low level of their morphological variability (10–15%), and a wide range in the CyA production level (80–170%) as compared to the original strain. This enabled us to select a number of clones with an increased CyA production level. The yield of the target metabolite was maximal in clone no. 13-27, where the CyA production level increased by 1.6–1.7 times.     

A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine

A 3-yr field trial was conducted on 8 commercial swine farms in Illinois to determine the effectiveness of a feline Toxoplasma gondii vaccine in reducing the exposure of swine to T. gondii. A vaccine consisting of live bradyzoites of the mutant T-263 strain, capable of preventing oocyst shedding by cats, was used in this study. Each farm was visited 3 times in 1994, 3 times in 1995, and once in 1996. Cats were trapped and inoculated with the T-263 oral vaccine during 1994 and 1995. On each visit, the following samples were collected: blood from pigs, cats, and mice for detection of serum antibodies to T. gondii, feces from cats to detect oocysts, and heart and brain tissues from rodents to determine the presence of T. gondii tissue cysts. The modified agglutination test (MAT), with a positive titer set at the 1:25 dilution, was used to determine serum antibodies. At first capture, 72.6% (61/84) of juvenile cats and 32.6% (31/95) of adult cats had no detectable antibodies (seronegative), indicating no prior exposure to T. gondii when they received their first vaccine. Of these first-time seronegative cats, 58.1% (18/31) of adult and 45.9% (28/61) of juvenile cats were recaptured and received a second dose of vaccine. Changes in the prevalence of T. gondii infection were evaluated from the prevaccination (1992, 1993) to the postvaccination (1996) period. Eleven cats (5%) were detected shedding oocysts between 1994 and 1996, of which 10 (90.1%) shed during 1994. The last detection of oocyst shedding by cats was during the first farm visit in 1995. There was a significant decrease in T. gondii seroprevalence for finishing pigs (P < 0.05, Wilcoxon sign rank test). There was a positive correlation (Spearman's p = 1.0, P < 0.0001) between the change in prevalence in juvenile cats and the change in prevalence in finishing pigs. The seropositivity rate (MAT > or = 1:25) in mice among all farms decreased from 4% in 1992-1993 to 0% in 1996. The mean prevalence of T. gondii tissue cyst isolation for mice on all farms decreased from 1.1% in 1994, to 0.8% in 1995, and to 0.5% in 1996. The results of this study suggest that the reduced exposure of pigs to T. gondii was due to the administration of the T. gondii vaccine to cats.     

Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome

The genetic heterogeneity of five influenza A (H3N2) strains isolated between 1968 and 1977 has been estimated by T1-oligonucleotide fingerprinting of 32P-labeled viral RNA. Assuming that the large T1-resistant oligonucleotides represent a random sample of the viral RNA, the genetic differences observed would affect 0.3 to 10.7% of the RNA positions of the genes studied, depending on the pair of viruses considered. A smaller degree of genetic heterogeneity was observed when six coetaneous viral samples were compared. The distribution of spontaneous mutations among the viral genes was studied by fingerprinting individual RNA segments isolated either by gel electrophoresis or hybridization with plasmids containing influenza-specific DNA sequences. No statistically significant differences were detected in the distribution of mutations among the viral genes studied. The mutation frequency at the hemagglutinin RNA region coding for the HA1 subunit was found to be two times higher than that at the region encoding that HA2 subunit. Our results suggest that the antigenic variability of influenza viruses may be a consequence of a general genetic variability which effects many of the viral genes.     

Effects of long-term rainfall variability on evapotranspiration and soil water distribution in the Chihuahuan Desert: A modeling analysis

We used the patch arid land simulator (PALS-FT) – a simple, mechanistic ecosystem model – to explore long-term variation in evapotranspiration (ET) as a function of variability in rainfall and plant functional type (FT) at a warm desert site in southern New Mexico. PALS-FT predicts soil evaporation and plant transpiration of a canopy composed of five principal plant FTs: annuals, perennial forbs, C₄ grasses, sub-shrubs, and evergreen shrubs. For each FT, the fractional contribution to transpiration depends upon phenological activity and cover as well as daily leaf stomatal conductance, which is a function of plant water potential, calculated from root-weighted soil water potential in six soil layers. Simulations of water loss from two plant community types (grass- vs. shrub-dominated) were carried out for the Jornada Basin, New Mexico, using 100 years of daily precipitation data (1891–1990). In order to emphasize variability associated with rainfall and fundamental differences in FT composition between communities, the seasonal patterns cover of perennials were held constant from year to year. Because the relative amount of year to year cover of winter and summer annual species is highly variable in this ecosystem, we examined their influence on model predictions of ET by allowing their cover to be variable, fixed, or absent.Over the entire 100-yr period, total annual ET is highly correlated with total annual rainfall in both community types, although T and E alone are less strongly correlated with rainfall, and variation in transpiration is nearly 3 times greater than evaporation and 2 times greater than variation in rainfall (CV of rainfall = 35%). Water use shows a relatively high similarity between the grass- and shrub-dominated communities, with a 100-yr average T/ET of 34% for both communities. However, based on a year-by-year comparison between communities, T/ET was significantly greater in the grass-dominated community, reflecting the fact that over the long term more than half of the rain occurs in the summer and is used slightly more efficiently (T¿E) by the C₄-grass community than the shrub community, although we found some rainfall patterns that resulted in much greater T/ET in the shrub community in a given year. Percent of water lost as transpiration (T/ET) suggests that while there is a general trend toward increased T/ET with rainfall in both community types, T/ET is extremely variable over the 100-yr simulation, especially for normal and below normal amounts of rainfall (T/ET values range from 1 to 58% for the grass-dominated site and 6 to 60% for the shrub-dominated site).These predictions suggest that because of the relatively shallow distribution of soil water, there is little opportunity for vertical partitioning of the soil water resource by differential rooting depths of the plant FTs, in contrast to the two-layer hypothesis of Walter (1971). However, functional types may avoid competition by keying on particular `windows' of moisture availability via differences in phenologies. We found very little differences in average, long-term model predictions of T, E, and ET when annual plant cover was variable, fixed, or absent. The results of our simulations help reconcile some of the disparate conclusions drawn from experimental studies about the relative contribution of transpiration vs. evaporation to total evapotranspiration, primarily by revealing the great year-to-year variability that is possible.     

Genetic influences on lipid metabolism trait variability within the Stanislas Cohort.

The contribution of 17 polymorphisms within 13 candidate genes on lipid trait variability was investigated by a multiplex assay in 772 men and 780 women coming for a health checkup examination. The studied genes were APOE, APOB, APOC3, CETP, LPL, PON, MTHFR, FGB, GpIIIa, SELE, ACE, and AGT. We found that APOB-Thr71Ile, APOE-(112/158), APOC3-1100C/T, and SELE-98G/T polymorphisms had a significant effect on lipid traits (P < or = 0.001 to P < or = 0.01). Genetic effects accounted for 3.5-5.7% of variation in apolipoprotein B (apoB)-related traits among men, and for 5.7-9.0% among women. The contribution of APOE polymorphism on apoB-related traits variability was two to three times more important in women than in men. We found suggestive evidence for interactive effects between genetics and age, smoking status, and oral contraceptives. Increase of LDL-cholesterol and apoB concentrations with age was stronger among the epsilon4 carriers in women, and apolipoprotein A-I (apoA-I) concentration decreased with age in epsilon4 male carriers. The effect of epsilon2 allele on LDL-cholesterol was more important in the oral contraceptive users. In nonsmokers only, the APOC3-1100C allele in women was related to lower apoB-related traits concentrations, and in men to higher apoA-I and HDL-cholesterol concentrations. In conclusion, this work, in addition to the reinforcement of the already known associations between APOB, APOE, and APOC3 genes and lipids, leads to new perspectives in the complex relationships among genes and environmental factors. The newly observed relationships between E-selectine gene and lipid concentrations support the hypotheses of multiple metabolic pathways contributing to the complexity of lipids variability.     

Litterfall as a measure of primary production in Mediterranean holm-oak forest

In this paper we discuss the use of litterfall as a method to measure primary production and its between year relation to climatic fluctuation. Seven years of study in a mediterranean holm-oak forest showed a moderate inter-annual variability of litterfall (C.V. 11%), while the annual variability of the actual evapotranspiration was three times higher (C.V.30%). Furthermore, the inter- and intra-annual variability of nutrient content in the various fractions are presented in relation to water availability. Monthly and seasonal variability was higher than the annual variability for all analyzed elements.     

Demographic modeling and monitoring cycle in a long-lived endangered shrub

Demographic monitoring is a well established tool for conservation managers. But, monitoring programs typically do not offer methods to explore the relationship between projected population trends and the probability of trend detection. Moreover, conservation biologists need to evaluate and incorporate the effect of variability and habitat perturbation on the efficiency of monitoring programs.We studied population demography of an endangered shrub endemic to East Central Spain, Vella pseudocytisus subsp. paui in order to understand its present demographic performance, how it is affected by increasing variability and perturbation and how these two factors are related to monitoring and monitoring thresholds.Using Lefkovich demographic matrices on six years of data, we produced 19 different population projections, and we compared these modeled projections against observed projection. We designated three conceptually important detection thresholds: an early warning; an unequivocal signal; and, a quasi-extinction threshold. Based on calculated detection times of all models for every threshold, we produced an averaged monitoring cycle (minimum visit frequency) to provide managers with a tool to design a consistent monitoring program for this plant.Our results indicate a relatively stable current population along with large detection times for critical threshold changes of modeled populations when considering small management changes (low population variability and low perturbation intensity). In contrast, model combinations incorporating high population variance and high perturbation produce disproportionally short times to detection of a significant population trend. In terms of designing studies to maximise results while minimising monitoring effort, designing a monitoring program capable of providing an early warning system of potential population failure requires more effort than detecting an unequivocal signal of decline.Finally, we propose two alternatives when calculating monitoring cycle (MC) values. One of them implies demographic MC if population perturbation and variation are high. For that, an optimal monitoring cycle is estimated based on a range of possible scenarios for population trends. Alternatively, we propose environmental MC, when low values of perturbation and variation are expected.     

Site stripping based on likelihood ratio reduction is a useful tool to evaluate the impact of non-clock-like behavior on viral phylogenetic reconstructions

The site stripping for clock detection procedure was implemented in the recently developed maximum likelihood framework for estimating evolutionary rates and divergence times in measurably evolving populations. The method was used to investigate the effect of rate variability on estimating divergence times in non-clock-like trees for human immunodeficiency viruses and hepatitis C viruses. We validate our approach by comparing dated coalescent nodes in molecular phylogenies with known dates of transmission. Our method was able to rapidly recover clock-like behavior and to indicate the presence and direction of a bias when estimates of divergence times using the unstripped data were flawed.     

The effects of exogenous testosterone on spatial memory in rats

Testosterone (T) is known to affect spatial abilities in men and women. Studies focusing on this relationship showed that both endogenous variability of T and administration of exogenous T, altered mental rotation and spatial visualization. Organizational and activational effects of T can be separately identified. The aim of our study was to evaluate the activational effects of exogenous T on spatial memory in male and female rats. T was administered 3 times a week over a two week period in either 1 mg/kg for low testosterone group or 10 mg/kg for high testosterone group. The Morris water maze was performed to assess the rat’s working and reference spatial memory. T and estradiol levels were measured in plasma. Increase in plasma T levels was confirmed in the experimental groups in comparison to the control groups (receiving sterile oil, 3 times a week over a two week period). Low dose T impaired working, but improved reference memory in female rats. In male rats the negative effects of T (both doses) on reference memory were shown. This experiment showed that the activational effects of exogenous testosterone on spatial memory of rats were gender and dose-dependent.     

Molecular markers (RFLPs and HSPs) for the genetic dissection of thermotolerance in maize

Summary Cellular membrane stability (CMS) is a physiological index widely used to evaluate thermostability in plants. The genetic basis of the character has been studied following two different approaches: restriction fragment length polymorphism (RFLP) analysis, and the effects of segregating heat shock protein (HSP) loci. RFLP analysis was based on a set of recombinant inbreds derived from the T32 × CM37 F₁ hybrid and characterized for about 200 RFLP loci. Heritability of CMS estimated by standard quantitative analysis was 0.73. Regression analysis of CMS on RFLPs detected a minimum number of six quantitative trait loci (QTL) accounting for 53% of the genetic variability. The analysis of the matrices of correlation between RFLP loci, either within or between chromosomes, indicates that no false assignment was produced by this analysis. The effect of HSPs on the variability of the CMS was tested for a low-molecular-weight peptide (HSP-17) showing presence-absence of segregation in the B73 × Pa33 F₂ population. Although the genetic variability of the character was very high (h ²=0.58) the effect of HSP-17 was not significant, indicating either that the polypeptide is not involved in the determination of the character or that its effect is not statistically detectable.     

Heterogeneity of times required for germination and outgrowth from single spores of nonproteolytic Clostridium botulinum

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t(germ)), time to emergence (t(emerg)), and times to reach the lengths of one (t(C1)) and two (t(C2)) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t(germ)), emergence (t(emerg) - t(germ)), cell maturation (t(C1) - t(emerg)), and doubling (t(C2) - t(C1)) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.