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.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Microbial Detection Method Based on Sensing Molecular Hydrogen

A simple method for detecting bacteria, based on the time of hydrogen evolution, was developed and tested against various members of the Enterobacteriaceae group. The test system consisted of (i) two electrodes, platinum and a reference electrode, (ii) a buffer amplifier, and (iii) a strip-chart recorder. Hydrogen evolution was measured by an increase in voltage in the negative (cathodic) direction and recorded on a strip-chart recorder. Hydrogen response curves consisted of (i) a lag period, (ii) a period of rapid buildup in potential due to hydrogen, and (iii) a period of decline in potential. A linear relationship was established between inoculum size and the time hydrogen was detected (lag period). Lag times ranged from 1 h for 10(6) cells/ml to 7 h for 10(0) cells/ml. For each 10-fold decrease in inoculum, length of the lag period increased 60 to 70 min. Mean cell concentrations at the time of hydrogen evolution were 10(6)/ml. Based on the linear relationship between inoculum size and lag period, these results indicate the potential application of the hydrogen-sensing method for rapidly detecting coliforms and other gas-producing microorganisms in a variety of clinical, food, and other samples.     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila.

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Comparison of two commercial preparations of buffered peptone water for the recovery and growth of Salmonella bacteria from foods

This study compared the performance of two commercial preparations of buffered peptone water. Performance was assessed in terms of ability to resuscitate and recover low numbers of stressed cells, buffering capacity, growth of Salmonella bacteria in pure culture and growth of Salmonella in food pre-enrichments. Although both the preparations of BPW had similar chemical compositions, differences in their recovery performance were found. Brand A recovered significantly higher numbers of heat-injured Salmonella (mean = 0.57 log10 cfu ml(-1) difference) in pure culture compared with brand B when dealing with very low inoculum levels. Although brand B had higher buffering capacity, the pH at the end of the pre-enrichment was found to be similar in both media, even in foods such as milk powder which showed the greatest decline in pH. Both brands were comparable in their ability to grow unstressed Salmonella from different food types. In unstressed cell studies, similar cell numbers were recovered at the end of a 24 h incubation period from both media, although brand B yielded a higher biomass. In the food study with unstressed cells, performance was related more to the food type and the likely association between this and the level and type of competitor organisms present, rather than to the brand of medium used.     

Conductance measurements of the lag phase of injured Salmonella typhimurium

The duration of the lag phase of Salmonella typhimurium injured by heating, freezing, acidification or drying was measured using the 'Malthus' conductance meter. Results confirmed those previously obtained by viable counting and additionally, revealed the very wide variation in lag between and within populations and the extreme length of lag that can occur in some severely injured cells. In an extreme example, the measured lag times of low numbers of bacteria taken from the same heat-injured population ranged from 16 h to 70 h. The implications for the detection of injured micro-organisms in food are discussed.     

Conductance measurements of the lag phase of injured Salmonella typhimurium

The duration of the lag phase of Salmonella typhimurium injured by heating, freezing, acidification or drying was measured using the 'Malthus' conductance meter. Results confirmed those previously obtained by viable counting and additionally, revealed the very wide variation in lag between and within populations and the extreme length of lag that can occur in some severely injured cells. In an extreme example, the measured lag times of low numbers of bacteria taken from the same heat-injured population ranged from 16 h to 70 h. The implications for the detection of injured micro-organisms in food are discussed.     

A comparison of the bioscreen method and microscopy for the determination of lag times of individual cells of listeria monocytogenes

Lag phase durations (tLag) of individual Listeria monocytogenes cells were analysed using the NightOwl Molecular Imaging System, and results were compared with mean individual cell lag times (tL) obtained from the detection time (td) method using Bioscreen. With Bioscreen, an average tL of 6.39+/-0.89 h was obtained from five separate experiments. With the NightOwl method, an average tLag of 2.73+/-0.06 h was obtained from three experiments consisting of eight total replicates. Lag values from the NightOwl and Bioscreen are related by the equation: tLag = tL + DT, where DT is the doubling time. The equivalent tLag mean value for the Bioscreen method was 7.11+/-0.84 h. Individual lag times measured by both methods were normally distributed (r2 for Bioscreen and NightOwl ranged from 0.951 to 0.999 and from 0.884 to 0.982, respectively). The results suggest that the NightOwl method can provide accurate estimates of individual cell lag times, which will facilitate the development of combined discrete continuous models for bacterial growth.     

Kinetics of single cells: observation and modeling of a stochastic process

The successive generation times for single cells of Escherichia coli K-12 were measured as described by A. Elfwing, Y. LeMarc, J. Baranyi, and A. Ballagi (Appl. Environ. Microbiol. 70:675-678, 2004), and the histograms they generated were used as empirical distributions to simulate growth of the population as the result of the multiplication of its single cells. This way, a stochastic birth model in which the underlying distributions were measured experimentally was simulated. To validate the model, analogous bacterial growth curves were generated by the use of different inoculum levels. The agreement with the simulation was very good, proving that the growth of the population can be predicted accurately if the distribution of the first few division times for the single cells within that population is known. Two questions were investigated by the simulation. (i) To what extent can we say that the distribution of the detection time, i.e., the time by which a single-cell-generated subpopulation reaches a detectable level, can be identified with that of the lag time of the original single cell? (ii) For low inocula, how does the inoculum size affect the lag time of the population?     

FURTHER OBSERVATIONS ON GROWTH OF SINGLE CELLS OF COCCOID BLUE-GREEN ALGAE

Initiation of growth (lag) and subsequent growth to visible colonies was examined for single cells of several coccoid blue-nreen algae under controlled incuba-tion conditions. At 30 or 39 C, with tungsten or fluorescent illumination, organisms such as Agmenel-lum quadruplicatum, strains PR-6 and strain BG-J, showed no evidence of lag associated with initiation of growth. The final colony count was within 20% of the expected number derived from cell counts and serial dilutions. For Anacystis nidulnns, Tx 20, a new medium C, 10 with EDTA as chelator was developed. In this medium groiuth of single cells at 39 C was excellent with quantitative recovery and no evidence of lag. At 30 C, however, Tx 20 showed anomalous behavior, lag and nonquantitative cell recovery. This behavior at 30 C is not yet understood.     

Predicting the growth of Salmonella typhimurium on beef by using the temperature function integration technique.

Lag and generation times for the growth of Salmonella typhimurium on sterile lean beef were modeled as functions of cooling time under various carcass-chilling scenarios. Gompertz growth models were fit to the log10 colony counts over time at each of six temperatures in the range of 15 to 40 degrees C. Lag and generation times were defined as the points at which the second and first derivatives, respectively, of each growth curve attained a maximum. Generation time and lag time parameters were modeled as functions of temperature by use of exponential-decay models. The models were applied to typical beef carcass-cooling scenarios to predict the potential growth of S. typhimurium during the cooling of beef. Validation studies indicated no significant difference between the observed and predicted bacterial populations on inoculated lean and fatty beef tissues cooled at either 6 or 9 degrees C/h.     

Predicting the growth of Salmonella typhimurium on beef by using the temperature function integration technique.

Lag and generation times for the growth of Salmonella typhimurium on sterile lean beef were modeled as functions of cooling time under various carcass-chilling scenarios. Gompertz growth models were fit to the log10 colony counts over time at each of six temperatures in the range of 15 to 40 degrees C. Lag and generation times were defined as the points at which the second and first derivatives, respectively, of each growth curve attained a maximum. Generation time and lag time parameters were modeled as functions of temperature by use of exponential-decay models. The models were applied to typical beef carcass-cooling scenarios to predict the potential growth of S. typhimurium during the cooling of beef. Validation studies indicated no significant difference between the observed and predicted bacterial populations on inoculated lean and fatty beef tissues cooled at either 6 or 9 degrees C/h.     

Comparison of biooxidation with carbon dioxide assimilation during bacterial growth on ferrous ion or elemental sulfur.

Biomass and oxygen uptake activity profiles of a mixed bioleaching culture were studied and compared at various temperatures. Bacteria were grown on ferrous ion or elemental sulfur in a Micro-Oxymax respirometer apparatus that allowed measurement of both oxygen consumption and carbon dioxide assimilation. Balanced growth was observed between 10 degrees C and 35 degrees C, with an optimum at 30 degrees C, on both energy sources. No significant growth was observed at the lowest temperature used, 5 degrees C, or at the highest temperature used, 40 degrees C. The oxygen to carbon dioxide molar yield was 50:1 when growing on ferrous ion but only 17:1 when growing on elemental sulfur. Upon transfer from a sulfide ore to a new energy source, greater numbers in the inoculum reduced the duration of the lag phase. Lag phase duration was also reduced by proximity to the optimum growth temperature. A longer lag phase decreased the achievable growth rate of the cells exponentially, significantly affecting biooxidation activity.     

Conditioned medium from Listeria innocua stimulates emergence from a resting state: not a response to E. coli quorum sensing autoinducer AI-2

The lag phase of the bacterial growth curve is an important determinant in speeding the detection of pathogens. It is affected by many factors including the prevailing growth environment and inoculum size, as well as specific signal molecules. The elucidation of growth-regulating signal molecules is further facilitated by culturing cells in defined growth media. In this study, a defined medium capable of supporting growth of Listeria innocua at similar levels as obtained using a complex brain heart infusion (BHI) media was developed. Further, the effects of conditioned medium (CM) on population lag time of L. innocua was investigated using a rapid parallel approach (with an automated microtiter plate reader). Importantly, the lag phase was shortened by up to approximately 50% by the addition of CM from L. innocua cultures obtained late in the exponential phase. Finally, while L. innocua were found to secrete bacterial signaling autoinducer, AI-2, tests using Escherichia coli based CM having a 90-fold difference in AI-2 level suggested that the observed decrease in lag phase was not due to E. coli-derived AI-2 and was instead due to elements specific to L. innocua. These findings indicate secreted signal molecules may be found in CM that speed detection of L. innocua.     

The effect of sublethal injury by heating, freezing, drying and gamma-radiation on the duration of the lag phase of Salmonella typhimurium

The duration of the lag phase of Salmonella typhimurium surviving heat, freezing, drying and gamma-radiation was used to indicate the time needed to repair sublethal injury. Following equivalent lethal treatments, heat and freeze-injured cells needed longer to repair than those injured by drying or gamma-radiation. Measurement of repair on membrane filters showed that in a heat-injured population having a lag time of 9 h, some individual cells needed up to 14 h to recover maximum tolerance to 3% NaCl.     

A comparison of solid and liquid media for measuring the sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media, and the time needed to recover resistance

Sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media was measured by viable counts in liquid and on agar-solidified versions of these media and on nutrient media. All solid media, including the supposedly non-inhibitory nutrient agar, were more inhibitory to injured cells than the corresponding liquid media. Catalase or pyruvate increased counts on nutrient agar to the level obtained in nutrient broth. Therefore nutrient agar plus pyruvate was the most suitable reference medium against which to compare recoveries on other media. Although recoveries of injured cells varied widely depending on the composition and physical state of the medium, this had a minor effect on estimates of repair time because resistance to all selective media was regained by the end of the lag phase.     

Estimating Bacterial Growth Parameters by Means of Detection Times

We developed a new numerical method to estimate bacterial growth parameters by means of detection times generated by different initial counts. The observed detection times are subjected to a transformation involving the (unknown) maximum specific growth rate and the (known) ratios between the different inoculum sizes and the constant detectable level of counts. We present an analysis of variance (ANOVA) protocol based on a theoretical result according to which, if the specific rate used for the transformation is correct, the transformed values are scattered around the same mean irrespective of the original inoculum sizes. That mean, termed the physiological state of the inoculum, , and the maximum specific growth rate, μ, can be estimated by minimizing the variance ratio of the ANOVA procedure. The lag time of the population can be calculated as λ = −ln /μ; i.e. the lag is inversely proportional to the maximum specific growth rate and depends on the initial physiological state of the population. The more accurately the cell number at the detection level is known, the better the estimate for the variance of the lag times of the individual cells.