Assessing the Contamination Potential of Freshly Extracted Escherichia coli Biofilm Cells by Impedancemetry

Planktonic bacteria passing to a sessile state during the formation of a biofilm undergo many gene expression and phenotypic changes. These transformations require a significant time to establish. Inversely, cells extracted from a biofilm should also require a significant time before acquiring the same physiological characteristics as planktonic cells. Relatively few studies have addressed the kinetics of this inverse transformation process. We tested one aspect, namely, the contamination potential of freshly extracted Escherichia coli biofilm cells, precultured in a synthetic medium, in a rich liquid growth medium. We compared the time between inoculation and the beginning of the growth phase of freshly extracted biofilm cells, and suspended exponential and suspended stationary phase cells precultured in the same synthetic medium. Unexpectedly, the lag time for the extracted biofilm cells was the same as the lag time of the suspended exponential phase cells and significantly less than the lag time of the suspended stationary phase cells. The lag times were determined by an impedance technique. Cells extracted from biofilms, i.e., biofilms formed in canalizations and broken up by hydrodynamic forces, are an important source of contamination. Our work shows, in the case of E. coli, the high potential of freshly extracted biofilm cells to reinfect a new medium.     

Parameter estimation for the distribution of single cell lag times

In Quantitative Microbial Risk Assessment, it is vital to understand how lag times of individual cells are distributed over a bacterial population. Such identified distributions can be used to predict the time by which, in a growth-supporting environment, a few pathogenic cells can multiply to a poisoning concentration level.We model the lag time of a single cell, inoculated into a new environment, by the delay of the growth function characterizing the generated subpopulation. We introduce an easy-to-implement procedure, based on the method of moments, to estimate the parameters of the distribution of single cell lag times. The advantage of the method is especially apparent for cases where the initial number of cells is small and random, and the culture is detectable only in the exponential growth phase.     

Interaction of Yeast Phosphoglyceromutase with Nucleotides

Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40 % O₂ were oxygen-resistant because when used as inocula for fresh 40 % O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40% O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.     

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.     

Effect of the development phase and growth rate of a Shigella sonnei population on the reproduction of homologous bacteriophage

This study showed that the minimum latent period (20 minutes) of the intracellular multiplication of dysentery bacteriophage S-9 in the population of S. sonnei substrate strain under the conditions of static heterogeneous surface batch cultivation was observed at the end of the lag phase and at the growth acceleration phase, in the first and second thirds of the exponential curve, while the maximum latent period (35-40 minutes) was observed at the stationary phase. The maximum yield of phage S-9 from one infected bacterial cell (628.3 +/- 116.8) was registered during the first third of the phase of the exponential growth of the bacterial population and the minimum yield (18.66 +/- 6.6), at the beginning of the lag phase. The significant direct correlation between the specific growth rate of the bacterial population and the yield of the phage from one infected bacterial cell at the end of the lag phase, at the growth acceleration and deceleration phases, as well as the significant inverse correlation between the yield of the phage and the time of the generation of the bacterial population at the growth acceleration phase were established.     

Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria

The susceptibility to bioremediation of the hydrocarbons contained in a waste from crude oil extraction was examined. Laboratory scale batch reactors were inoculated with indigenous bacteria and biodegradation was followed for 45 days. The total hydrocarbon content was reduced to 70% of its initial value at the end of the experiments. Saturated and aromatic hydrocarbons were the most readily degraded fractions with, respectively, 70% and 60% of the fraction remaining at the end of the experiment. A minor degradation was observed in the resins fraction (20%), whereas the asphaltenes fraction remained almost constant.The substrate preferences of the natural population towards various fractions of the crude oil were determined by both the length of the lag phase and the slope of the exponential growth in a mineral salt-base medium containing either of the different hydrocarbon fraction as the sole source of carbon. The highest consumption rate for every fraction during the time course experiments was in agreement with the shortest lag phase and the greatest exponential growth slope in the corresponding selective media, indicating changes in the population composition.     

Population growth lags in introduced species

When introduced to new ecosystems, species'' populations often grow immediately postrelease. Some introduced species, however, maintain a low population size for years or decades before sudden, rapid population growth is observed. Because exponential population growth always starts slowly, it can be difficult to distinguish species experiencing the early phases of slow exponential population growth (inherent lags) from those with actively delayed growth rates (prolonged lags). Introduced ungulates provide an excellent system in which to examine lags, because some introduced ungulate populations have demonstrated rapid population growth immediately postintroduction, while others have not. Using studies from the literature, we investigated which exotic ungulate species and populations (n = 36) showed prolonged population growth lags by comparing the doubling time of real ungulate populations to those predicted from exponential growth models for theoretical populations. Having identified the specific populations that displayed prolonged lags, we examined the impacts of several environmental and biological variables likely to influence the length of lag period. We found that seventeen populations (47%) showed significant prolonged population growth lags. We could not, however, determine the specific factors that contributed to the length of these lag phases, suggesting that these ungulate populations'' growth is idiosyncratic and difficult to predict. Introduced species that exhibit delayed growth should be closely monitored by managers, who must be proactive in controlling their growth to minimize the impact such populations may have on their environment.     

Evaluation of growth hormone production from GH1 cells in vitro: Effect of culture media and time in culture

Summary Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).     

Recovery of exocellular acid phosphatase activity on Saccharomyces mellis after treatment of the organism with reagents that affect the cell surface

Derepressed cells of Saccharomyces mellis were treated in one of several different ways to either elute or inactivate the exocellular enzyme, acid phosphatase. The enzyme was either (i) eluted from resting cells with 0.5 m KCl plus 0.1% beta-mercaptoethanol, (ii) eluted from exponential phase cells by growing the organism in derepressing media containing 0.5 m KCl, or (iii) inactivated on exponential phase cells by adding sufficient acid or base to growth media to destroy the enzyme but not enough to kill the cells. These treatments did not affect viability. Treated cells were transferred to fresh growth media or some other reaction mixture, and the kinetics of recovery of acid phosphatase activity was studied. In these reaction mixtures, enzyme was synthesized only by actively growing cells. Treated resting cells were indistinguishable from untreated, repressed resting cells in that the organism inoculated into complete growth medium remained in the lag phase for approximately 6 hr before both growth and enzyme synthesis began. Exponential phase derepressed cells treated by method (ii) or (iii) were transferred to fresh medium under conditions that allowed growth to continue. The cells immediately started to manufacture enzyme at a rate greater than normal until the steady-state level was reached, thus demonstrating a feedback control system. Exponential phase repressed cells were also transferred to fresh derepressing media under conditions which sustained growth. Though these cells began to grow immediately, there was a lag before acid phosphatase synthesis began followed by a lengthy inductive period. The length of the period of induction could be correlated with the polyphosphate content of the cells. As the supply of polyphosphate neared exhaustion, the rate of synthesis increased rapidly until it was greater than normal; this differential rate was sustained until the steady-state concentration was reached. When derepressed cells grow in a medium containing 0.5 m KCl, some acid phosphatase activity is found free in the culture fluid and some remains firmly attached to the cells despite the presence of the salt. The bound activity is subject to feedback control, but the steady-state level of this activity on the cells is only one-third that of the acid phosphatase on cells growing in nonsaline media. The extracellular phosphatase is produced at a rate that is several-fold greater than that of the exocellular enzyme in a nonsaline medium. The synthesis of the extracellular enzyme does not seem to be controlled by a feedback mechanism but is produced at a maximal rate as long as the cells are growing.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

The Effect of Nonanal on Dipodascus aggregatus II. Influence of the Prehistory of the Inoculum and of the Presence of Ethanol

Nonanal, added in ethannlic solution, in concentrations lower than 40 to 80 μM did not affect the growth of Dipodascus aggregatus, provided the inoculum had been harvested from the exponential phase of growth. Growth could even be inhibited by 80 μM. If the inoculum had been grown to the exponential phase and then for another period, to the acceleration phase, in fresh liquid medium, growth was strongly promoted by 80 μM nonanal. If cells from the exponential phase were grown for another period in the supernatant fluid of centrifuged cultures from the exponential phase, 80 μM affected growth in the following way: in five different experiments growth was not stimulated, in one experiment undoubtedly promoted, and weakly stimulated in another one. The growth of cultures inoculated with cells grown only on malt agar was not affected by 80 μM nonanal. Pretreatment of cells, harvested from the acceleration phase, with nonanal (80 μM) in the presence of ethanol did not diminish the growth-promoting action of nonanal on the cultures inoculated with these cells. Nonanal, in the absence of ethanol, in a concentration of 10 μM did not affect the growth of cells, harvested from the acceleration phase, whereas 100 μM nonanal strongly inhibited growth. An attempt is made to explain the results starting from the endogenous metabolism.     

Hazard analysis applied to microbial growth in foods: development of mathematical models describing the effect of water activity

Mathematical models have been developed which describe the effect of lowering the water activity on the growth kinetics of Staphylococcus aureus and Salmonella typhimurium. By treating the lag phase and exponential phase kinetics separately predictions can be made on the extent of microbial growth over successive time/temperature cycles. Staph. aureus was far more tolerant than Salm. typhimurium to lowered water activity and under near growth limiting conditions of water activity and temperature was showing lag periods as long as ca 40 d. The maximum lag period observed for Salm. typhimurium was ca 5 d. Under these conditions the predicted generation times for Staph. aureus were 2-3 d and for Salm. typhimurium.     

Growth and Enzyme Activity of Myxococcus virescens in Liquid Medium

The object of this work was to develop a method for determination of the growth of Myxocuccus virescens in liquid medium. The bacteria were grown in N III-B medium in 100 ml Kjeldahl flasks, which were fixed on a disc forming an angle of 50 degrees with the horizontal plane. The disc was rotated two full revolutions per minute. The total nitrogen content of the washed swarms, developing on the glass walls of the flasks, was used as an expression for the myxobacterial growth. After a lag the bacteria grown in total darkness had a growth phase, approximately exponential, of about 270 hours, which was followed by a steep phase of decline. When the bacteria were illuminated daily for a short period, a lag of 50–200 hours appeared in the middle of the exponential growth phase, after which a new exponential growth phase began. This second growth seemed to depend on variants insensitive to light induced lysis. The increase of enzymes, active on casein and autoclaved aerobacter cells, closely followed the first part of the growth curve. However both activities began to decrease before the growth maximum. No sign of proteolytic activity or lytic activity on autoclaved aerobacter cells could be detected after about 700 hours' incubation. In illuminated flasks it is shown that the production of yellow pigments in culture solution is sharply increased at the end of the exponential growth phase. The lytic enzymes of M. virescens seem to be extracellular, secreted during the exponential phase of growth. No activity was exhibited by washed cell swarms, even if they were sonically disintegrated.     

Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen")

Lankford, C. E. (The University of Texas, Austin), James R. Walker, James B. Reeves, N. H. Nabbut, B. R. Byers, and R. J. Jones. Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen"). J. Bacteriol. 91:1070-1079. 1966.-When cells of Bacillus megaterium, grown on Brain Heart Infusion Agar, were inoculated into a chemically defined medium, they exhibited a division lag which was an inverse function of inoculum size. The addition of filtrates of cultures from the same medium eliminated the inoculum-dependent component of lag, but not an inoculum-independent residual lag of constant duration. Culture filtrate of B. subtilis var. niger not only eliminated its inoculum-dependent lag but also was required to sustain exponential division. Dose-response and "growth time" bioassays, developed to measure lag-reducing activity of filtrates, demonstrated accumulation of active filtrate factor to a "critical" concentration prior to division initiation. Addition of this concentration to cultures eliminated the inoculum-dependent lag. Accumulation of the factor ceased temporarily at onset of division, but excretion was resumed later during exponential growth. Accumulation of a lag-reducing, cell-associated factor followed a similar course. Chromatographic and bioautographic analyses of culture filtrates of B. megaterium indicated that a single substance was primarily responsible for their activity. Results of dose-response tests for reciprocal activities of filtrates of different Bacillus species and strains suggested production of different factors by some, and of different quantities of similar factors by others. It is proposed that such endogenous factors which are synthesized and accumulate to a population-dependent concentration as a requisite to initiation and maintenance of division be designated as "schizokinens."     

Hazard analysis applied to microbial growth in foods: Development of mathematical models describing the effect of water activity

Mathematical models have been developed which describe the effect of lowering the water activity on the growth kinetics of Staphylococcus aureus and Salmonella typhimurium. By treating the lag phase and exponential phase kinetics separately predictions can be made on the extent of microbial growth over successive time-temperature cycles. Staph. aureus was far more tolerant than Salm. typhimurium to lowered water activity and under near growth limiting conditions of water activity and temperature was showing lag periods as long as ca 40 d. The maximum lag period observed for Salm. typhimurium was ca 5 d. Under these conditions the predicted generation times for Staph. aureus were 2–3 d and for Salm. typhimurium 1–4 d.     

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.     

Indirect measurement of the lag time distribution of single cells of Listeria innocua in food

The distribution of log counts at a given time during the exponential growth phase of Listeria innocua measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55 degrees C for 45 min or at 62 degrees C for 2 min, and an unheated batch was incubated at 4 degrees C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62 degrees C were greater than those of the cells treated at 55 degrees C. Three batches of paté samples were heated at 55 degrees C for 25 min, 62 degrees C for 81 s, or 65 degrees C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15 degrees C. The distribution of the lag times of the cells heated at 55 degrees C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62 degrees C and 65 degrees C, the lag duration was longer and its variance greater.     

Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum

It has been found that Dictyostelium discoideum cells from the exponential growth phase of axenically grown cultures are cohesive, whereas those from stationary phase are not. These differences in cohesiveness are seen in phosphate buffer and in axenic medium. Stationary phase medium inhibits the aggregation of log phase cells; stationary phase cells inoculated into freshly prepared medium regain their cohesiveness. Stationary phase medium may contain an inhibitor of cell cohesion. pH differences between the two types of medium are not entirely responsible for loss of cohesiveness.