Continuous Drip Flow System to Develop Biofilm ofE. faecalisunder Anaerobic Conditions

Purpose.To evaluate a structurally matureE. faecalisbiofilm developed under anaerobic/dynamic conditions in anin vitrosystem.Methods.An experimental device was developed using a continuous drip flow system designed to develop biofilm under anaerobic conditions. The inoculum was replaced every 24 hours with a fresh growth medium for up to 10 days to feed the system. Gram staining was done every 24 hours to control the microorganism purity. Biofilms developed under the system were evaluated under the scanning electron microscope (SEM).Results.SEM micrographs demonstrated mushroom-shaped structures, corresponding to a matureE. faecalisbiofilm. In the mature biofilm bacterial cells are totally encased in a polymeric extracellular matrix.Conclusions.The proposedin vitrosystem model provides an additional useful tool to study the biofilm concept in endodontic microbiology, allowing for a better understanding of persistent root canal infections.

The Frequency of Persisters in Escherichia coli Reflects the Kinetics of Awakening from Dormancy

ABSTRACT A genetically homogenous bacterial population may contain physiologically distinct subpopulations. In one such case, a minor part of an otherwise antibiotic-sensitive bacterial population maintains a nondividing state even in a growth-supporting environment and is therefore not killed by bactericidal antibiotics. This phenomenon, called persistence, can lead to failure of antibiotic treatment. We followed the development of sensitivity to killing by ampicillin and norfloxacin when Escherichia coli cells were transferred from a stationary-phase culture into fresh growth medium. In parallel, we monitored growth resumption by individual bacteria. We found that bacteria in a population resumed growth and became sensitive to antibiotics at different times after transfer to fresh medium. Moreover, both growing and dormant bacteria coexisted in the same culture for many hours. The kinetics of awakening was strongly influenced by growth conditions: inocula taken from the same stationary-phase culture led to very different persister frequencies when they were transferred into different fresh media. Bactericidal antibiotics kill cells that have woken up, but the later-awakening subpopulation is tolerant to them and can be identified as persisters when the antibiotic is removed. Our observations demonstrate that persister count is a dynamic measure and that the persister frequency of a particular culture is not a fixed value.

New Insights into Methyl Bromide Cooxidation byNitrosomonas europaea Obtained by Experimenting with Moderately Low Density Cell Suspensions

ABSTRACT We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (∼6 � 107 cells ml−1) of the NH3-oxidizing bacterium Nitrosomonas europaea. In the presence of 10 mM NH4 + and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH4 +, 18% of the NH4 +, and 35% of the NH4 +, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH4 +-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells−1). Approximately 90% of the NH3-dependent O2uptake activity and the NO2 −-producing activity were lost after N. europaea was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO2 −production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO2 − accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO2 −-producing and MeBr-oxidizing activities could not be attributed directly to NH4 + or NH3 limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO2 − and formaldehyde) in the medium. Additional cooxidation-related studies of N. europaea are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH3-oxidizing activity.

Changes in Cell Size and DNA Content inSulfolobus Cultures during Dilution and Temperature Shift Experiments

ABSTRACT Stationary-phase cultures of different hyperthermophilic species of the archaeal genus Sulfolobus were diluted into fresh growth medium and analyzed by flow cytometry and phase-fluorescence microscopy. After dilution, cellular growth started rapidly but no nucleoid partition, cell division, or chromosome replication took place until the cells had been increasing in size for several hours. Initiation of chromosome replication required that the cells first go through partition and cell division, revealing a strong interdependence between these key cell cycle events. The time points at which nucleoid partition, division, and replication occurred after the dilution were used to estimate the relative lengths of the cell cycle periods. When exponentially growing cultures were diluted into fresh growth medium, there was an unexpected transient inhibition of growth and cell division, showing that the cultures did not maintain balanced growth. Furthermore, when cultures growing at 79°C were shifted to room temperature or to ice-water baths, the cells were found to “freeze” in mid-growth. After a shift back to 79°C, growth, replication, and division rapidly resumed and the mode and kinetics of the resumption differed depending upon the nature and length of the shifts. Dilution of stationary-phase cultures provides a simple protocol for the generation of partially synchronized populations that may be used to study cell cycle-specific events.

Removal of glycosaminoglycans from cultures of human skin fibroblasts

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.

Evidence of heterogeneity of protein-turnover states in cultured cells

Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847—859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, ‘chased’ for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267—283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.