Hyperthermic radiosensitization of thermotolerant Chinese hamster ovary cells

Synchronous G1 cells were given a priming dose of heat (45.5 degrees C for 15 min) and then heated and irradiated 6-120 h later. Compared to heat radiosensitization for cells irradiated 10 min after the priming heat dose (thermal enhancement ratio, TER of 2.6 for a 10-fold reduction in survival), heat radiosensitization 18-24 h after the priming heat dose was less (i.e., TER of 1.6 for radiation at 24 h compared with heat-radiation at 24 h). A thermotolerance ratio (TTR) at 24 h was calculated to be 2.6/1.6 = 1.6. TERs at 100-fold or 1000-fold reduction in survival and ratios of slopes of radiation survival curves also showed that the cells developed a similar amount of thermotolerance for heat radiosensitization at 18-24 h. Furthermore, since the TER for heat radiosensitization increased with heat killing either from the priming heat dose or the second heat dose in a similar manner for single or fractionated doses, the TER for nonthermotolerant and thermotolerant cells was the same when related to the heat damage (i.e., amount of killing from heat alone). When the radiation response of cells heated and irradiated 6-120 h after the priming heat dose was compared with the response of cells receiving radiation only, changes in TER as a function of time after the initial priming heat dose were shown to involve: recovery of heat damage interacting with the subsequent radiation dose, thermotolerance for heat radiosensitization, and redistribution of cells surviving the first heat dose into radioresistant phases of the cell cycle. In fact, redistribution resulted in a minimal TER at 72 h for heat-radiation compared with radiation alone, instead of at 24 h where maximal thermotolerance for heat killing was observed [P. K. Holahan and W. C. Dewey, Radiat. Res. 106, 111 (1986)]. These observations are discussed relative to clinical considerations and similar results reported from in vivo experiments.     

Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique

Abstract Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. Thus, after 40 days only 0.02–0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non-culturable bacteria in soil.     

Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.     

Escherichia coli K-12 cell-cell interactions seen by time-lapse video.

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.     

Population Heterogeneity of Lactobacillus plantarum WCFS1 Microcolonies in Response to and Recovery from Acid Stress

Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium Lactobacillus plantarum grows, it acidifies the growth medium to a low pH. We have examined the growth of L. plantarum microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS-Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.     

Lipopolysaccharide O-Chain Core Region Required for Cellular Cohesion and Compaction of In Vitro and Root Biofilms Developed by Rhizobium leguminosarum

The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.     

Hyperthermic survival of Chinese hamster ovary cells as a function of cellular population density at the time of plating

The survival of synchronous G1 or asynchronous Chinese hamster ovary cells in vitro to heat treatment may depend on the cellular population density at the time of heating and/or as the cells are cultured after heating. The addition of lethally irradiated feeder cells may increase survival at 10(-3) by as much as 10- to 100-fold for a variety of conditions when cells are heated either in suspension culture or as monolayers with or without trypsinization. The protective effect associated with feeder cells appears to be associated with close cell-to-cell proximity. However, when cells are heated without trypsinization about 24 hr or later after plating, when adaptation to monolayer has occurred, the protective effect is reduced; i.e., addition of feeder cells enhances survival much less, for example, about 2- to 3-fold at 10(-2)-10(-3) survival. Also, the survival of a cell to heat is independent of whether the neighboring cell in a microcolony is destined to live or die. Finally, if protective effects associated with cell density do occur and are not controlled, serious artifacts can result as the interaction of heat and radiation is studied; for example, survival curves can be moved upward, and thus changed in shape as the number of cells plated is increased with an increase in the hyperthermic treatment or radiation dose following hyperthermia. Therefore, to understand mechanisms and to obtain information relevant to populations of cells in close proximity, such as those in vivo, these cellular population density effects should be considered and understood.     

Sequence of Events in the Digestion of Fresh Legume Leaves by Rumen Bacteria

When fresh whole leaves of six different species of forage legumes were suspended in an artificial rumen medium and inoculated with rumen bacteria, bacterial adhesion and proliferation were noted at the stomata, and penetration of the stomate by these bacteria was documented by electron microscopy. The invading bacteria adhered to surfaces within the intercellular space of the leaf and produced very extensive exopolysaccharide-enclosed microcolonies. After some of the legume leaf cell walls were disorganized and ruptured by bacterial digestion, these cells (notably, parenchyma and epidermal cells) were invaded by bacteria, with subsequent formation of intracellular microcolonies. However, other cells were neither ruptured nor colonized (notably, stomata guard cells and vascular tissue). At all stages of the digestion of intact legume leaves, the rumen bacteria grew in microcolonies composed of cells of single or mixed morphological types, and a particular ecological niche was often completely and consistently occupied by a very large microcolony of cells of single or mixed morphological types.     

On tests of equal effect per fraction in microcolony assays of survival after fractionated irradiations

H. D. Thames, Jr. and H. R. Withers [Br. J. Radiol. 53, 1071-1077 (1980)] propose a test of an equal effect per fraction in microcolony assays after fractionated radiation, in which the total effect is measured by counting microcolonies derived from surviving cells in a tissue. The factors considered to influence the cytocidal effect per fraction are incomplete repair, repopulation, and synchrony. The statistics used in the method are criticized and conditions are given under which the test should not be used. An alternative method of testing for an equal effect per fraction is proposed. The pros and cons of each test are discussed and compared using some mouse jejunal crypt cell survival data.     

Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development

Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas. Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies. In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony. Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract. In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell.     

Rapid species and strain differentiation of non-tubercoulous mycobacteria by Fourier-Transform Infrared microspectroscopy

Rapid identification of non-tuberculous mycobacteria (NTM) species is important in clinical laboratories to stipulate the appropriate therapy and to offer a comprehensive infection control. We applied Fourier-Transform Infrared microspectroscopy to evaluate, whether the most frequent species of NTM can be rapidly and uniformly identified by this method using microcolonies of NTM growing on solid nutrient agar plates. To establish a standardized protocol, the heterogeneity of cell growth within the microcolonies and the reproducibility of measuring the IR spectra from whole mycobacterial microcolonies were first studied. Hierarchical cluster analysis applied to spectra obtained by linear mapping across microcolony imprints from fast- and slow-growing NTM revealed only little spectral variance between the various microcolony zones. In parallel, when repetitive measurements were performed on independently grown whole single microcolonies with diameters of 80 and 140 mum, excellent reproducibility could be achieved, verifying that mycobacterial microcolonies are well suited for FT-IR-based identification. Twenty-eight different and well-defined strains, comprising the most frequent species of NTM isolated in clinical laboratories, were used to create a classification system based on FT-IR spectra from single microcolonies. Hierarchical cluster analysis allowed the assignment of all isolates measured in replicates to their correct species-specific clusters. Additionally, a clear separation of all strains into strain-specific sub-clusters was observed. These results demonstrate the potential of FT-IR microspectroscopy to rapidly differentiate NTM at the species and strain level. The data so far obtained suggest that an extended spectral database, containing more NTM strains and covering a broader biological variance, may provide a practical solution to rapidly identify unknown NTM isolates in routine clinical-microbiological laboratories with the additional possibility to type these microorganisms at the sub-species level.     

Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.     

Regeneration and dose-response characteristics of irradiated mouse dorsal epidermal cells.

A microcolony technique is described for measuring epidermal cell survival 3 days after whole-body X-irradiation. This assay provides a cell D0 value of 233 +/- 11 rad and a zero-dose extrapolate of 1-23 x 10(4) cells/cm2 for mice irradiated in oxygen 20 hours after hair plucking. The microcolony cellularity had an apparent doubling-time of 25 hours which may be an upper limit at least for some clones. The clones appeared to fragment continually and form new daughter clones, suggesting that few would form macroscopic nodules. Many of the clones were also apparently associated with hair follicles.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

FT-IR microspectroscopy for microbiological studies

In this article we present an infrared microspectroscopic investigation on Candida albicans microcolonies, taken as a model system for studies on other microorganisms. Excellent Fourier transform infrared (FT-IR) absorption spectra from 4000 to 850 cm(-1) have been collected in only 20 s from sampling areas of 100x100 microm(2) in microcolonies, which had been transferred from the agar plate onto zinc selenide (ZnSe) windows. When different regions within a single microcolony were investigated, absorption spectra with important differences in the carbohydrate absorption (from 1200 to 850 cm(-1)) were detected for the cells in the center and in the periphery of the colony. Results obtained on microcolonies grown on solid agar with increasing dextrose concentrations indicated that the observed spectral heterogeneity was related to differences in dextrose uptake, which was lower for the old cells in the center of the colony than for the metabolically active cells at the periphery. Although it is otherwise difficult to quantitatively evaluate the dextrose uptake in a microcolony, FT-IR absorption microspectroscopy offers a new and rapid method for the analysis of this process. The possibility of studying highly absorbing colonies by attenuated total reflection (ATR) by means of an ATR microscope germanium objective is also presented here for the first time. An evaluation of the contact area sampled by this technique is reported with a discussion of the spatial resolution, the quality and the potential of the ATR measurements.     

Chromosome aberrations and radiation-induced cell death. II. Predicted and observed cell survival

The surviving fraction of synchronous V₇₉ Chinese hamster cells was measured in two post-irradiation generations after a 300-rad X-ray dose in G₁ by comparing the colony multiplicity in irradiated and control cultures. In addition, the ability of the irradiated population to form colonies was measured immediately after G₁ irradiation or at 6, 32, 75 or 96 h after X-irradiation. Formulae were used in conjunctionwith previously observed transmission and survival parameters of chromosome aberrations² to predict the amount of cell death at any given time after irradiation. The results indicate that the survival pattern of these cells can indeed be predicted on the basis of cell loss from chromosome aberrations.It is likely that an asymmetrical chromosome exchange (dicentric, centric ring, or tricentric) and a chromosome deletion are equally capable of causing cell death, whereas translocations or inversions apparently do not lead to inviability. Furthermore, cell death is rapid: 45% of the total observed death occurs in the first two post-irradiation generations. The initial decrease in viability is caused predominantly by the formation of anaphase bridges, while cell death from fragment loss becomes increasingly important in later generations. In fact, it is probable that, on the average, a cell that loses a single acentric fragment will survive one generation.     

Role of Microcolony Formation in the Protistan Grazing Defense of the Aquatic Bacterium Pseudomonas sp. MWH1

A BSTRACTThe defense strategy of the aquatic bacterium Pseudomonas sp. MWH1 against flagellate grazing was investigated in chemostat and batch experiments. The influence of predation on the Pseudomonas population was studied in the absence and presence of a potential competitor ( Vibrio sp. CB5), as well as under starvation conditions and in a situation of unlimited growth. In the competition experiment the two bacterial strains were distinguished by immunofluorescence microscopy. When the Pseudomonas strain was cultured in the absence of the predator Ochromonas sp. DS, only mobile single cells were detectable. Grazing by this bacterivorous flagellate resulted in all experiments in the occurrence of a Pseudomonas subpopulation, which grew as floclike, suspended microcolonies. These microcolonies consisted of up to approximately 1,000 cells and were, because of their large size, protected against flagellate grazing. The microcolony subpopulation dominated the total Pseudomonas population in situations of high grazing pressure at a wide range of bacterial growth conditions. Thus, the formation of the microcolonies is interpreted as a successful grazing-defense strategy, which is effective under several growth conditions, allowing for the survival of the strain even when substrate depletion is combined with strong grazing pressure. Batch culture experiments demonstrated that the change in morphology of Pseudomonas sp. MWH1 is not controlled by growth rate, although no formation of microcolonies was observed after the addition of 0.2-&mgr;m-filtered flagellate cultures to Pseudomonas cultures, indicating that a chemical trigger released by the flagellate is not involved in the control of this defense mechanism.     

Bacterial microcolony--a possible approach for a rapid differentiation of bacteria

A method is developed for cultivating and observing bacteria in an early phase of their growth, when microcolonies were formed. The morphology of the microcolonies, including form, structure, characteristics of its periphery and center, as well as the mode of arrangement of bacteria in it and to each other proved to be typical for a species and often permitted its differentiation from the other species. Photographs are presented of typical microcolonies of S. aureus and of E. coli. A series of photographs is presented also as an illustration of the possibility to differentiate some species in the genus Streptococcus. The microcolonies observation is made 3 hours after material inoculation, that may permit a rapid bacteriological diagnosis. It is believed also, that the microcolony technique could be useful in the characterisation and identification of the species in the general bacteriology and taxonomy.     

Microcolony imaging of Aspergillus fumigatus treated with echinocandins reveals both fungistatic and fungicidal activities

Background

The echinocandins are lipopeptides that can be employed as antifungal drugs that inhibit the synthesis of 1,3-β-glucans within the fungal cell wall. Anidulafungin and caspofungin are echinocandins used in the treatment of Candida infections and have activity against other fungi including Aspergillus fumigatus. The echinocandins are generally considered fungistatic against Aspergillus species.

Methods

Culture of A. fumigatus from conidia to microcolonies on a support of porous aluminium oxide (PAO), combined with fluorescence microscopy and scanning electron microscopy, was used to investigate the effects of anidulafungin and caspofungin. The PAO was an effective matrix for conidial germination and microcolony growth. Additionally, PAO supports could be moved between agar plates containing different concentrations of echinocandins to change dosage and to investigate the recovery of fungal microcolonies from these drugs. Culture on PAO combined with microscopy and image analysis permits quantitative studies on microcolony growth with the flexibility of adding or removing antifungal agents, dyes, fixatives or osmotic stresses during growth with minimal disturbance of fungal microcolonies.

Significance

Anidulafungin and caspofungin reduced but did not halt growth at the microcony level; additionally both drugs killed individual cells, particularly at concentrations around the MIC. Intact but not lysed cells showed rapid recovery when the drugs were removed. The classification of these drugs as either fungistatic or fungicidal is simplistic. Microcolony analysis on PAO appears to be a valuable tool to investigate the action of antifungal agents.