CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy

A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized.     

Changes in the biofilm microflora of limestone caused by atmospheric pollutants

Historic limestone materials in urban environments are continually exposed to air pollutants, including sulfur compounds and hydrocarbons. We investigated the effects of air pollution on the biofilm microflora of historic limestone gravestones located at two locations Massachusetts, USA. Our data showed that the culturable populations of chemolithotrophic and heterotrophic bacteria, and fungi were suppressed in the polluted habitat comparing with the unpolluted location. The diversity of the microflora was also reduced in the surface biofilms on gravestones in the city contaminated by air pollution. However, both the sulfur-oxidizing and hydrocarbon-utilizing microflora were enriched in the biofilms exposed to air pollution. In a laboratory study, low concentrations of the polluting chemicals stimulated growth of these bacteria, and resulted in rapid acid production. Scanning electron microscopy demonstrated that the biofilms of both the sulfur-oxidizing bacteria and the hydrocarbon-degrading microflora penetrated into the limestone. The enrichment of sulfur- and hydrocarbon-utilizing bacteria in the biofilms may contribute to dissolution of the stone. However, further research is required to determine the effects of specific metabolites of these microorganisms on stone deterioration.     

Synthetic Consolidants Attacked by Melanin-Producing Fungi: Case Study of the Biodeterioration of Milan (Italy) Cathedral Marble Treated with Acrylics

Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.     

Synthetic consolidants attacked by melanin-producing fungi: case study of the biodeterioration of Milan (Italy) cathedral marble treated with acrylics

Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.     

Distribution of microbes producing antimicrobial epsilon-poly-L-lysine polymers in soil microflora determined by a novel method

We developed a simple and sensitive screening method to investigate the distribution of microbes producing an antimicrobial poly(amino acid), epsilon-poly-L-lysine (epsilon-PL), in microflora. An acidic dye, Poly R-478, incorporated in an agar plate detected epsilon-PL producers by electrostatic interaction with the secreted basic polymers. All epsilon-PL producers, isolated after careful and sufficient screening of soil microflora, belonged exclusively to two groups of bacteria of the family Streptomycetaceae and ergot fungi. They were characterized based on the density and diameter of the concentric zone formed by the secreted polymers. The density depended on each isolate. The increase in the diameter of the concentric zone per unit of time varied among isolates and was negatively correlated with the molecular weight. Although the distribution of epsilon-PL producers was extremely limited, their products were structurally varied. The molecular masses of the secreted polymers among the isolates ranged from 0.8 to 2.0 kDa. There were also isolates producing unknown polymers inconsistent with the correlation or producing a mixture of polymers with original and modified structures. A chemically modified polymer was an epsilon-PL derivative, as determined by mass spectrometry. Since the structural variations had no relation to the phylogenetic position of the isolates, it is possible that enzymes involved in the synthesis diversified after putative horizontal transfers of relevant genes.     

Biodeterioration of natural stone with special reference to nitrifying bacteria

An evaluation of field data from historical buildings in Germany showed that chemoorganotrophic bacteria are the most numerous microorganisms in building stones, followed by fungi and nitrifying bacteria. Chemoorganotrophic bacteria and fungi were present in almost every sample. Ammonia and nitrite oxidizers were found in 55 and 62% of the samples, respectively. Within months, natural stone was colonized by chemoorganotrophic microorganisms. The highest cell numbers were usually found near the surface. The colonization of natural stone by nitrifying bacteria took several years. The highest cell numbers were in some cases found underneath the surface. Nitrifying bacteria showed a preference for calcareous material with a medium pore radius between 1 and 10 m. Cell numbers of nitrifying bacteria did not correlate to the nitrate content of the stone material. We demonstrated that the stone inhabiting microflora can cause significant loss of nitrate by denitrification. Our data strongly suggested that microbial colonization of historical buildings was enhanced by anthropogenic air pollution. Samples taken from stone material with a pore radius 1 m had significantly higher cell numbers when they were covered with black crusts. A comparison of samples taken between 1990–1995 from buildings throughout Germany showed that in eastern Germany a significantly stronger colonization with facultatively methylotrophic bacteria and nitrifying bacteria existed. The same was true for natural stone from an urban exposure site when compared to material from a rural exposure site. Data from outdoor exposure and laboratory simulation experiments indicated that the colonization of calcareous stone by nitrifying bacteria was enhanced by chemical weathering.     

Evaluation of the effect of some biocides against organisms isolated from historic monuments

On the surfaces of monuments and buildings, organic and inorganic pollutants accumulate, as well as various microbial communities which contribute to stone decay. In order to control these organisms, we have tested some chemical products with biocide and water-repellent properties. Some of these products were tested in an agar diffusion test and on limestone slabs. Efficacy of the products and the microbial inhibition were studied with scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) techniques. This revised version was published online in June 2006 with corrections to the Cover Date.     

Airborne microorganisms in a subterranean archaeological area of the basilica of San Lorenzo in Lucina (Rome)

The basilica of San Lorenzo in Lucina in Rome was built in the fifth century on the foundations of a Roman insula used for dwellings and commercial activities, where the first Christian communities had held their meetings. This archaeological area is still rather isolated since it can only be visited by groups for one/two hours once a month. Such a situation is ideal for studying both the quantity of microorganisms in the air under undisturbed conditions and/or conveyed by visitors. The biodeterioration of subterranean remains is determined by the development of bacteria, cyanobacteria, actinomycetes, algae and fungi. On the walls of this underground archaeological area the main form of alteration is the efflorescence. In order to compare the stone and airborne microflora, the microorganisms living on stone and the airborne ones have been detected. Air samples were taken both during the visits and under non-turbulent conditions utilizing three sampling methods: gravitational, intake by multi-stage impact and intake by single-stage impact. The results have made it possible to determine the microbial content of the air, as well as the sampling schedule required for rarely visited underground environments. Furthermore, a comparison of the three methods used showed that the adoption of one or the other sampling method gave different complementary information on airborne microflora. This revised version was published online in June 2006 with corrections to the Cover Date.     

Incidence of marine and mangrove bacteria accumulating polyhydroxyalkanoates on the mid-west coast of India

The tropical marine and mangrove microflora from the mid-west coast of India were found to be diverse with respect to enzymatic activities. The bacterial counts and the enzyme activities of the mangrove microflora were found to be influenced by the seasonal variations. The microflora of these ecosystems also possessed a high potential to accumulate important polymers such as polyhydroxyalkanoates (PHA). Out of a total of 866 bacterial cultures isolated from this region, 337 cultures were scored positive for PHA production. Amongst these isolates, seven cultures accumulated more than 1 g of PHA per litre of culture broth. The TLC profiles of the methyl esters of PHA from the seven cultures showed varied profiles. The sediment samples also showed the presence of PHA with five different monomeric units.     

Validation of fluorescent in situ hybridization combined with flow cytometry for assessing interindividual variation in the composition of human fecal microflora during long-term storage of samples

This work was conducted to assess the accuracy of in situ hybridization to show differences in human microflora composition between volunteers and to optimize the storage of fecal samples to allow delayed analysis of gut microflora composition in humans. Fecal samples from 25 healthy subjects (14 women, 11 men aged 24-51) were collected. The samples were fixed in 4% Paraformaldehyde (PFA) solution at 4 degrees C overnight and stored at -70 degrees C. Twenty samples were analysed to quantify the variation due to interindividual differences in the composition of fecal microflora. The five remaining samples were stored either after PFA fixation or directly frozen at -70 degrees C and were monitored on a 12-month period. The fecal microflora was analysed by in situ hybridization combined with flow cytometry detection. Ribosomal RNA-targeted probes were used to assess the relative proportions of four phylogenetic groups: Clostridium coccoides-Eubacterium rectale (Erec 482), Bacteroides (Bac 303), Faecalibacterium prausnitzii (Fprau 645) and Bifidobacterium (Bif 164). Our results demonstrated that the method used is adapted to detect significant differences in fecal microflora composition in humans. Moreover, samples stored in PFA solution demonstrated a stable composition even after 8 months of storage. Conversely, frozen samples were less stable as the Bifidobacterium and C. coccoides-E. rectale groups showed significant differences after 2 months of storage. In conclusion, the fecal microflora composition can be analysed up to 8 months after 4% PFA fixation and storage at -70 degrees C. It represents an extended time compared with the 2-month period currently recommended. This will give more flexibility for applying this technology in epidemiological studies including a large number of samples.     

Microbial interaction in cooked cured meat products under vacuum or modified atmosphere at 4 degrees C

AIMS: To investigate the antagonistic activity of two lactic acid strains against the spoilage microflora in cooked cured meat products, vacuum or modified atmosphere packed at 4 degrees C and to determine the inhibitory capacity of their bacteriocins. METHODS AND RESULTS: Frankfurter-type sausages and sliced cooked cured pork shoulder were inoculated with Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 or with their bacteriocins. The microbial, physico-chemical (pH, L- and D-lactate, acetate and ammonia) and colour changes were studied. Results under vacuum packaging showed that in the uninoculated samples of the pork product the spoilage microflora grew but in the inoculated ones the spoilage microorganisms (e.g. Brochothrix thermosphacta and enterococci) reduced during the storage. This observation was more pronounced in the samples with the addition of bacteriocins. In the frankfurter-type sausages the spoilage microflora did not grow in the uninoculated and inoculated samples. In the modified atmosphere enriched in CO2 the population of spoilage microflora remained at low levels in both products, indicating that CO2 has an effect on the spoilage microorganisms' growth. In the pork product the concentrations of acetate and d-lactate increased while L-lactate decreased, but in the frankfurter-type sausages increase of acetate and D-lactate was not observed. CONCLUSIONS: Lactic acid strains had an effect on the spoilage microflora growth but did not affect, negatively, the organoleptic properties of the products. These strains may be used as biopreservative cultures or their bacteriocins could be an important contribution to microbiological quality of meat products. SIGNIFICANCE AND IMPACT OF STUDY: Establishment of biopreservation as a method for extension of shelf life of meat products.     

Fountain Flow cytometry, a new technique for the rapid detection and enumeration of microorganisms in aqueous samples.

BACKGROUND: Pathogenic microorganisms are known to cause widespread waterborne disease worldwide. There is an urgent need to develop a technique for the real-time detection of pathogens in environmental samples at low concentrations, <10 microorganisms/ml, in large sample volumes, > or =100 ml. METHODS: A novel method, Fountain Flowtrade mark cytometry, for the rapid and sensitive detection of individual microorganisms in aqueous samples is presented. Each sample is first incubated with a fluorescent label and then passed as a stream in front of a laser, which excites the label. The fluorescence is detected with a CCD imager as the sample flows toward the imager along its optical axis. The feasibility of Fountain Flow cytometry (FFC) is demonstrated by the detection of Escherichia coli labeled with ChemChrome CV6 and SYBR Gold in buffer and natural river water. RESULTS: Detections of labeled E. coli were made in aqueous suspensions with an efficiency of 96% +/- 14% down to a concentration approximately 200 bacteria/ml. CONCLUSIONS: The feasibility of FFC is demonstrated by the detection of E. coli in buffer and natural river water. FFC should apply to the detection of a wide range of pathogenic microorganisms including amoebae.     

Quantitative assessment of vaginal microflora during use of tampons of various compositions

Although the effect of vaginal tampons on the microbial flora during menstruation has recently been studied by several investigators, quantitative effects attributable to particular tampon fibers have received less attention. The purposes of the present study were (i) to determine and then to compare the effects of polyacrylate rayon tampons and viscose rayon tampons on the normal vaginal flora, (ii) to compare quantitative bacterial counts obtained from these tampons with those obtained from concomitant vaginal swabs, and (iii) to determine whether either of these tampon types alters the vaginal microflora when compared with the microflora in the same women using all-cotton tampons or external catamenial pads. Tampon and swab samples were obtained at predetermined times from 18 women for an average of seven menstrual cycles. Samples consisting of swabs from women wearing menstrual pads were compared with swab and tampon samples taken at predetermined times during the menstrual cycle from women using cotton, polyacrylate rayon, or viscose rayon tampons. Samples were analyzed for total aerobic, facultative, and anaerobic bacterial counts. Statistical evaluation of the results indicated that, on the whole, total bacterial counts decreased during menstruation and that the numbers of bacteria in tampons tended to be lower than those in swab samples taken at the same time. The tampon type had little effect on the vaginal microflora.     

Quantitative assessment of vaginal microflora during use of tampons of various compositions.

Although the effect of vaginal tampons on the microbial flora during menstruation has recently been studied by several investigators, quantitative effects attributable to particular tampon fibers have received less attention. The purposes of the present study were (i) to determine and then to compare the effects of polyacrylate rayon tampons and viscose rayon tampons on the normal vaginal flora, (ii) to compare quantitative bacterial counts obtained from these tampons with those obtained from concomitant vaginal swabs, and (iii) to determine whether either of these tampon types alters the vaginal microflora when compared with the microflora in the same women using all-cotton tampons or external catamenial pads. Tampon and swab samples were obtained at predetermined times from 18 women for an average of seven menstrual cycles. Samples consisting of swabs from women wearing menstrual pads were compared with swab and tampon samples taken at predetermined times during the menstrual cycle from women using cotton, polyacrylate rayon, or viscose rayon tampons. Samples were analyzed for total aerobic, facultative, and anaerobic bacterial counts. Statistical evaluation of the results indicated that, on the whole, total bacterial counts decreased during menstruation and that the numbers of bacteria in tampons tended to be lower than those in swab samples taken at the same time. The tampon type had little effect on the vaginal microflora.     

The influence of air pollution on the phyllosphere microflora composition of Tillandsia leaves (Bromeliaceae)

The effect of air pollution on total phyllospheric microflora from two species of the epiphytic neotropical genus Tillandsia (Bromeliaceae) was studied by comparing unpolluted plants living in a forest (Escazú, San José) with polluted ones from an urban site of Costa Rica (San José city). Dilutions of homogenized leaf samples were plated on media suitable for each microbial group. For each microorganism group, total counts were performed and purified strains of randomly chosen colonies were identified. There was a global reduction in the number of living microorganisms due to pollution effects, especially yeasts and bacteria, while nitrogen-fixing microorganisms and fungi were less affected. Our results showed that the phyllosphere microflora of Tillandsia plants living in a tropical urban environment changes in terms of number and species composition of yeasts and bacteria with respect to plants living in unpolluted environment.     

An uncooked vegan diet shifts the profile of human fecal microflora: computerized analysis of direct stool sample gas-liquid chromatography profiles of bacterial cellular fatty acids.

The effect of an uncooked extreme vegan diet on fecal microflora was studied by direct stool sample gas-liquid chromatography (GLC) of bacterial cellular fatty acids and by quantitative bacterial culture by using classical microbiological techniques of isolation, identification, and enumeration of different bacterial species. Eighteen volunteers were divided randomly into two groups. The test group received an uncooked vegan diet for 1 month and a conventional diet of mixed Western type for the other month of the study. The control group consumed a conventional diet throughout the study period. Stool samples were collected. Bacterial cellular fatty acids were extracted directly from the stool samples and measured by GLC. Computerized analysis of the resulting fatty acid profiles was performed. Such a profile represents all bacterial cellular fatty acids in a sample and thus reflects its microflora and can be used to detect changes, differences, or similarities of bacterial flora between individual samples or sample groups. GLC profiles changed significantly in the test group after the induction and discontinuation of the vegan diet but not in the control group at any time, whereas quantitative bacterial culture did not detect any significant change in fecal bacteriology in either of the groups. The results suggest that an uncooked extreme vegan diet alters the fecal bacterial flora significantly when it is measured by direct stool sample GLC of bacterial fatty acids.     

Effects of low electric treatment on yeast microflora

AIMS: To contribute to the understanding of phenomena related to different intensity electric current treatments on the growth and metabolism of selected micro-organisms using laboratory samples of pure and co-cultures (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermondii strain 465). METHODS AND RESULTS: Low electric current (10, 30, 50 and 100 mA) was applied to prepared samples. Parameters, such as polarity, treatment duration (18-48 h) and type of inoculum yeast, were varied one at a time to highlight their cause-effect relationships. The effects on cell activity as well as microflora viability were assessed. Bioindicators capable of describing the phenomena caused by the electric current on the microflora were identified. CONCLUSIONS: Results demonstrated that a low voltage treatment using graphite electrodes had a greater effect on the viable S. cerevisiae strain 404 microflora. There was less bactericidal activity in the S. cerevisiae strain 404 than in the H. guilliermondii strain 465. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be of significant importance in the development of new technological processes in the fields of agriculture and food, particularly new fermenting process controls.     

Characterization and Distribution of Water-repellent, Self-cleaning Plant Surfaces

During the last 20 years, a wealth of data dealing with scanningelectron microscopy of plant surfaces has been published. Theultrastructure of epidermal surfaces has been investigated withrespect to taxonomic, as well as functional aspects. Withinthe latter, water-repellency has received much attention andhas been well documented. Water-repellency is based on surfaceroughness caused by different microstructures (trichomes, cuticularfolds and wax crystals), together with the hydrophobic propertiesof the epicuticular wax. In addition, contaminating particlesare carried away by water droplets, resulting in a cleaned surface(Lotus-effect). Therefore, rough, waxy leaves are not only water-repellentbut anti-adhesive with respect to particulate contamination.Based on 200 water-repellent plant species, the present papersurveys micromorphological characteristics of anti-adhesiveplant surfaces. Leaves that are permanently water-repellentcan be differentiated by distictively convex to papillose epidermalcells and a very dense layer of epicuticular waxes. Leaves thatare water-repellent for only a limited period of time have onlyslightly convex epidermal cells and often have a less densewax layer. Finally, an overview is given on the occurrence ofwater-repellency among different life forms and within differenthabitats. Water-repellency is concentrated in herbaceous species,while it is rare in trees. Among different habitats, subtropicalregions, wetlands and disturbed areas appear to have more specieswith water-repellent leaves. The importance of roughness andwater-repellency, respectively, as the basis of an anti-adhesive,self-cleaning surface, in comparison to other functions of microstructures,is discussed.     

Intestinal Microflora Associated with Enteritis of Early-Weaned Pigs

Studies were conducted on the enumeration of 7 groups of fecal microflora including total aerobes, total anaerobes, coliforms, lactobacilli, staphylococci, streptococci and yeasts and molds of 18-day old piglets. These pigs were early weaned (21 days) on different modifications of an early-weaning ration. The above mentioned microflora were enumerated again when some of the pigs in a replicate started scouring. The occurrence of diarrhea was always associated with significant increases in the numbers of coliforms and corresponding decreases in the lactobacilli counts. No other single group of fecal microflora differed in the scouring and non-scouring animals. The composition of the early-weaning ration offered to the animals did not, in itself, influence the fecal microflora to any appreciable extent. In another series of experiments, enumeration of coliforms and lactobacilli was conducted on samples obtained from different segments of the intestinal tracts of scouring and non-scouring pigs. Increased numbers of coliforms and decreased numbers of lactobacilli were observed at all levels of the intestinal tracts of the scouring animals. However, these changes were more marked in the duodenal samples than in those obtained from other parts of the intestine.     

Diversity of probiotic adhesion genes in the gastrointestinal tract of goats

Gastrointestinal (GI) microflora is an important system in the host, as it has both pathogenic and probiotic bacteria. Most of the studies were focused on the human gut microflora and the available information on the intestinal microflora of goats was limited. This urged the need to inspect the impacts of the goat's gut microflora. Metagenomic investigation of probiotic bacteria in the GI tract of goat is one of the challenging streams because of the less available data of the uncultivable bacteria. In our report, comparative analysis of metagenomic and enrichment samples of goat intestinal content was done and this approach will be helpful in analyzing the identification of uncultivable and cultivable probiotic bacteria. This study mainly focused on three key probiotic adhesion genes, such as EF-Tu, mapA, and mub. The GI of four different goats were investigated for these genes. The data from this study showed that there is a wide diversity of these genes among goat intestinal samples.