text:
- Research article
- Open Access
Diversity and seasonal dynamics of bacterial community in indoor environment
© Rintala et al; licensee BioMed Central Ltd. 2008
- Received: 09 October 2007
- Accepted: 08 April 2008
- Published: 08 April 2008
We spend most of our lives in indoor environments and are exposed to microbes present in these environments. Hence, knowledge about this exposure is important for understanding how it impacts on human health. However, the bacterial flora in indoor environments has been only fragmentarily explored and mostly using culture methods. The application of molecular methods previously utilised in other environments has resulted in a substantial increase in our awareness of microbial diversity.
The composition and dynamics of indoor dust bacterial flora were investigated in two buildings over a period of one year. Four samples were taken in each building, corresponding to the four seasons, and 16S rDNA libraries were constructed. A total of 893 clones were analysed and 283 distinct operational taxonomic units (OTUs) detected among them using 97% sequence similarity as the criterion. All libraries were dominated by Gram-positive sequences, with the most abundant phylum being Firmicutes. Four OTUs having high similarity to Corynebacterium-, Propionibacterium-, Streptococcus- and Staphylococcus- sequences were present in all samples. The most abundant of the Gram-negative OTUs were members of the family Sphingomonadaceae, followed by Oxalobacteraceae, Comamonadaceae, Neisseriaceae and Rhizobiaceae.
The relative abundance of alpha- and betaproteobacteria increased slightly towards summer at the expense of firmicutes. The proportion of firmicutes and gammaproteobacteria of the total diversity was highest in winter and that of actinobacteria, alpha- and betaproteobacteria in spring or summer, whereas the diversity of bacteroidetes peaked in fall. A statistical comparison of the libraries revealed that the bacterial flora of the two buildings differed during all seasons except spring, but differences between seasons within one building were not that clear, indicating that differences between the buildings were greater than the differences between seasons.
This work demonstrated that the bacterial flora of indoor dust is complex and dominated by Gram-positive species. The dominant phylotypes most probably originated from users of the building. Seasonal variation was observed as proportional changes of the phyla and at the species level. The microflora of the two buildings investigated differed statistically and differences between the buildings were more pronounced than differences between seasons.
- Clone Library
- Indoor Environment
- House Dust
- Dust Sample
- Bacterial Flora
We spend most of our lives in different indoor environments; in homes, day-care facilities, schools and workplaces, and are continuously challenged by the microbial content of these environments. Traditionally, infections are considered as the main health effect of bacteria; however, these micro-organisms can affect our health in many other ways. During infancy, contact with bacteria in the environment leads to development of the indigenous human microflora, the importance of which for human health has been acknowledged but is still not fully understood [1]. Microbial exposure in childhood also prevents the development of allergic conditions [2, 3]. Later in life, microbial exposure in indoor environments can also affect human health; for example, moisture damage in a building can lead to increased indoor microbial levels and this can have adverse health effects [4].
New questions concerning environmental microbial exposure have emerged lately. For example, the numbers of community acquired MRSA infections among healthy individuals have risen rapidly in recent years [5], showing that there are reservoirs of this pathogen outside of hospitals. Although infectious agents normally cannot persist for a long time in air, a recent study has shown that antibiotic-resistant Staphylococcus aureus is common in indoor air of normal residential buildings [6]. Against this background, it is surprising how little we know about the composition of the microbial communities in indoor environments. The information is fragmentary and mostly based on cultivation methods, although it has been claimed that only approximately 1% of the microbes in indoor environments are viable [7].
Most of the literature about bacteria in normal indoor environments is concerned with cultivation of air samples. Indoor dust samples have rarely been investigated by cultivation or methods permitting identification of bacterial species. In healthy offices, Enterococcus, Staphylococcus, Pantoea and Pseudomonas were the most abundant bacterial genera found in carpet dust [8]. Based on the results of cultivation methods, it seems that both Gram-positive and Gram-negative bacteria are common in indoor air of residential settings, office buildings and hospitals [8–11]. There are several frequently encountered taxa; Gram-positive cocci, corynebacteria, bacilli, in addition to Gram-negative species, such as Acinetobacter and Pseudomonas.
However, Gram-positive bacteria dominate the flora, at least in studies using culture methods, comprising up to 75% of the bacteria present in the indoor air [9]. Certain Gram-positive bacteria with strong immunogenic properties or potential toxin producers, such as mycobacteria, streptomycetes or Nocardiopsis spp., have also been shown to be present in indoor environments [12–14]. In a mouse model, these bacteria induced inflammation in vitro and in vivo [15, 16].
Cultivation methods can only detect a small fraction of microbial flora, depending on the culture conditions being used and the viability of the microbes. With respect to indoor environments, the cultivability has been reported to vary between 0.03% to 100%, depending on the sampling method and microbial species, for example [7, 17]. Bacteria are less resistant to sampling stress than fungi. It would be predicted that application of molecular methods will change the current picture of what bacteria are present in indoor environments. Sequencing of 16S ribosomal DNA clone libraries has been successfully used for characterisation of bacterial flora in soil, marine habitats and human intestine [18–20]. The advantage of this approach is that all microbial species should be detected equally and the limitations of culture conditions can be overcome. One major benefit is that one can make a relatively exact phylogenetic placement of the detected DNA sequences with respect to reference sequences in public databases. Recent advances in statistical analysis methods of clone libraries [21–23] allow one to conduct a more sophisticated assessment of the differences in microbial communities between samples.
Recently, two studies using culture-independent methods to investigate bacterial flora in indoor environments have been published [24, 25]. Lee et al. (2007) investigated the bacterial diversity in a child-care facility using both culture-based and molecular methods. In that study, Pseudomonadaceae and Oxalobacteraceae were the most dominant families in the clone libraries, though Gram-positive genera, such as Bacillus, Streptococcus and Staphylococcus were also present. The most commonly cultured bacteria were Bacillus spp. and Staphylococcus spp. The authors found 29 putative bacterial species among the isolates, compared to 141 distinct OTUs using 3% sequence divergence in the clone libraries. Kelley et al. (2004) studied biofilms present on shower curtains using 16S rDNA clone libraries and found that this microenvironment maintains a diverse microbial community. In that study, sequences representing Sphingomonas spp. and Methylobacterium spp. were the most often encountered. Already these two examples show that the microbial community in indoor environments is more diverse than would have been suspected from reports of culture studies. In a study of the outdoor air microbes present in two cities in USA using molecular methods, the most common bacteria found in the outdoor air were acidobacteria, verrucomicrobia, bacilli, clostridia and some proteobacteria, such as Sphingomonas-species [26].
Indoor microbial samples can be taken from surfaces as swab samples, from indoor air or dust. House dust is a reservoir for all kinds of pollutants and can be considered as a long-term integrated sample of indoor particulate matter that has, at least partly, been airborne [27]. Dust is relatively easy to collect for analysis and in the last years, dust samples have been extensively used in epidemiological studies to characterise human exposure to environmental microbial material [28, 29], hence dust samples are widely used and provide a good picture of the total microbial exposure in indoor environments. In many epidemiological studies, the endotoxin level in dust samples has been used as surrogate for exposure to environmental microbes. However, measurement of endotoxin activity does not provide an accurate assessment of the total microbial, or even the bacterial exposure. Therefore, more information about the actual species diversity in indoor dust is needed to allow a better assessment of the importance of indoor bacteria for human health. It has been shown that fungal concentrations in indoor air and dust vary temporally [30–32] and thus, the sampling time and season can affect the results of an indoor microbial analysis. Seasonal variation of cultivable levels of bacteria in indoor air has also been observed; though no clear pattern has emerged [9]. Seasonal changes in the bacterial flora of house dust have not been investigated earlier; this is a gap in the fundamental knowledge needed for exposure assessment.
The aim of this study was to investigate the species level diversity and seasonal dynamics of bacterial flora in indoor dust by sequencing of 16S rDNA clone libraries. Dust samples were collected from hard indoor surfaces including floor, tables and shelves by vacuum cleaning. Office rooms in two buildings were sampled at different times during one year to obtain four samples per building, one for each season. Eight 16S rDNA clone libraries were constructed and approximately 100 sequences per library analysed. The results indicated a high amount of Gram-positive species and human-derived sequences in indoor dust and furthermore that variation between buildings was more pronounced that variation between seasons. To our knowledge, this is the first study where full length 16S clone libraries have been used to describe in detail the diversity of indoor bacterial populations.
Bacterial diversity in house dust
Characteristics of the clone libraries.
No. of clones
S obs
S Chao
Coverage ACE (%)
Coverage Chao (%)
1 all clones
2 all clones
All of the sequences have been submitted to the European Molecular Biology Laboratory (EMBL) sequence database. The accession numbers are the following; sequences from Building 1, winter sample (clones BF0001A001-102): AM696714–AM696815, Building 2, winter sample (clones BF0002A001-159): AM696816–AM696974, Building 1, spring sample (clones BF0001B001-76): AM696975–AM697050, Building 2, spring sample (clones BF0002B001-109): AM697051–AM697159, Building 1, summer sample (clones BF0001C001-144): AM697160–AM697303, Building 2, summer sample (clones BF0002C001-82): AM697304–AM697385, Building 1, fall sample (clones BF0001D001-104): AM697386–AM697489, Building 2, fall sample (clones BF0002D001-117): AM697490–AM697606.
Seasonal dynamics
Statistical comparison of the clone libraries.
ΔCXY Libshuff
p XY Libshuff
ΔCYX Libshuff
p YX Libshuff
p Unifrac
Comparison of seasons:
1W vs. 1Sp
< 0.001*
1W vs. 1Su
< 0.001*
1W vs. 1F
< 0.001*
1Sp vs. 1Su
1Sp vs. 1F
1Su vs. 1F
< 0.001*
2W vs. 2Sp
2W vs. 2Su
2W vs. 2F
2Sp vs. 2Su
2Sp vs. 2F
2Su vs. 2F
Comparison of buildings:
1W vs. 2W
< 0.001*
1Sp vs. 2Sp
1Su vs. 2Su
1F vs. 2F
< 0.001*
The seasonal fluctuation in the actual bacterial flora within one building at the species level was large; less than one percent of the OTUs were found in more than two seasons and over 75% of all OTUs in one building were found in only one sample. Therefore, differences at the higher taxonomic levels, such as the class level, were investigated. The percentual proportion of low GC Gram-positive and actinobacterial OTUs remained almost the same during all seasons in Building 2. With the exception of a slight decrease in alphaproteobacterial and an increase in the gammaproteobacterial diversity in the winter sample, the differences between seasons were small. In Building 1, the seasonal differences in the proportions of different bacterial classes were clearer. The amount of actinobacterial OTUs was higher in the spring sample and that of alpha- and betaproteobacteria in the summer sample. The numbers of alpha- and betaproteobacterial OTUs were highest in spring and summer samples in both buildings.
Comparison of the two buildings
When combining all sequences obtained from one building into a composite sample and comparing the two buildings with each other with the Libshuff program, the libraries were statistically different (Table 2). This difference was confirmed by both Libshuff and Unifrac in comparisons of the buildings over the individual seasons, with the exception of spring (Table 2). In the Unifrac cluster environments and PCA analyses, the buildings were clearly separated from each other (Figures 3 and 4). The Shannon and Simpson diversity indices pointed a higher diversity in Building 1 throughout the year, except in winter.
From the 283 OTUs found, 56 were present in both buildings; 15 of them were of actinobacterial origin, 28 firmicutes, six were alphaproteobacteria, five OTUs were affiliated with betaproteobacteria and two with gammaproteobacteria. The actinobacterial OTUs comprised sequences with similarity to coryne- (98–99%), propioni- (99%), and microbacteria (96–97%) as well as micrococci (96–99%), Actinomyces viscosus (98%), Nocardioides oleivorans (99%) and Friedmanniella spumicola (99%). The OTUs affiliating with the phylum Firmicutes included mostly OTUs having their closest matches among staphylo- (99%), strepto- (99%) and peptostreptococci (96–99%), lactobacilli (99%) and uncultured species from human colon (97–99%), in addition to some OTUs having 99% sequence similarity to Ruminococcus, Enterobacterium, Enterococcus, Selenomonas and Veillonella species. The alphaproteobacterial sequences present in both buildings were similar to Methylobacterium organophilum (97%), Rhizobium (97–98%) and Sphingomonas species (98–99%), betaproteobacterial sequences affiliated with Neisseria spp. (99%), Variovorax spp. (99%) and Janthinobacterium spp. (97%) and gammaproteobacterial OTUs had a high sequence similarity to Shigella sonnei (99%) and Stenotrophomonas maltophilia (99%). Four OTUs were observed throughout the year in both buildings. These had 99% sequence similarity to Corynebacterium spp., Propionibacterium acnes, Staphylococcus epidermidis and Streptococcus thermophilus (see Additional file 1).
Differences in the bacterial flora between the two buildings were mainly due to OTUs consisting of one sequence; in all samples, there were 14–27 singletons that explained 66–74% of the variation between buildings. If it is assumed that occurrence of one sequence type in several libraries from the same building indicates that it originates from an indoor source, there were few such OTUs present, and none of them were present in all four samples. With respect to the sequence types that were present during more than one season, 14 were detected only in Building 1. These were clones affiliating with Microbacteriaceae, unclassified Frankinae, Rhizobiaceae, Oxalobacteraceae, Clostridiaceae, Staphylococcaceae and uncultured Firmicutes. The only phylotype in Building 1 detected in three samples had 99% sequence similarity to Bacillus cereus. In Building 2, there were 13 OTUs detected only in that building in more than one sample; for four of them their closest matches were Kocuria palustris (99%), Aerococcus viridans (99%), uncultured Deinococcus (96%) and uncultured bacterium from human vagina (98%), were detected in three samples.
The molecular diversity of the bacterial flora in indoor environment was monitored in two buildings over all four seasons using 16S rDNA clone libraries derived from dust samples. As far as we are aware, this is the first study to have used 16S clone libraries in the characterization of the indoor dust bacterial flora. Two other papers have been recently published, one investigating shower curtains and one studying cotton swab surface samples from a child-care facility [24, 25]. In this work, dust was chosen as the sample material because it is widely used in studies involving indoor environments and can be considered as a long-term integrated sample of the pollutants that have been airborne [27]. The disadvantage of dust samples is that they are a complex mixture of microbes originating from many different sources including the outdoor air. However, this study intended to gather basic information about the bacterial flora present in an indoor environment with all the possible sources, and dust samples serve that purpose well. Dust was collected only from hard surfaces, such as hard floor, tables and shelves, since carpets and textile covered furniture can retain microbes and interfere with the assessment of seasonal variation. Altogether 893 clone sequences were analyzed and within them, 283 distinct OTUs were detected using 3% sequence difference as the criterion. Lee et al. (2007) sequenced 453 clones in cotton swab samples from a child-care facility and found 141 bacterial species using the 3% divergence level, i.e. almost exactly the same proportion as in our study. The Shannon and Simpson diversity indices suggested highest diversity in the spring samples and lowest in the winter samples in both buildings. The Shannon index gives more weight to the rare species and Simpson to the dominant [33], but in this case they were concordant. The ACE and Chao estimators did not agree with Shannon and Simpson in all cases. The Chao estimator takes into account only singletons and doubletons, ACE uses OTUs having one to ten clones each [34]. The ACE and especially Chao are dependent of the amount of singletons and the discrepancies with the diversity indices are most probably due to different amounts of singletons in the libraries. The estimated coverage of the libraries varied between 19 and 40% when using the ACE estimator. The coverage values are higher than those reported for clone libraries from soil, where library coverages from 7 to 16% have been described [18, 35]. In addition, higher coverages have been reported with libraries from human sources, e.g. 76–86% [36] and as high as 99% [20], which may be due to the larger number of sequenced clones in the latter studies.
In both buildings investigated in this work, the estimated OTU number was about 500 using 97% sequence identity as the criterion in DOTUR (Distance-based OTU and richness), and the pooled sequence data from all four samples taken from that building. The ACE estimate for the individual libraries varied from 147 to 305. The individual libraries harboured many sequence types unique to that library, so the pooled data set including all seasons provides a better estimate of the total diversity in that building than a single sample. In clone libraries, with increasing numbers of sequences, the number of OTUs increases, until saturation is reached. For the pooled datasets, the saturation in the estimated total OTU number was more or less achieved, for Building 1 better than for Building 2, as shown in the collector's curves. Thus, 500 OTUs represent a relatively good estimate of the number of bacterial species in house dust.
Bacterial diversity in house dust
In our study, Gram-positive bacteria, especially bacteria of the phylum Firmicutes dominated the flora. This is in accordance with culture-based studies made in indoor environments. The most frequently encountered sequences in this work originated from species of the genera Corynebacterium, Propionibacterium, Staphylococcus and Streptococcus. In a study of indoor air of residential buildings, staphylococci accounted for 16–37% of the culturable bacteria, depending on the room where samples had been taken [9], i.e. the proportion was highest in the bedroom and lowest in the basement. Bouillard et al. (2005) investigated healthy office buildings, and found that the two most frequently found species in air samples were Micrococcus spp. and Staphylococcus spp. and in addition, bacteria of the family Streptococcaceae were present in dust samples. These abundant bacteria most probably originate from the users of the building as these species are typical representatives of the normal flora of human skin, outer ear and oral cavity [37–40]. Moreover, there was a multitude of phylotypes affiliating with species or phylotypes abundant in human colon or feces, such as Bacteroidetes, Clostridium, members of the family Peptostreptococcaceae and a number of species not yet cultured. This emphasizes the major human impact on the indoor dust microbiota, at least in these two buildings. The high diversity of bacteria from human sources may also reflect the high occupancy of the buildings by many different persons. Although the sampled rooms were only used by the personnel, carryover from the rooms used by patients may also have occurred. A molecular survey of aeroplane bacterial contamination revealed that the same Gram-positive genera; Streptococcus, Staphylococcus, Corynebacterium Propionibacterium and Kocuria that are common in indoor dust are present in the indoor air and surfaces of aeroplanes [41].
Approximately 40% of the OTUs were of Gram-negative origin. The most abundant families in this study were Sphingomonadaceae, Xanthomonadaceae, Oxalobacteraceae and Rhizobiaceae. These bacteria are common in the soil and rhizosphere, however, some species, such as S. maltophilia, are potential pathogens. In the study of Bouillard et al. (2005), Gram-negative species accounted for approximately one-third of the total bacterial strains isolated from air, dust and surface samples of an office building, with the two dominant species being Pantoea sp., S. maltophilia and Pseudomonas putida. On the contrary, the culture-independent studies have suggested that Gram-negative species are more abundant than the Gram-positive species. Lee et al. (2007) found that Pseudomonas- and Oxalobacteraceae-like sequences were the most abundant in their clone libraries from a child-care facility. This may be due to the different DNA extraction method used; in our study, we used bead-beating, whereas Lee et al. used enzymatic cell lysis, which is a more gentle method and may favour extraction of the DNA from gram negative bacteria. The bead beating time used in the DNA extraction protocol may also affect the observed community composition [42]. Gram-negative bacteria of the orders Burkholderiales and Sphingomonadales were more readily detected with a microarray after 5s bead beating. In contrast, Gram-positive bacteria were better detected after 45s bead beating [42]. The differences can also be attributable to the different sample types; the swab samples taken from toy and furniture surfaces in the study of Lee et al. (2007) probably harboured mostly bacteria able to form biofilms on these surfaces. In addition, Kelley at al. (2004) reported that Sphingomonas spp. and Methylobacterium spp. were frequently present in biofilms on shower curtains. These genera were also found in this study, in both buildings, which suggests that they are members of the normal microflora of buildings.
Seasonal dynamics
Information about the seasonal variation is important for any exposure assessment. To date, most of the published information about seasonal variation of microbial flora in indoor environments has concentrated on viable counts of fungi, and to a lesser extent, on bacteria. Viable fungal concentrations in house dust are known to vary between seasons [32], however the seasonal variation of the viable flora at the species level is not very clear [31]. The first study using molecular methods to investigate the seasonal variation of fungal flora in indoor environments has been recently conducted by Pitkäranta et al. [43]. Seasonal variation of indoor bacterial concentrations has been studied using air samples. The study by Reponen et al. (1992) showed that although the indoor air counts of fungi were significantly lower in wintertime than during other seasons, airborne bacteria did not exhibit an equally clear seasonal pattern [44]. Moschandreas et al. (2003) found that total concentrations of cultivable bacteria in indoor air of 20 homes in the Chicago area were highest in summer and fall [9].
We investigated the seasonal variation of the bacterial flora using statistical tools. The two programs used for the analyses, web-Libshuff and Unifrac-significance, gave rather similar outcomes and only in a few cases contradictory results. In Building 1, only the winter sample differed from the other samples when using the Libshuff analysis. Unifrac detected a statistically significant difference between all seasons, except for spring and summer. In Building 2, both programs detected differences between some seasons, but they were not highly significant. These findings are supported by the Unifrac cluster environments and PCA analyses, in which the seasons were not clearly distinguished apart from the Building 1 winter sample Building 2 summer sample. The clear distinction of the Building 1 winter sample is mainly due to dominance of one OTU type in the library having 99% sequence similarity to Serratia fonticola, a member of the enterobacterial group, and so represented a building-driven trend, rather than seasonal. The dominance of this OTU may reflect a temporary source present at the time of the sampling, or it may be a bias inherent in polymerase chain reaction (PCR)-technology. It is known that some sequences may be preferentially amplified in PCR resulting in a higher dominance of these sequences in the library [45]. To prevent this phenomenon, all amplifications were done in 10 replicates and the number of cycles was kept low. Since S. fonticola was observed only in one sample, we assume that it represented a temporary source. In the PCA analysis, 19% of the variation between samples was explained by the season and 15% by the building. However, the seasonal difference is driven by the winter sample of Building 1, which has the above mentioned problems and otherwise the differences were not very large. The comparisons between buildings were all statistically significant and the seasons of the same buildings also clustered together, thus it does seem that the differences between buildings are more consistent. Although the seasonal variation could not be clearly demonstrated with the statistical methods used, a strong seasonal variation existed at the species level, since more than 75% of the OTUs detected in one building were present in only one season. The statistical methods are based on evolutionary distances; Libshuff uses a distance matrix and Unifrac a phylogenetic tree as the input, and probably reflect better changes in the community structure than differences on species level.
Comparison of the samples at a higher taxonomic level to some extent revealed an increase in the relative abundance of alpha- and betaproteobacteria towards summer at the expense of low GC Gram-positive bacteria. The proportion of low GC Gram-positive bacteria and gammaproteobacteria in the total diversity was highest in winter, that of actinobacteria, alpha- and betaproteobacteria in spring or summer, and finally the diversity of bacteroidetes peaked in the autumn. This may reflect the diverse sources of these bacteria and their fluctuating impact on the indoor microbial flora. The species of the phyla Firmicutes, Gammaproteobacteria and Bacteroidetes detected in this study contained mostly those that are normal inhabitants of the human body [1]. Since the proportion of low GC Gram-positive bacteria and gammaproteobacteria was highest in winter, it seems that the human impact on the microbial flora is highest in winter. Alternatively, the human impact is constant, but during other seasons outdoor factors have a greater influence on the microbiota. Some of the actinobacterial phylotypes encountered, such as P. acnes and some corynebacteria, are most probably of human origin, but other types detected in spring and summer samples, such as OTUs similar to the genera Frigoribacterium, Subtercola and Plantibacter may have originated from outdoor sources. This, as well as the increasing number of Alpha- and Betaproteobacterial sequences in spring and summer perhaps reflected the higher impact of outdoor sources in spring and summer. The results are concordant with the seasonal variation of fungal flora in the same samples [43]. In subarctic climate, the ground is covered by snow in winter and microbial concentrations in the outdoor air are lower than during other seasons. Because of the cold, the windows are also not opened in winter as much as during other seasons. So, the outdoor air affects less the indoor environments in winter, and the indoor sources (human, etc.) can be better detected.
Comparison of the buildings
The two buildings chosen for the study were similar in age, building frame, ventilation type, use and rural location. The main difference between the buildings was their different status with respect to moisture damage and perceived indoor air quality. A statistical comparison of the two buildings suggested that there were significant differences in the microbial flora of the buildings, apart from the spring samples. The differences in the microbiota of the buildings were mainly driven by singletons. Against that background we cannot say if the observed differences are a reflection of a too small number of clones sequenced, or real differences. Both buildings had bacterial taxa that were typical for that building, and which were detected during more than one season. Some of these are likely linked with the inhabitants of the building (Staphylococcus, Clostridium, Aerococcus) and some are known to associate with plants (Frankinae, Rhizobiaceae) and thus, may be from outdoor sources or indoor plants. B. cereus and the family Oxalobacteraceae present in Building 1 were the only OTUs that could be associated with the moisture damaged building. To date, the sole bacterial group that has been associated with moisture damage is the group of spore-forming actinomycetes [46, 47]. Spore-forming actinomycetes were found in this study as singletons in some of the libraries; and thus it was not possible to draw a direct connection to the building. B. cereus has been isolated from indoor dust of schools and day-care centres [48]. Bacilli and oxalotrophic bacteria are known to colonise fungal hyphae in soil [49]. In theory, these bacteria could be evidence of fungal growth in the building, but for the present, this remains only a hypothesis. In general, because only two buildings were investigated in this study, no conclusions can be drawn regarding the effect of moisture damage on the bacterial flora in this material.
In conclusion, this work demonstrated that the microbial flora of indoor dust was complex and dominated by Gram-positive species. The dominant phylotypes most probably originated from users of the building. A seasonal variation was observed, this being reflected in the proportional changes of the microbial phyla as well as at the species level. Statistical methods did not detect clear differences between seasons. The microflora of the two investigated buildings differed statistically and differences between the buildings were more pronounced than the differences noted between seasons.
This work provided basic information about bacterial diversity in indoor dust and its seasonal dynamics and hence, qualitative information about the total bacterial exposure in indoor environments. Future work should include a characterisation of the different sources of microbes in indoor dust and a quantitative assessment of the different microbial taxa.
Dust sampling
Dust samples were taken from office rooms of two buildings located in small towns in central Finland about 100 km apart. The buildings were chosen based on their similar age, structure, usage and willingness to participate in the study. A technical inspection was performed in both buildings by a trained civil engineer. Both buildings were brick-framed, had two floors in addition to a basement floor, which was partly underground, and a mechanical exhaust ventilation system. Both were used as nursing homes for the elderly. The sampling was done during the year 2003, and the temperature varied in central Finland as follows: in winter (January-March) the average monthly temperature was -15°C to -2°C, in spring (April-May) +1°C to +10°C, in summer (June-August) +13°C to +21°C and in fall (September-November) +1°C to +11°C. The values are average monthly temperatures measured at Kuopio airport. The Buildings 1 and 2 were located within 60 km radius from the weather station at the airport.
Building 1 was built around 1920, and no substantial repairs had been done since that time. The basement floor had undergone some repairs after detection of moisture and microbial damage on the floor and outer walls in 1999. There were also local signs of moisture and microbial damage in the bathrooms in first and second floor. The first floor of the building served as a nursing home and the second floor as a ward of the health centre, housing mostly elderly patients. The employees in the building complained of building-related symptoms and indoor air problems.
Building 2 was a nursing home built around 1940 and which had undergone a thorough restoration 1982. No visible signs of moisture or microbial damage were detected, apart from minor signs in the washroom. The users of the building did not report any problems related to the building or indoor air.
The sampling was performed in the office rooms located on the second floor in both buildings. In both buildings, the sampled rooms had workplaces for two or three persons; however, several other individuals visited the rooms every day. For one sample, settled dust was collected for 2 months, twice a week, from the hard floor and other hard surfaces, such as bookshelves and the tops of cupboards, with a vacuum cleaner Miele S371, (Miele & Cie. KG, Gütersloh, Germany). Separate vacuum cleaners were used for each building, and the tubing and nozzle were cleaned between samples with 70% ethanol. Four samples were collected from each building, corresponding to the four seasons. Hair and larger particles were removed by passing the dust through an autoclaved tea sieve. The remaining fine dust was divided into aliquots and stored at -20°C.
DNA isolation
Duplicate DNA isolations were conducted from 25 mg of dust using GenElute™ Plant Genomic DNA Miniprep Kit (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany). The sample was weighed into a 2 ml screw cap tube with 0.5 g of 0.1 mm glass beads (BioSpec Products Inc., Bartlesville, OK, USA), 400 μl of lysis solution was added, and the cells were disrupted with Mini Beadbeater-8 (BioSpec Products Inc., Bartlesville, OK, USA) for 1 min at maximum speed. The released DNA was purified with the kit according to the manufacturer's instructions. An additional purification step was performed with a Wizard DNA Clean up column (Promega, Madison, WI, USA) and the DNA was eluted in 50 μl of nuclease free water. The replicates were combined to give 100 μl of DNA originating from 50 mg of dust.
Construction of clone libraries and sequencing
Universal PCR amplification of the full length bacterial 16S rRNA gene was carried out using primers pA and pH', which target the regions 8–24 and 1522–1542 of the E. coli 16S rRNA gene, respectively [50]. Ten parallel reactions and negative controls containing nuclease free water instead of DNA in a volume of 50 μl were carried out under the following conditions: 1 × Biotools buffer (B&M Labs, Madrid, Spain), 200 μM each dNTP, 0.2 μM each primer, 5% Dimethyl sulfoxide (DMSO), 0.5 mM Betaine, 2.5 U Biotools DNA polymerase (B&M Labs, Madrid, Spain), 0.05 U Pfu polymerase (Promega, Madison, WI, USA) and 1 μl DNA template. PCR amplification was carried out under the following cycling parameters: initial denaturation at 95°C for 3 min, followed by 25 cycles of 30 s at 96°C, 45 s at 55°C, and 2 min 30 s at 72°C, followed by a final extension at 72°C for 10 min. PCR products were purified with Wizard PCR preps (Promega, Madison, WI, USA) and checked in agarose gel electrophoresis.
Immediately before ligation the purified PCR products were incubated in 72°C for 30 min in the following reaction mix: F-516 DyNAzyme PCR Buffer (Finnzymes, Espoo, Finland), dNTPs and DyNAzyme DNA Polymerase (Finnzymes, Espoo, Finland) in concentrations mentioned above. Using PCR Cloning Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, 4 μl aliquots of PCR products were ligated and cloned into the competent E. coli provided in the kit. Minimum 192 white colonies per library were picked and grown overnight in 1 ml of LB-broth [51] containing 150 μg/ml ampicillin. Aliquots of clone cultures were stored with 15% glycerol at -80°C and plasmids were extracted from the remaining culture using MultiScreen 96PLASMID Plates (Millipore, Billerica, MA, USA). Inserts were re-amplified with universal forward and reverse primers targeting the vector sequences flanking the insert. PCR products were visualised in 1% agarose gel with ethidium bromide staining and purified with MultiScreen 384PCR-plates (Millipore, Billerica, MA, USA).
Purified fragments were sequenced using the BigDye Terminator cycle sequencing kit version 3.1 (Applied Biosystems, Foster City, CA, USA) with internal primers pD', pE and pF'. Sequencing reactions were run on ABI3700 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).
Sequence analysis
Sequences were edited and assembled using Pregap and Gap4 programs of the Staden Package [52]. Full length sequences excluding primer sites were aligned against EMBL DNA databases using Fasta 3.4 [53]. Sequences having less than 95% similarity to known sequences were checked for chimeras using the chimera detection available at the Ribosomal Database project website [54]. Multiple alignments of the sequences were constructed using AlignX program of the VectorNTI package (InforMax Inc., Bethesda, USA). ClustalW and DNADIST program available in the PHYLIP package [55] were utilised to construct a distance matrix that was used as an input in the DOTUR-program, which was used for defining OTUs at a distance level of 3%.
Sample coverages and species richness estimates were calculated using the DOTUR program [23]. The program calculates various diversity indices and species richness estimators at different distance levels. The estimates were calculated separately for each season and building sample as well as for a pooled sample containing the libraries from both buildings collected during one season and thus, representing that season.
The sequence libraries obtained from each building and season were compared using web-Libshuff version 0.96 and Unifrac. Web-Libshuff is a web-based version of the Libshuff program [21]. The program compares two sequence libraries to determine if they differ significantly from each other. The program calculates a homologous and a heterologous coverage curve for the libraries, uses Cramér von Mises statistic to calculate the distance between the two curves and Monte Carlo test procedure to compare them. Unifrac can compare several libraries at the same time and can also be used to visualise relationships between environments [22]. The two buildings were compared for each season and the seasons were compared within each building.
The authors wish to thank Mari Koski for skilful technical assistance. The work was financially supported by the Finnish Technology Agency (Tekes), FINE-technology program; grant 40035/04.
Authors' Affiliations
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text:
siskel & ebert on a favorite movie of mine
Really Bad Movies
a bard's eye view of love, life and psychotronic cinema
Friday, February 26, 2016
Thursday, February 25, 2016
yakuza apocalypse [2015]: .5 review
anna and nick didn't get home late this evening which allowed me a bit of time to turn on the roku and surf the offerings of our various streaming services. okay okay, i admit i was on youtube for a while because i wanted to finish watching a short documentary on the late chilean avant-garde poet juan luis martinez [do check out his work. all his books are available on his website]. then after the docu i went to amazon prime and found this bloodbath by none other than prolific filmmaker takashi miike.
anna and nick came home half way thru this pic so this is half a review. let me tell you my impressions. takashi miike does not let plot get in the way of his spectacular visual language. in fact the director has yet to meet a plot he can't say fuck you. i'm sure this film would make sense if you dropped a bit of acid. or toned down the noise of your mind and just be with this movie.
the plot concerns a yakuza boss who is much loved and feared in a small japanese city. a young man apprentices himself to the boss. the young man is a bit of an outrider for yakuza; he refuses to get tattooed on account of his 'sensitive skin.' but this kid can take a punch and is loyal to a tee. then his world is pulled inside out.
because his boss is a vampire! yes, a bloodsucker, undead , motherfucker! before you can go all what the fuck there is an earlier scene with a bunch of elderly men locked and chained in a basement set to knitting. yes, knitting! why? i don't fucking know. it is an odd, cruel part of the universe of this film. the elderly men's clothing is shit-stained. they are tortured. still they knit.
later a man dressed in a priest frock who carries a baby coffin on his back and speaks english faces off the boss in the street. an indian who speaks hindi? who is in league with the coffin wearing padre karate chops the boss in to oblivion. our young man witnesses this butt kicking and tries to protect his employer. no avail. for the boss is beheaded. the priest and his henchman disappear.
the young apprentice comes to after being knocked out and holds the head of his boss. the head reanimates bites our young man and turns him into a vampire. oh no; the shit doth hitteth the fan.
what then? that's when i turned off the movie. anna and nick came home. the second word in the title is apocalypse. a showdown between vampire yakuza and just plain ass weird yakuza is coming with arterial spray and lots of bodies. oh, yes, a goblin appears and warns the plain ass weird yakuza that another vampire is in their midst. this goblin is the freakiest damned thing of pure nightmare.
i'd forgotten how much fun a takashi miike movie is. balls out, splatterific, odd and goofy fun. watching this flick is like opening the window on a stuffy room to a warm spring day. it clears the mind and sharpens the senses. or warps the mind and distorts the senses. i'll go for the former. watching a really bad movie is one of the reasons to stay alive.
Sunday, February 21, 2016
Friday, February 19, 2016
i just caught the tail end of a program broadcast on the national geographic channel about gen x
i am a gen xer so i saw the tv talk about the mis-perception of young people in the '90s
we were called 'twenty-nothings' and 'slackers'
nirvana spoke to generation x according the documentary
& how we are so anti-corporate that we are difficult to market to
i would like to see a program about gen xers in middle age
how slack did we become how marketable did we get
generations matter to the people for we all like to fit in with our group
i like being a gen xer
but then people are human no matter what age they are born
i suppose i would like being a baby boomer had i been born a decade earlier or a millennial had i been born a couple decades later
i wasn't given the choice of which decade or century to be born in
each group of young people discover for the first time everything like sex love responsibilities drugs friendships jobs etc etc
music speaks to our generation your generation too & i have listened enjoyed even loved the music of your generation
& & & i have read generations upon generations of poetry like rimbaud catullus horace ikkyu issa loy cavafy stein
i am these generations
& i am a man of this time who is confused & delighted by life
Thursday, February 18, 2016
Wednesday, February 17, 2016
anticipating rain i gathered my two cloth shopping bags -- trader joe's -- & walked to the supermarket
i am rarely home during the weekday i wonder who are all these people what are they doing at home they are probably wondering the same thing about me
no rain indeed it is cloudy warm with shafts of sunlight peaking thru
i make it to the store pick up a hand cart & go about my business i buy the usual things like bananas nuts bread etc the products & actions of habit & daily ritual
i use the self-checkout kiosk i'm sure these devices are the same where you live swipe the bar code from the item across the scanner then set down your purchase but don't bag it yet for the sensor will freak out thinking there is a foreign object
bananas don't have a bar code neither do tomatoes but the damn scale on the damn self-check machine won't work
i hit the help button a red light flashes above me i stand there because i am a fool
oh technology the world is remade in your image
the store employee comes over & helps me
another item won't scan i punch in the bar # into the machine but the machine doesn't recognize the digits
i hit the help button again i've been at this machine for several minutes now other customers waiting their turn see an old grey-haired man & think oh yes old people don't know their tech
the store employee kindly calmly explains how to enter the sequence of digits into the machine i am back in school at the blackboard solving for x
only then when i am done scanning my items then swiping the store card then swiping my credit card & after the computer indexes tallies & adds up the dollar amounts can i bag my groceries
& when i start bagging the sensor of the machine freaks out because there is a foreign object on it
but the walk home was nice the streets packed with people doing their thing the sky clouds over again as i wait for rain
Tuesday, February 16, 2016
chasing ufos
i don't believe in alien abduction
little green men
or crop circles
& i've been waiting
for them
all my life
Monday, February 15, 2016
i found myself eyeballing the books used as filler in dept. store displays today
the store specialized in containers
stuff to buy when you need to organize the abundance of your stuff
the books in these containers were quite good, including a collection of poems in draft facsimiles by john keats
i wondered who picked the books for these displays could it be a person with an advanced degree in the humanities even an mfa who took a job in corporate u.s.a. whose compromise with capitalism is vouchsafed by a selection of literary works to be used in store displays
or the selections of books could be quite by accident the books are remainders and so are cheap to buy
there was a floating remaindered bookseller back in the '90s
i would become a kid at christmas when i found one of these remaindered bookstores
i have no idea if books are valued or devalued i do know people regular people ordinary people the kind like you and me do read and often a lot
the books at the store are certainly valued at least by me and i know many others
i don't worry about the fate of books and literacy in our present age i worry about our viability as a species
language survives its making so does books so does poetry
language will not survive us when we die so does language
until then we shall continue and record and express our amazement at being alive in these wild times
Sunday, February 14, 2016
Thursday, February 11, 2016
quote unquote
I write because I am entranced by dusk, confounded by the government, at once scared and beguiled by the future, envious of children, forgetful of the past, exasperated by myself, uncomfortable in crowded rooms, curious, ignorant, egotistical, good with vegetables, selfish, lost in the snow and by the sea, hopeful, human.
--martin stannard 'why I write' [conversations with myself: selected reviews and notes 1984 - 1998; stride, 1999]
for look at this graph of the history of the universe from the big bang to the present & find your place in it
Sunday, February 07, 2016
a few years ago i read an article about l.a. based artist billy al bengston in my favorite, now closed down, magazine shop, the newsbeat. i loved the newsbeat. i would stop in at least three times a month from the early 2000s to its closure. the store had a wonderful selection of periodicals including a large choice of art mags. that is how i discovered billy al bengston. and i wondered where the hell he'd been all my life. he's the kind of dude who created a kind of abstract pop art that incorporated suburban malls and asian influence. i still haven't seen a bengston painting in person. but, below is a video presentation of billy al bengston show from 1969. hopefully the artist gets an art book of his own soon.
fox mulder, played by david duchovny, is having his faith tested in middle-age. his sense of wild wonder and discovery has left him. he says so, in last weeks episode of the newly rebooted the x-files: mulder & scully meet the were-monster. half a lifetime chasing ufos and things that go bump in the night and not getting closer to THE TRUTH can wear a person down.
so dana scully, played by gillian anderson, is wondering where the old mulder went. because now she gets a sad, self-lacerating, self-pitying man who thinks he has wasted his life. scully, the ever reasonable side of the duo, wants the old mulder back.
but does he return to scully, and to us, the viewers, who like our mulder as a slightly paranoid conspiracy-theory laden freakonicus?
i don't want to spoil the episode for you if you haven't seen it. this episode was played light and for laughs as much as scares. mulder's mid-life plight is similar to many 50-somethings: how to retain mystery and wonder and the love of life in middle-age.
well, it can be done. mulder might be discovering how. life is a freaking wild adventure even in its most domestic form. scully and mulder reminds us to lift up the curtain of disbelief and look with our crazy eyes upon our fathomless world. for it shall astonish us.
Wednesday, February 03, 2016
quote unquote
Have a good look -- stop the breath, peel off the skin, and everybody ends up looking the same. No matter how long you live, the result is not altered. Cast off the notion that "I exist." Entrust yourself to the windblown clouds, and do not wish to live forever.
This world
Is but
A fleeting dream
So why be alarmed
At its evanescence?
Your span of life is set and entreaties to the gods to lengthen it are to no avail. Keep your mind fixed on the one great matter of life and death. Life ends in death, that's the way things are.
--ikkyu 'poem with skeletons' [barbaric vast & wild: a gathering of outside and subterranean poetry from origins to present ed. by jerome rothenberg & john bloomberg-rissman; black widow press, 2015]

subset: commoncrawl


text:
Although it would be nice if every divorce could go peacefully and amicably, we understand that's not usually the case. This is why the attorneys at the Law Office of Andres R. Guevara have spent years practicing law, so we can better serve our clients when they need us.
We come up with creative solutions and aggressive strategies to ensure our clients are content and secure when they part ways with their spouses.


text:
The permanent exhibition in just a few words:
The permanent exhibition retraces the history of civilization with a chronological presentation that covers some five thousand years since the discovery of writing, in contrast to the fossils displayed at the museum's entrance that are some 170 million years old.
Through selected examples of papyri, manuscripts, incunabula, original editions and autographic documents, visitors can truly take the measure of these creations of the mind, which shaped humanity and so fascinated Martin Bodmer.
Not all of the Foundation's contents can be exhibited in the permanent collection display, which has been remodeled twice since the museum's opening, in 2008-2009 and 2011-2012. The audio guide text is designed around the configuration that was put in place in 2012. The display of the permanent collection is being revised and freshly installed throughout 2015.
For more of the story (in French):
Les origines de la civilisation | Hellas et Roma | La Bible et la Chrétienté
Moyen Age et Renaissance | Traditions orientales | Siècles classiques
Goethe, le siècle des Lumières | Sciences antiques et modernes
De l'Europe romantique | Aux temps modernes


text:
How One Entrepreneur Is Bringing About Global Change By Solving A Local Problem
There's no escaping the fact that there is a certain underlying degree of elitism in entrepreneurship.
After all, most entrepreneurs want to solve big problems, disrupt age-old processes, and as Steve Jobs put it, put a dent in the universe.
We tend to think globally and then try to apply our ideas locally. While this makes for a catchy slogan, it's often easier said than done.
In fact, I've found that the opposite is often true. Organic ideas that solve a local problem often are the most impactful when applied at scale.
By thinking locally and then scaling globally, entrepreneurs can create a thriving business and still make a splash on the global stage.
No one I've met embodies this more than a Colorado-based entrepreneur Lani Dolifka. You might not have heard of her, but chances are you've come across her business.
Lani is the co-founder and CEO of Watermill Express, the largest drive-up pure drinking water and ice provider in the nation. While the economics of the business are impressive, Lani has also been able to make a splash (no pun intended) on the global stage through her advocacy work.
Entrepreneur, heal thyself
If I've learned anything during my time as an entrepreneur, it's that trying to solve a problem you haven't experienced first hand is far more difficult than solving a problem you do know.
For example, when I first launched BodeTree, I was attempting to make it easier for small business owners to analyze their finances. It was a problem that I thought others experienced, but to be honest, I had never struggled with it firsthand.
Lani, on the other hand, developed the concept for Watermill Express as a direct response to problems she faced in her own backyard.
She lived in Colorado, near the Rocky Mountain Arsenal, where 600 documented chemicals were discovered in the local groundwater.
The Watermill story began in the mid-80's when she and her husband were young newlyweds. Money was tight, and Lani developed a habit of carefully reviewing every bill that came through her hands.
One day, while reviewing her city water bill, she noticed a warning printed on the back of the invoice. It stated that pregnant women or children under six years of age should not drink city water.
It was a mind-bending moment for the young newlywed. Here she was, paying a bill for water that was of questionable quality and safety.
Rather than complain or take on the local government, Lani, her husband, a family member and a close friend decided to solve the problem themselves. Through research, trial and error, and pure determination, they developed a revolutionary water purification unit that could provide clean drinking water at an affordable rate.
According to Lani, "If you see something that needs to be fixed, fix it yourself. Don't wait for others to take action."
A pragmatic approach to scale
Shortly after the first units were developed and installed, the Dolifkas heard from a family friend in South Texas that their community was also experiencing water quality problems.
In response, they built another unit, loaded it onto a trailer, and drove it down to Texas. The community embraced the concept wholeheartedly, leading Lani to realize that the need for safe, affordable drinking water was an issue throughout the country.
As young entrepreneurs, Lani and her husband didn't have the capital or resources to continue expanding the business on their own. The sought out partners, investors, and joint ventures to start expanding into different markets.
The real breakthrough, however, came when they decided to franchise the concept.


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SteadyGROWpro grow medium is specifically formulated for the germination, propagation, and growth of plants. It's cell structure allows for optimal water retention and air flow, thanks to an aromatic-based system of thermoset carbon based polymers.
Unlike rockwool, SteadyGROWpro is eco-friendly, and can be safely ground for disposal or incinerated with no harm to the environment.


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Ah, well thank you for being honest and helping to get this far already! I think I'm going to cheat my way through it and forget about trying to make it open up altogether, as you said it would need a modelling expert and I'm definitely not that. Outsourcing could be a good idea, however, the cost would be a factor I would have to consider. Thank you for your support and advice, its greatly appreciated! Andy
*These also should have been included in the first post - my mistake on this! I followed the method above and it produced very good results! However, I've tried to model the back section of the soap dispenser (box section) by selecting points at the back an extruding them - would this be the best way to go about creating the back section? 1) 2) These are my result so far by extruding it: Is there a way I am able to make the rear box section edges less rounded without altering the main dispenser body (dispenser body is in a SDS)? I assume its settings that could be altered within this SDS or by bevelling edges maybe? In addition to modelling this section, I also have the task of modelling the inner details and making it open and close. Advice on the best way to go about doing this too? All suggestions appreciated, Andy
You are a genius! Thank you very much for your help - I'll let you know how it goes!
andyIIId started following Modelling a Soap Dispenser
Modelling a Soap Dispenser
andyIIId posted a topic in Modelling / SculptingHi all, I'm trying to model the shiny section in the centre of the soap dispenser but I have no clue on how. This is what I'm working with so far: Any ideas or suggestions? Much appreciated!


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Alien Creeps TD for Windows 7/8/8.1/10/XP/Vista/MAC OS/Laptop
They're Here! The aliens have arrived on Earth and they are not coming in peace – this is a full-out war!
The perfect setting for a tower defense game, Alien Creeps TD, for free from Outplay Entertainment, full of dynamic animations and colorful graphics, mighty towers, frantic action and masses of enemies! You have been chosen to defend the earth from the invaders!
A Massive Arsenal And Host Of Heroes At Your Command
Rockets, lasers, machine guns, tesla coils and more – your towers have it all. Send out infantry troops to help hold back the incoming hordes while your towers zap, burn and blast the enemies to bits and pieces. Deploy your brave heroes into battle and upgrade them along with your towers, using coins earned during battle, for more much needed firepower.
Fight The Alien Forces
The enemies come in all sizes, shapes and colors and are fully equipped to take over the world. Take on the enemy waves across many harsh terrains with your towers and heroes in explosive, action-packed battles!
Short And Sweet Action
Alien Creeps is perfect for players who like to play quick games, and in Alien Creeps TD, with a story-driven campaign of rounds only lasting a few minutes compared to the average TD game that takes forever to get going, and three different gameplay modes, you can squeeze some fast action in during any time of your day, whether on the toilet or during your study breaks. You can even fast forward for breakneck speed. Be careful not to get addicted!
Join A Global Legion Of Defenders Now
Alien Creeps TD for Android and iOS is the #1 strategy game in over 30 countries, with millions of players fighting to protect our planet Earth. The creeps are coming…are you going to pick up arms as well? Join the global defense force now and share airstrikes and reinforcements with your friends!
How To Play/Download Alien Creeps TD for PC
Follow the instructions below, it's really easy and it takes about 5-10 minutes to run the game.
- Download Bluestacks from their official site
- Run and Install it
- Open the bluestacks app and setup your Google Play Store with your gmail, sometime it asks for phone verification
- After that search for "Alien Creeps TD"
- Click the Install button next to game icon
- When it's done, you can find and play Alien Creeps TD in "All Apps" or in "My Applications" sections
Download Alien Creeps TD for Mobile and Tablet Devices
Google Play Store Link
Apple App Store Link
Alien Creeps TD Trailer
More Alien Creeps TD Tips Using Bluestacks
- If Bluestacks don't work for you for some reason, you can try Andyroid
- Sometimes when playing Alien Creeps TD, bluestacks ask you fee to pay but gives you option to download some apps. Every time it asks you just select "Install apps" and continue using it free.
- You can configure your keys inside bluestacks. You can map specific key to specific location. And there is options for tilt, swipe, tap, move up/down/right/left…etc
I hope you enjoyed this guide, please rate and share. If you have problems running Alien Creeps TDon desktop or any other issues, leave a comment below.


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Learn how Quest Data Protection solutions help more than 8,000 businesses and non-profit organizations around the globe monitor, manage and protect IT resources and data, both on-premises and in the cloud.
Duration: 01:37
Functional IT silos may be detrimental to your data and you might not even know it. Learn more about breaking down these silos. You'll not only save time and money, you'll also be able to focus on innovation, demonstrating to other stakeholders that IT is an awesome, strategic business partner.
Duration: 02:15
Battle the dark forces that threaten IT systems with Nick the IT Ninja and learn how Quest KACE makes it possible for him to master proliferating endpoints, security threats, system downtime, and compliance issues.
Duration: 00:40
See how Quest minimizes your storage footprint and helps you to easily manage and secure your on and off-prem data.
Duration: 01:05
Join the Innovation with Quest.
Duration: 00:30
Your data is growing faster than you expected. But with Quest Data Protection solutions, your backups will never be older than five minutes, restoration will be just 15 minutes away, and your storage requirements will see a reduction of up to 93%. See how in this short video.
Duration: 02:15
Watch the Channelnomics MSP Awards 2018 Winner interview to learn why Quest was awarded Best Backup and Continuity Offering.
Duration: 02:45
Learn how the Quest Partner Circle helps our partners to drive profits and increase capabilities while making it easy to do business with us.
Duration: 02:06
Simplifique o acesso, a preparação e o provisionamento de dados.


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Your Internet service provider might have provided you with a modem/router combo for your house. You may have also connected a second router to this as an Access Point or as a totally separate router to put off its own Wi-Fi network.
The Circle device will only manage one network at a time. Make sure that the Circle device is paired with whichever router the devices you want managed are also connected to. Below we've listed the ways that two routers can be setup on your network and how to best connect your Circle device to them.
Bridging to your main router
Your Internet service provider or yourself might have setup your router/modem combo in bridge mode, and then connected your main router to it via an ethernet cord. In this scenario your router/modem combo won't be putting off its own Wi-Fi network so feel free to pair your Circle device with your main network. If you want, you can plug in your Circle device via ethernet afterwards to the router that is putting off Wi-Fi and not in bridge mode.
Router and access point
If you've got your router/modem combo setup as the DHCP server, and set the second router up in Access Point mode, connect the Circle device via ethernet to the router/modem combo after pairing and it will see all devices that connect to your network. If your router/modem combo is putting off a different SSID then the Access Point we recommend making them the same, or that the Circle device is paired with the Wi-Fi put off by the router/modem combo.
Two separate routers
Some households might have two routers setup, both with their own DHCP servers running on the router and putting off their own networks. This isn't a setup we recommend, but if that's how your house is set up, make sure your Circle device is paired with the WiFi network that you want to manage, that the devices you want to manage are connected only to that network, and that they don't switch over to the other network. (You can make sure devices stay on the right network by changing the second network's password, and only allowing the correct devices to connect to that second network.) If you're plugging the Circle device in via ethernet, make sure that it is connected to the router that the devices you want to manage are also connected to.
For more about advanced network setups and the Circle device, check out this help article.


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Ready to launch your career as a software developer, or just want to know if this is the right path for you? Check out our courses below to find out where you should start, what you will learn, and what you can expect after you successfully complete each course. Get started in HTML, CSS, and JavaScript with beginner-level courses, or take your training to the next level with intermediate courses available now!
Find out if a career in tech and our Code 201 course are right for you.
Code 101: Is this career path really for you? Spend a Saturday with us to build your first web page and test your new skills to see if you enjoy the prospects and process of software development.
Code 201: Get used to writing syntax and pushing code as you learn the basics of web development through HTML, CSS, Javascript, and the tools and best practices used by software developers around the world.
Code 301: What's the technology behind launching a web app and working with a software development team? In this course, you will go beyond HTML, CSS, and JavaScript syntax and learn how to launch a web app, collaborate on projects, use advanced frameworks and libraries, and work with basic algorithms and data structures.
Get training that will prepare you for an internship at a tech company or our Code 301 course.Learn More
Become a web producer, product manager, or entry-level website builder, or continue on into a Code 401 course.Learn More
Get advanced training in a specific programming language and land a high-paying job in the industry.Learn More
Code 401: These are the most intense courses offered at Code Fellows. Students come in with various backgrounds and leave with the training and skills they need to excel as professional software developers. To test straight into Code 401, students will need previous experience in software development, whether through Code Fellows courses, self-study, previous education, or on-the-job training.
We're here to give you all the tools you need to succeed in your new programming career.How to Apply Contact Us


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When setting up the parameters for your Forex trades, you can choose between the standards of stops and expirations or, you can now set a binary option. Forex binary options are not available with all trading systems but are fast becoming a standard. There are advantages to these parameters that can work well for you, but only if you have a firm grasp of the limitations of your resources. If you are bundled into a group trading package via a brokerage, setting the parameters may not be something you have much input in deciding. However, understanding what they are will help you in understanding the choices and actions of your brokers.
Stops and Expirations
The standard trading parameters on the Forex market, and common to the stock market, are to set a stop amount that tells brokers when to sell your shares. This is a predetermined value of the unit for how low it can go before you trade, or high it can rise before you do the same. This is done to minimize risk. While the inclination may be to stay with a rising value, the rise only lasts for so long before investors cash in to access the profit and drive the value down. Conversely, a dip in value may not be something a currency or commodity can recover from and it is time to begin trading to minimize loss and move on. Expirations are time limits to how long you want to park your money on a certain risk. This allows you to see what it might do, but not abandon your funds to it indefinitely. Stops and expirations are fixed amounts or time limits that have little flexibility. They trigger selling when their marker is hit.
Forex binary options introduce a greater element of flexibility in setting parameters with investments. Rather than setting a strict time limit, these settings allow for fluctuation before triggering a trade marker. It works by the investor setting a greater than or less than payout option. This hedges the risk against a loss better than a direct stop. If a direct stop tells the system to sell if the value goes below 1, then the trade is made and the investor is out. If twenty minutes later the value jumps to 1.2, the investor has lost out. With binary options, the investor or brokers can set a profit payout percent should the value become less than 1. Then, only a percent of the funds are traded out, returning funding to the account but leaving a mitigated risk for loss or, allowing a risk for gain should the value rise. Binary options remove total financial risk for the investor. There is risk of loss still involved, but it is potentially less than a straight trade out. This type of option is more flexible with market fluctuation than an expiration because the parameter is not randomly selected from a time frame, but a percentage of value context.
Low Risk, but high return?
Binary options are considered the "money market" approach to Forex trading. They are low risk because the percentage parameter you set pre-determines how much you are willing to lose and prevents you from going beyond that. This option focuses on developing your return on investment (ROI) potential for standard trades. The trade-off is that the potential for high return is very low. The percent triggers will not pay out more if you exceed the parameter. One of the best uses for them are short term trades. They can generate profit in hours whereas standard approaches may take several days before the stops are reached and profit percentage points can be assessed. As low-risk as this all may be, it is not a good beginners trading strategy. You must be willing to lose to learn how the Forex market behaves. Taking your experience, you can now begin to use the different options available to maximize the return on your investment. This approach is also well suited for a seasoned trader who wishes to explore, but not risk, with new units of value.
Using Options Effectively
Forex binary options are not a way to make a large return on your trades. They are best used to help you navigate the market to know when to jump in. You can set time limits for them that go by hour, day or week. Then, you can gauge from the ATR (Average True Range) reported from the time frames what the potential for an exchange is shaping into. Binary settings are a means to test the waters, not something to build an entire foundation on. By mixing this technique with the traditional stops and expirations, you can manage trading your funds better and lessen your overall financial risk.


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Click to sign up for our mailing list!
Click above to see what the artists at the Village Theatre have in store for you!
Performance Dates
Tickets are available in advance from any cast or crew member or via online ordering. Remaining tickets for the show will be available at the doors.
Auditions and Performances are held at:
The Laurel Grange
21 Garden Street
West Newbury, MA.
(About one mile from exit 58 off of Route 95)
Click above to use Google Maps for Directions


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A few weeks ago I heard a news report that made all New York single girls breathe a sigh of relief and plan their next girl's night out. The long-standing belief that there are more single women than single men in New York is a myth. At 27, and my biological clock constantly ringing in my ears, I jumped for joy and began to prepare for another journey into the New York dating wilderness.
Here is the ugly truth about New York; it is a cesspool of men that will make your skin crawl, cause you to consider buying stock in the Kleenex company, wonder how far up your potential guy can get up his bosses' ass and have you running to your local library to compare your new guy to Oedipus.
However, like the hunt for the needle in the haystack, I am ABSOLUTELY POSITIVE there HAS to be a guy that won't have me or any of the females in New York planning their stay at the local convent.
Where are all the Ryan Goslins that break up fights in the middle of the street? Can anyone point me in the direction of the Matt Damons that have a brilliant career but still manage to have a wife and adorable children? Lord, can you please shine a light on where my very own Adam Levine has been hiding?
With that said, I'm really not sure where Carrie, Charlotte, Miranda and Samantha found all their attractive dates but it certainly isn't in any of the places I've ventured to. I'm almost positive you single girl's haven't found them where you are going either.
Wouldn't it just be easier if there was an honest review of places where are a girl can go to find a potential Mr. Right?
That's where this blog comes in. I am going to spare you the trauma of going to a really nice place to find that the possible Mr. Right Nows that frequent that place will have more in common with your dad then they ever would have with you.
Each week I will review one or several haunts in the city and post them here. Yes, I'll be sure to describe the decor, the drink prices, etc. More importantly, I'll review the kind of guys that go there because life is WAY too short to waste months on someone who is more interested in his suit than the color of your eyes.
Feel free to suggest places I can write about and any feedback is greatly appreciated. For a more in-depth look into what dating disasters this other little experiment will lead me to, please follow my sister blog somuchforthefairytale.wordpress.com


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Redcar is situated on the north east coast of the UK, our blog lets you know the best places to eat, stay and have fun in Redcar. We plan to keep everyone updated about the latest events and news in Redcar. In our blog about Redcar you will find everything from the best bed and breakfast in Redcar to the best fun days at Redcar.
Monday, 29 June 2015
Sunday, 28 June 2015
Tuesday, 23 June 2015
Monday, 22 June 2015
Tuesday, 16 June 2015
Monday, 15 June 2015
Friday, 12 June 2015
Saturday, 6 June 2015
AAA Band Reunion, All your family are welcome. Great packed Funday
Please share this to everyone, we want to get in touch with as many old band members, people that hosted the Lakewood Ohio Band members and also those who went to Canada, This includes mums, dads, or friends of people that were in the band.
All your family are welcome. Great packed fun day and Evening do is on the cards.
** TICKETS CAN BE GOT VIA GORDON WITH CHEQ OR ME VIA PAYPAL HERE OR EMAIL FOR THE DETAILS** email is below on poster......... afternoon, BBQ, bouncy castle, face painting, Adult games, Base Ball, and in memory of Stan Peverly any monies raised will go to the Redcar Youth Club.
Contact Vanessa Hardy nee London. 07789 867 638 after 7pm or Gordon Sanderson 07837 654 697
Monday, 1 June 2015
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That tangy Secret Stadium Sauce has been described in many ways, from "a combination of barbecue sauce and sauerkraut juice" to "a mix of clam juice and sweetened tomato sauce."
One day in the early 1970s at Milwaukee County Stadium, condiments were running low, so a concoction of ketchup, barbecue sauce, mustard, smoked syrup and some other stuff was thrown together, and Secret Stadium Sauce was born.
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Uniting the Anthem family around the mission God has entrusted us with, and equipping each other for the mission God has given each one of us.
2016 Theme: INTENTIONAL MISSION IN OUR EVERYDAY LIVES
Our focus this year is to turn our attention and training towards how the gospel and mission of Jesus informs our regular, day-to-day lives and brings greater intentionality to the mission of making disciples that God has given to every believer.
How then will they call on him in whom they have not believed? And how are they to believe in him of whom they have never heard? And how are they to hear without someone preaching? And how are they to preach unless they are sent? As it is written, "How beautiful are the feet of those who preach the good news!" –Romans 10:14-15
Every believer is transformed by the the work of Christ, empowered by the Holy Spirit, and sent by the Father to go out into all the world and proclaim the gospel to the whole of creation.
For the single mom, the business owner,for the bagger at Trader Joes, the barista, the VP of a company... we want to see the body of Christ at Anthem, equipping each other to be empowered to fulfill their calling right where God has them.
We will unite together in our 'main sessions'; worshipping together, praying together, learning from different speakers, and mobilizing as a church for the year ahead. The body will equip each other throughout the day in 'breakout sessions,' where different people, with different life experiences will be sharing their stories, training and equipping the body for life and the work of ministry.
Friday night will gather to kick off the summit, and we'll return Saturday for an all day combination of main sessions and breakout sessions led by different facilitators. All are welcome to the Friday night session and only those registered will be able to attend Saturday. Childcare available for children 6 months and up!
Interesting in volunteering at this year's summit? Fill out this quick form to let us know by clicking here.


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Groundlings - Fri & Sat Nites
Larry is currently in the house band at the legendary Groundlings Theater on Melrose Ave. Los Angeles, California.
See him perform there for the Friday and Saturday night shows.
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Best Hostels in Guanajuato, Mexico
9 Hostels, 1 Cities
All Listed Hostels in Guanajuato, Mexico
Al Son de los Santos 22647Guanajuato, Mexico
Al Son de los Santos is one of the most charming places with a traditional Mexican artisan style, with a picturesque and nice intimate touch. We have common areas like a kitchenette and terrace where you can share experiences during your stay. We have 6 different and pleasant rooms for all taste»
Min bed price
Hostel Guanajuato 23090Guanajuato, Mexico
Hostel Guanajuato is located in the city of Guanajuato in the historical center is a small property where we have 4 private rooms, a male dorm and a female dorm, common areas as our TV room, kitchen, terrace and internet. Our house dates from 1875 and maintained its structure until 1927 when it w»
Min bed price
Hostal Galeria Cafe 1810 22012Guanajuato, Mexico
Hostal Galería Café 1810 opens its doors to provide, in the colonial city of Guanajuato, a hosting service comfortable, clean, safe and economical. We have shared and private rooms with seating for four and six people and we also have a room for eight and six people. With prices starting at $ 9…»
Min bed price
Hostel Alonso 23390Guanajuato, Mexico
Hostel Alonso, is a house that has been authentically Mexican Hostel open to all amenities and facilities necessary to make your stay in our beautiful city, all a wonderful experience. We have 5 private rooms and 1 bedroom for men and a women's dormitory. We also have a fully equipped kitche»
Min bed price
Hostal-Inn 23529Guanajuato, Mexico
On your visit to Guanajuato El Catrín opens its doors for you that you come in order to meet , relax and enjoy your trip . Offering to our customers private rooms with private bathrooms, private rooms with shared bathroom and a big dorm. The safety, cleanliness and attention are present within eac»
Min bed price
La Hosteria 22922Guanajuato, Mexico
Welcome to La Hosteria, we are a design , modern, fully redecorated Hostel offering cheap accommodation in the center of Ganajuato Capital. La Hosteria is a warm and friendly Hostel that welcomes travelers from all over the world. We have a wealth of experience in the industry and know what trav»
Min bed price
Hostel Casa del Angel 22400Guanajuato, Mexico
Hostel Casa del Angel is located in the very heart of Guanajuatos historic district. Less than two blocks from the Plaza de la Paz and public bus access, its perfectly situated within walking distance of the Mueso de Pueblo, the Diego Rivera Museum, the University of Guanajuato and the many other»
Min bed price
La Casa del Tio 23406Guanajuato, Mexico
La Casa del Tio is recommended by Lonely Planet, Let,s Go and other travel guides because it has a great location, cleanliness, security and good value. Those characteristics are the keys of success of this small hostel and bed & breakfast located inside the historic Guanajuato, a few steps of…»
Min bed price
La Casa de Dante 25167Guanajuato, Mexico
A wonderful panoramic view and our now famous great Mexican breakfast await you at La Casa de Dante, because many travelers checks up the 165 steps of our lane to stay with us on a picturesque private room or in our spacious dormitory. In addition to good service and comfort, with us you will fin»
Dorms: € 14.34
Privates: € 15.82


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Soccer Guard
Power Soccer Shop Plastic Guard & 13″ Ball
The Power Soccer Shop Plastic Guard is sturdy and light and adjustable to most power wheelchairs (attaches to leg-rest). It mounts with nuts, bolts and clamps. Northeast Passage is happy to consult on the best setup for your chair. The 13" ball is used in the game of Power Soccer (the guard maneuvers the ball around the course). We rent the guard and ball separately or together. Long term rentals available. Please inquire about availability.


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Bing Crosby (seated) listens to Phil Harris carry on during the 1955 Crosby Pro-Am at the Pebble Beach Golf Links on January 15, 1955. (Monterey Herald Archives)
MPC basketball camp
7 hours ago
Monterey County has a rich and colorful history. The Monterey County Herald has been there to cover the names and places that make our county unique. With some "never seen before" images from our archives, The Herald hopes to jar your memory and to participate in this ongoing look back on the history of Monterey County.
Phil, for those who don't know, was one of the most popular voices on the radio at the time. He had been Jack Benny's bandleader, and a character on Benny's radio show. He married singer / actress Alice Faye, and the pair had a hit "I Love Lucy" style radio program together into the 50s.
Harris went on to voice Baloo the Bear for Disney's The Jungle Book and the animated Thomas O'Mally cat in the Aristocats.