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Millions of biological samples, including cells of human, animal or bacterial origin, viruses, serum/plasma or DNA/RNA, are stored every year throughout the world for diagnostics and research. The purpose of this review is to summarize the resources necessary to set up a biobanking facility, the challenges and pitfalls of sample collection, and the most important techniques for separation and storage of samples. Biological samples can be stored for up to 30 years, but specific protocols are required to reduce the damage induced by preservation techniques. Software dedicated to biological banks facilitate sample registration and identification, the cataloguing of sample properties (type of sample/specimen, associated diseases and/or therapeutic protocols, environmental information, etc.), sample tracking, quality assurance and specimen availability. Biobank facilities must adopt good laboratory practices and a stringent quality control system and, when required, comply with ethical issues.
Introduction: why biobanking
The majority of relevant studies on microbial pathogenesis, infectious disease etiology and epidemiology, and environmental microbiology are based on obtaining biological samples. Biobanking, intended as the process of collecting, treating, and long-term storing biological samples, represents an essential tool for biological, biomedical and industrial research and for laboratory diagnostics. The characteristics of an ideal specimen bank were described by Lee in 1990 [1] as having a secure funding source, a cryogenic storage facility, developed criteria for selection of the best samples to be stored; at the same time each facility must develop an ongoing research to optimize sample collection/processing and storage conditions. Because biobanking has gained an emerging importance in diagnostics, research, and epidemiology, many organizations have now their own biobanking facilities, characterized by different preserving techniques, their own functional protocols, and, ideally, their own bioinformatic procedures. For these reasons, although it requires huge investments in personnel, automation and storing facilities, biobanking is becoming a part of biomedical and environmental national scientific programs.
Millions of human, animal and microbiological samples are stored each year for diagnostic and research purposes, including applications in microbiology and infectious diseases, genetics, oncology, etc. In the microbiological setting, the most important reasons for biobanking could be summarized as follows:
- (a)
To realize epidemiological studies intended to compare samples of human or animal origin within the same epidemic episode or from episodes occurring at different points in time or at distant locations. Stored samples offer unique opportunities to study the genetic characteristics of microorganisms, to establish transmission modalities in the local or worldwide settings, or to perform additional analysis on old samples when new questions arise or new pathogens are suspected to appear. Infection control plans, including the development of vaccines or of adequate containment procedures are often based on these information.
- (b)
To ensure progresses in diagnostic procedures by comparing samples from the same individual at different points in time or from different subjects with similar diseases, and by analyzing stored samples with new analytical methods that may increase sensitivity or specificity of infectious disease detection.
- (c)
To perform research studies requiring large number of samples collected in different geographical locations or requiring multiple parameters to be analyzed in specialized laboratories throughout the world.
- (d)
To constitute repositories of human or animal cell lines or microorganisms used for diagnostic and research procedures (i.e., isolation of viruses in well characterized cell lines), to set up programs checking the quality in diagnostic and research laboratories or to provide reference (state of the art) reagents for research.
- (e)
To establish collections of microorganisms aiming at characterizing microbial diversity and microbial evolution in the world. Microorganisms are essential parts of the biosphere; they can be also used in production of drugs, as biocontrol agents, and for many other beneficial purposes. Studying and safeguarding microbial diversity for future use and exploitation is therefore of fundamental importance [2]. Microbial culture collection faces an immense task: for instance, over 1.5 million fungal species are estimated worldwide, but less than 100,000 are described [3]. At the current rate of discovery, it will take 700 years to describe them all. Biological banks may thus be, in the near future, an invaluable tool for accelerated discovery and characterization of microorganisms and for promoting their beneficial uses to mankind.
Current situation
Presently, thousands of laboratories of microbiology (including medical, veterinary and environmental microbiology), clinical chemistry, pathology, epidemiology, and genetics have their own ongoing programs for sample collection and biobanking. There are, however, some public (government) and private non-profit organizations pursuing nation-wide or international programs for biobanking. At national level, the Swedish National Biobank Program (http://www.biobank.se) is a joint national program on functional genomics. The main objectives of this program are to increase the knowledge and the quality of the Swedish biobanking system, to increase usability and availability of stored samples and to increase ethical awareness. Another important nation-wide program, the United Kingdom Biobank (http://www.uk.biobank.ac.uk) aims at building a major resource to support a diverse range of research, which will, in turn, improve the prevention, diagnosis, and treatment of illnesses. Private non-profit reference culture collections were established more than 40 years ago. The World Directory of Collections of Cultures of Microorganisms (http://wdcm.nig.ac.jp), an activity of the World Federation for Culture Collections, holds an excess of 1 million microbial strains, of which 44% are fungi, 43% bacteria, 2% viruses, 1% cells, and 10% others. Two additional important organizations devoted to the acquisition, preservation and distribution of microorganisms and cell lines are the German Collection of Microorganisms and Cell Cultures (DSMZ, http://www.dsmz.de) and the American Type Culture Collection in the United States (ATCC, http://www.atcc.org). Both these organizations constitute invaluable tools, providing state of the art reagents, reference microorganisms and cell lines for research and diagnostic purposes. For an exhaustive list of the major international culture collections the reader is referred to Smith and Ryan [4].
When faced with financial constraints, some academic centers recently decided to transfer their genomic banks to private organizations; this type of collaboration may pose serious problems in the protection of human subjects, in property rights, and in the possible commercial use of future benefits to the community. To avoid these threats, the "Charitable trust" was recently suggested as a potentially effective model for genomic biobanks, Because it complies with privacy rules and with benefits to the community, without losing its value in biological research and in epidemiology [5].
Purpose of the review
The purpose of this review is to summarize the resources necessary to set up a biobanking facility; the challenges and pitfalls of sample collection and the most important techniques used for separation and storage of samples will also be presented and discussed. The review will finally deal with principles of electronic data management and of accurate quality control procedures. The factors affecting the quality and the future use of biological samples will be discussed with particular attention to the nature of samples to be stored (i.e., blood, bacteria, fungi, etc.). This review considers some statements that may generally apply to biobanks, but are not for all biobanks. The purposes of the repository (research, diagnosis, epidemiology, industry, etc.), the type of specimen, the availability of personnel and equipment as well as other factors, deeply influence the characteristics of each biobank.
Staff and equipment
A functional biobanking facility requires adequate resources in terms of personnel, space, laboratory instrumentation, computers, and of quality system, including protocols and reference reagents. If human samples are stored, ethical issues must be solved. In literature, there are few indications on minimum personnel requirements for a biological bank [6]. The personnel may be a part of the laboratory performing all the routine or research work of the laboratory itself, while in turn holding the biological bank; in alternative, the personnel may be dedicated solely to biobanking, as it may happen in centralized facilities. Although a detailed analysis of the biobanking process and its throughputs is necessary before deciding the type of organization and its needs, two to three laboratory technicians may be considered as the minimum requirement to ensure a continuous biobanking service by processing, aliquoting, storing the samples, and holding the sample archive in all its aspects. Informatics greatly improves the management of a biological bank. Commercially available software packages may be satisfactory in small to medium biobanks, but ideally each facility should develop its own, dedicated software. In this case, a computer engineer or a computer programmer is required to develop and improve the software, in provision of the fact that systems should be amended continuously to meet the changing needs of the laboratory staff [6]. The coordinator of the biobank could be a component of the medical or PhD staff of the lab, who dedicates a part of his/her time to organize the biological bank, ensures the respect of the legal and ethical issues, keeps the contacts with the scientists who request the samples stored in the bank for their own diagnostic or research purposes, and defines with the administration space, personnel and equipment resources necessary to perform biobanking. Laboratory requirements include a processing room with a class II biological safety cabinet, a centrifuge and a microcentrifuge; this room may contain a personal computer with software dedicated to biobanking or a paper-based archive. An additional room is dedicated to the storing facilities. Samples are cryopreserved in freezers at −20 or −80 °C and/or in liquid nitrogen freezers that incorporate remote alarm systems; freeze–dryers may also be used.
Sample collection and processing
The investment in biobanking, in terms of facilities, personnel, and expertise is very important, and a cost-effective rationale is required for its maintenance. A high degree of consistency of the sample, of processing quality and of reproducibility of experimental results obtained with stored specimens can be gained by a centralized biobank facility (i.e., in one department or even in a single institute, university or hospital). When compared to a centralized approach, the distributed organization would require more staff, more hardware and facility space; it has been recently assessed that the cost of a distributed organization was about twice that of the centralised approach (http://www.ukbiobank.ac.uk). Despite these advantages, usually the need to collect and store different types of specimens (microorganisms, human or animal blood or tissues) for completely different research or diagnostic purposes, and the lack of an institutional-based, rather than a single laboratory-based approach, make centralization of a biobank very difficult to achieve.
Before sample collection, ideally each research or diagnostic staff must define the potential parameters to be studied or, more in general, the study design, so that appropriate collection and processing protocols can be designed. However, samples stored in a biological bank are often used to answer to questions arisen after the initial studies have been completed or to test old questions with new techniques. In order to satisfy future research needs, the original sample may be divided into separate aliquots appropriate for different purposes, such as several vials containing the same material (i.e., several vials containing small aliquots of serum or one bacterial strain) and/or vials containing different materials obtained from a single sample (i.e., intact cells, separated DNA and/or RNA, proteins, etc.). For these reasons, sample collection and processing must be optimized to avoid approaches that may preclude future analysis, and to ensure preservation of the integrity of the whole cell and of its components. Automation procedures can be a tool for the improvement of biobanking throughputs, of quality control and may decrease costs. A great effort has been initially devoted in automating the mechanical aspects of specimen manipulation. In fact, the presence in many laboratories of automated liquid handling and sample dispensing systems constitutes an important aid for appropriate specimen processing and storage [7]. In the last few years process control software supporting the laboratory hardware have greatly improved the automation of biobanking: However, transferring samples between different preparation processes still requires, at least partially, human intervention. Future trends of automation will therefore probably include the integration of a greater number of functions in a single system. In order to produce proper statistical analysis for experimental and epidemiological studies, samples representative of the study protocols should be collected together with adequate controls. Guidelines to collect samples and to evaluate the quality of submitted specimens are therefore the first essential step to set up a biological bank. An appropriate sampling procedure may assure that the sample is correctly identified, that it has been collected and sent to the biobanking facility in order to maximize the recovery of microorganisms or cells and that, if required by the study protocol, the accompanying forms containing the patients' data and that other relevant information have been filled without errors or omissions. For a reliable sample collection, establishing clear communications between researchers and the staff collecting the specimens is essential. After the standardization of the collection step, the researchers may optimize sample processing by running preliminary tests to assess the best conditions for preserving sample integrity. For this purpose, researchers usually compare the effects of different preservation regimens on viability, pathogenicity, morphological or physiological parameters and on the genomic stability of cells or strains before and after preservation [8]. In fact, a major concern of storage protocols regards the stability of biological samples [9]. The factors known to influence stability include: (a) The use and type of preservants, or anticoagulants in the case of blood and urine; (b) temperature range during the time between sample collection and processing, and during short or long-term storage; human and animal cells are quite stable at room temperature for up to 48 h, while viruses and bacteria show very different survival time, according to species-specific characteristics. The appropriate temperature to avoid sample degradation thus depends on the cells or cellular components to be processed and stored; (c) timing before initial processing and storage; this parameter may vary considerably depending on the sample to be stored. Guidelines published by the National committee for clinical laboratory standard [10] stipulate that defined clotting and centrifugation protocols are important to obtain appropriate serum specimens. Furthermore, serum or plasma should be physically separated from blood cells within a two hour maximum from time of collection, while no indications are presently available for human or animal cells or microorganisms. However, in general, chapters considering the effects on recovery of the starting material and of the individual characteristics of bacterial and viral species are contained in laboratory manuals [11, 12]; (d) sterility during specimen collection and processing is essential if the processing protocols include the isolation of RNA or the culture of cells or viruses from the sample; (e) the presence and activity of endogenous degrading or inhibiting substances in the sample itself. For instance, proteins are sensitive to degradation by proteases, while RNA is very sensitive to degradation by the widely distributed RNAses (see below), so separation protocols must include specific procedures to avoid each type of degradation [8].
Several issues of safety arise when biological samples are manipulated in the laboratory. Therefore special precautions must be taken during collection, processing and storage of the materials used for biobanking. The personnel must be trained in handling infectious materials, and the laboratories must adopt biological safety level II or III, according to the pathogens handled. The description of the safety procedures is beyond the scope of this review and good sources for guidelines related to biosafety issues are available from the Center for Disease Control (Biosafety in Microbiological and Biomedical Laboratories, IV Edition, http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm) and from the World Health Organization (Laboratory Biosafety Manual, II Edition, http://www.who.int, Document WHO/CDS/CSR/LYO/2003.4).
Principles of cryopreservation and freeze–drying
Specific conditions are required for the long-term storage of human, animal, and microbial whole cells or for separated cellular components such as nucleic acids. The most widely used systems for storage include cryopreservation and freeze–drying. Cryopreservation means that materials are stored at low (from −20 to −80 °C in freezers) or ultra-low temperatures (−150 °C in liquid nitrogen containers); in the latter case, cryopreservation takes place in the liquid or in the vapor phase of nitrogen. Liquid nitrogen devices permit storage at quite constant temperatures, while mechanical freezers are subjected to temperature variations that may compromise the quality of samples stored in the upper compartments of upright front-loading freezers [13]. Because of their increased storage capacities liquid nitrogen devices are more suitable for banking facilities with thousands of stored samples, while −80 °C freezers support small and medium-sized biological bank facilities. Due to the possibility of cross contamination of non-infected samples with stored infectious materials harboring hepatitis B or other pathogenic viruses [14], modern nitrogen freezing systems are based on vapor-phase, rather than on liquid-phase cryopreservation.
The scientific rationale of using ultra-low temperatures is that, below −139 °C for pure water or below −130 °C for culture media, molecules do not move, thus preventing any chemical reaction and reducing cell damage, occurring during cryogenic procedures [9]. In fact, a major drawback of cryopreservation consists in severe damage of the cells exposed to freezing. Cryoinjury is a lethal event, depending on the formation of intracellular ice, on the efflux of water outside the cell, and on an increase in the concentration of intracellular salts in the solution. The most critical range at which thermal shock occurs is between +15 and 0 °C, while minor events occur at temperatures below 0 °C [15]. The modern era of cryobiology started with the discovery that glycerol protects eukaryotic cells against freeze damage; after this initial observation, several cryoprotectants were discovered and are now widely used to cryopreserve cells, including dimethylsulfoxide (Me2SO), glycerol, blood serum, ethylene glycol, methanol, skimmed milk, yeast extracts, tripticase soy, etc. (for an excellent review on cryoprotectants, see [16]). A recent survey showed that dimethylsulfoxide was used in 314, glycerol in 308, and blood components in 238 scientific papers involving cryopreservation of microorganisms [16]. These agents may provide protection from cryoinjury by binding intracellular water, thus preventing the formation of ice crystals and the excessive cellular dehydration, or by reducing the concentration of intracellular salts. Non-permeable cryopreservants (polysaccharides, proteins, dextrans) are particularly suitable for microorganisms, because they adsorb on the microbial surface and form a viscous layer that is highly effective in protecting microbial cell walls and membranes [16]. The cooling rate of the sample plays also a major role in cellular preservation after freezing, with different cell types having different survival rates depending on the rate used for their cooling. Generally, a slow cooling rate, in the order of 5–10 °C/min, does not allow intracellular crystallization and results in a high cellular viability after storage. A recent paper suggested that ultra high cooling rates (>5000 °C/min) may also result in high viability [17], but this solution is technically demanding and not available in the majority of biobanking facilities. Freezers with computer-driven programmed cooling/thawing rates are available on the market, allowing a strict and effective control and optimization of the cooling process. After storage, and immediately before the desired use, gentle thawing at 37 °C in a water bath can rapidly reawaken cells. Automation is increasing also in the field of cryopreservation and covers everything from normal lab freezers to large integrated archives. Industrial companies have developed fully automated storage and retrieval systems, which operate in low temperature freezers (−80 °C) and contain computer-driven robotic systems that scan and sort cryogenic vials or microplates from the freezers [7]. At the moment, due to their extreme sensitivity to minimal temperature variations, peripheral blood and tissue cells processing is not feasible with automated storage/retrieval systems. Cryopreservation is the most widely used method for the long-term storage of cells and cellular products; major disadvantages are the relative high costs of equipment and reagents and, in the case of liquid nitrogen, the need of a constant supply, as any interruption in flux causes the loss of stored samples.
Freeze–drying, also called lyophilization, is an alternative method for the long-term storage of biobanked samples. Freeze–drying is a three-step process. In the first step, the material is frozen to convert the water into ice. In the second step, the ice formed after freezing is removed by conversion from solid to vapor form. In the third step, the additional water left in the sample because of its strong bound to the solid components of the sample is converted into vapor and removed. The lyophilized material is then stored in sealed vacuum ampoules that are used throughout the entire freeze–drying process. Before utilization, the samples can be restored by opening the ampoules and by the addition of sterile water. Freeze–drying causes a form of damage that is termed lyoinjury. Intracellular proteins compensate the loss of hydrogen bonding with water by protein–protein interactions that lead to denaturation and loss of their activity. In membranes, water loss causes phase transitions from the biologically active fluid to gel phase, with possible alteration of their functions [18]. Bacteria and yeasts are quite tolerant to lyophilization and therefore freeze–drying of these cells can be carried out satisfactorily, while cells with higher structural complexity are less suitable for lyophilization [19, 20]. Freeze–drying requires a special apparatus (a freeze–dryer), but, after the process, the samples can be kept at temperatures >5 °C, so freezers are not necessary. Although the majority of biological banks rely on cryopreserved samples, freeze–drying in general ensures a satisfactory long-term viability, avoids contamination during storage (ampoules are tightly sealed) and requires simple storage and distribution. Major disadvantages of lyophilization are: it is a time consuming process not suitable for all cell types (i.e., some eukaryotic cells do not tolerate lyophilization) and it requires detailed definition and optimization of specific protocols for individual cell types or microbial species.
Storage of viable microorganisms
In most laboratories, bacterial or fungal strains, parasites and viruses need to be maintained for epidemiological purposes, quality control, teaching, and research. In the last few years, a special scientific requirement has arisen regarding the long-term viability and the retention of selected properties of genetically modified microorganisms.
Storage of bacteria
Bacteria are recovered from clinical, environmental or from other specimens by incubation in media routinely used for their isolation. Cultures are allowed to mature to late growth or stationary phase before being harvested [21]. After confluence, bacterial cultures are suspended in liquid media and processed for storage. Because of their cellular organization and regrowth capacities, bacterial prokaryotic cells can be either cryopreserved or freeze–dried. The majority of bacterial stock isolates can be easily kept at −80 °C, although the supporting material, the concentration of the initial inoculum, and the type of cryopreservative used may have a significant impact on bacterial survival and regrowth capacities [22–24]. Small glass or plastic beads can also be used before freezing; the bacteria will coat the beads and then individual beads can be removed after storage, avoiding thawing of the entire sample. After storage, a rapid thawing of cultures and a quick transfer of bacteria to an appropriate growth medium is recommended. In general, bacteria can be stored at −20 °C for one to three years, at −70 °C for one to ten years, while freezing in liquid nitrogen preserves bacteria for up to 30 years [21]. It has been shown that, after 12–18 months, viability of common pathogenic bacterial species stored without preservants dropped down to <20% of the initial inoculum, while the addition of these substances raised viability to >80–90%[16, 22]. Experimental infections using stored bacteria have shown that virulence properties are usually maintained after lyophilization or freezing at −70 °C [25]. After storage and thawing, however, some "fastidious" bacterial species may pose special problems in terms of viability and stability of antigenic, molecular and biochemical properties (Table 1). Neisseria gonorrhoeae and Helicobacter pylori are prototypic "difficult to grow" bacterial species, whose characteristics have been studied after preservation [26, 27]. Harbec et al. [26] showed that N. gonorrhoeae strains could be stored at −20 °C or at −70 °C by using specific cryogenic protocols. After 18 months, strains were viable and retained their original antimicrobial susceptibilities, making both methods suitable for epidemiological purposes (i.e., comparison of antibiotic resistance profiles) or for research protocols. Helicobacter pylori is another bacterial species that is very sensitive to storage conditions. To adequately preserve the viability and the genetic and physiological characteristics of helicobacters, Shahamat et al. [27] suggested that, when cryopreserving Helicobacter pylori, the initial inoculum should be greater than 5 × 106 cells/ml and at least 90% of the cells should be vegetative, i.e., spiral shaped cells at microscopic observation. They also tested the effects of different cryopreservative media for bacterial storage at −70 °C or in liquid nitrogen. Although most of the strains did recover within 24 months, longer periods of storage significantly lowered the percentage of viable strains after thawing (around 60%). These data confirm that selected bacterial species are particularly fragile after long-term storage and that specific protocols aiming at improving their survival are still required. Mycoplasmas and chlamydiae differ from the bacteria commonly encountered in biological and environmental specimens on account of their cellular composition, their lack of staining with the Gram stain, and their special in vivo and in vitro growth characteristics. A few scientific papers have demonstrated that, despite their peculiar growth and metabolic requirements, mycoplasmas and chlamydiae can be successfully stored in cryopreserved or lyophilized vials for up to ten years [28, 29]. However, a loss of 95% in infectious elementary bodies of Chlamydia species may significantly reduce recovery after storage, suggesting that cryopreserving these species may require adjustment in the initial inoculum and optimization of the freezing protocols [29]. The survival rate of bacteria after freeze–drying has been measured by Miyamoto-Shinohara et al. [30]. They showed that the survival rate after ten years of storage varied significantly according to the species tested; in fact, the survival rate of gram-positive bacteria was generally higher than that of gram-negative bacteria, probably because of their greater resistance to drying due to the structure of their surface components.
|Bacterial species
|Neisseria gonorrheae
|Helicobacter pylori
|Haemophilus influenzae
Storage of fungi
Unusual fungal infections are emerging as important morbidity and mortality causes in immunocompromised human hosts and pose special problems to diagnostic laboratories; in addition, fungi possess an enormous potential to provide solutions in agriculture, environment, human and veterinary medicine. These reasons explain the growing interest in the characterization and preservation of these microorganisms. Many protocols have been suggested to be suitable for preservation of fungi, although no individual preservation technique has been successfully applied to all fungi [4, 31–33]. As first step to preservation, fungal cultures are obtained by conventional sampling techniques and are further grown on Sabouraud agar or on other media widely described in literature [11]. Usually, a cellular amount sufficient to establish a biobank for yeasts is obtained after a 48–72-h incubation in aerobic atmosphere, while molds may require 7–12 days of incubation. Although some cryoprotective agents have been shown to facilitate the dispersal of fungi, the difficulty in obtaining homogeneous suspensions after culture may compromise the possibility to prepare a fungal suspension adequate for freezing [32]. Cryopreservation in liquid nitrogen and freeze–drying (lyophilization) are the methods recommended by the American Type Culture Collection for fungal repositories [34]. Other techniques may be appropriate for organisms that cannot be cryopreserved or lyophilized, such as storage in soil, water or mineral oil [4, 35]. Cryopreservation at temperatures <−140 °C is the preferred technique in most microbial biological banks because cultures can remain stable for long periods due to little metabolic activity occurring at that temperature. The technique of freeze–drying is most suitable for members of the Ascomycota, Zigomycota and some Basidimomycota, with survival in excess of 30 years for some isolates [4]. The main advantage of this technique is that sealed ampoules offer a consistent protection against air dispersal of fungi during storage or during packaging and shipping to distant laboratories. The main disadvantage of freeze–drying is that lyoinjury and genetic damage may occur during the cooling and drying stages [36, 37]. Because both cryopreserved and lyophilized fungal cultures present problems of viability after reconstitution, it is very important to check viability before and after preservation independently of the technique used. Experimental protocols on stored fungal cultures established that acceptable viability for fungi is the germination and development >75% of propagules/cells [4]. Although the majority of fungi can be preserved with the above-mentioned techniques, some "preservation recalcitrant fungi" cannot be. These fungi often include those that do not sporulate in culture (Oomycota, Basidiomycota) and others, which are difficult to maintain in culture (Diplocarpon) or are facultative pathogens (Table 2) [4].
|Site of isolation
|Saprolegnia spp.
|Fish pathogen
|Aphanomyces spp.
|Water, vegetables
|Fish pathogen
|Serpula lacrymans
|Human alveolitis
|Vegetables, flowers
|Site of isolation
|Saprolegnia spp.
|Fish pathogen
|Aphanomyces spp.
|Water, vegetables
|Fish pathogen
|Serpula lacrymans
|Human alveolitis
|Vegetables, flowers
Storage of parasites
Cryopreservation at ultra low temperatures represents an acceptable choice for the long-term storage of parasites; however, the majority of the conventional methods for the propagation and preservation of parasites have some limitations, including the length of the procedures used, difficulties in initial isolation, the loss of strains, bacterial and fungal contamination during manipulations and changes in the original biological and metabolic characteristics [38]. The technical procedures used to cryopreserve parasites do not differ significantly from those used for other microorganisms. In general, glycerol and dimethylsulfoxide have the highest cryoprotective effects, but glycerol permeates the cell more slowly than dimethylsulfoxide and requires a period of equilibration [38]. The optimal cooling rate is highly variable depending on the parasitic species, while faster thawing rates produce usually best infectivity or motility [39]. Few other important peculiarities of parasitic cryopreservation have been described. Firstly, it has been shown that blood protozoa can be frozen without cryoprotectants or a precise control of the cooling rate [38]; secondly, developmental stages within the same parasitic species may be selectively affected by different freezing protocols [40]. Additional information is available on the cryopreservation of selected parasitic species. Morphological studies on Trypanosoma species documented ultrastructural changes in frozen cells (membrane injuries, denaturation of chromatin, occurrence of large vacuoles), while functional experiments demonstrated that the infectivity of Trypanosomes [41] and of Strongyloides stercoralis was partially maintained [42]. Fayer et al. [43] demonstrated that oocysts of Cryptosporidium parvum can retain viability and infectivity at temperatures between −10 and −20 °C. Vaccinology and diagnostic immunology constitute important fields taking advantage of the capacity of long-term storing techniques to provide parasitic products that keep their stability through time. In fact, the preservation of Toxoplasma gondii bradyzoites has been successfully applied to make a live bradyzoite-based vaccine [44], while cryopreserved plasmodia were a source for the preparation of parasitic antigens to be used in diagnostic or research immunoassays [45]. Preliminary experiments have demonstrated that also the lyophilisation technique might be useful for the preservation of apicomplexan parasites, especially those with a potential for being used as live vaccines, such as Babesia and Plasmodium[46].
Storage of viruses
The methods by which viruses may be stored are similar to those employed for the other microorganisms and the majority of literature using viral repositories store viruses by cryopreservation or freeze–drying. Their infectious and pathogenic nature and the absence of therapeutic options able to eradicate many viral infections, require special safety precautions for handling biological samples. First, the laboratories handling viruses must adopt biosafety level II or biosafety level III facilities, depending on the characteristics of the viruses handled; secondly, biobanks having viral stocks require experienced personnel adopting written, internationally accepted guidelines for biosafety. The recent episodes of fatal infections during the laboratory manipulation of SARS coronavirus has evidenced that in many laboratories safety precautions are less than optimal [47]. Exhaustive biosafety manuals are found in the WHO and CDC web sites (see Section 5). The appropriate selection of the specimen represents an important issue in virology. The specimen or the collection site must permit recovery of the virus under study, avoiding contamination with undesired microorganisms, and it must represent the process to be investigated by diagnostic or research protocols. Several papers have demonstrated that this goal may be sometimes difficult to achieve. A prototypic example includes the poor association between the detection of human papillomavirus DNA in oral exfoliated cells as compared to biopsies of cancers of the oral tract and the presence of an oral tumor, indicating that exfoliated cells contain a mixed human papillomavirus population that does not accurately reflect the viral population involved in the development of the tumor [48].
An excellent state of the art article published in 1999 [49] punctualizes some important general rules necessary for an adequate preservation of viruses: (a) Freeze–dried viral preparations can be maintained for decades at 4 °C; (b) viruses stored in liquid nitrogen retain their pathogenic properties. It must be kept in mind that viral stocks stored in liquid nitrogen containers may cross-contaminate other samples archived in the same container and, therefore, vapor phase containers are best indicated. It has been suggested that including individual vials containing viral preparations in a second, heat shrinkable tube may protect from cross contamination and from accidental ruptures in liquid nitrogen containers [49]. Storing conditions may vary according to the final destination of banked materials: if retention of virus infectivity is not essential, for instance when the sample is used to obtain antigen preparations, it can be safely stored at −20 °C; (c) proteins added to biobanked viral stocks provide protection of virus infectivity in stored samples by mechanisms that are not completely understood. One hypothesis suggested that proteins provide buffering capacities against pH changes, and reduce processes that damage nucleic acids; (d) virus infectivity is best maintained when samples are preserved in small volumes, because freezing and thawing the samples occur much quicker and viruses are much less damaged when preparations contain high viral titres. Viruses are quite stable at low temperatures, although the genetic material of RNA viruses may present a certain degree of fragility when inappropriately handled. The small size of the virus and the absence of free water are largely responsible for this stability; viruses with lipid envelopes are often less stable than non-enveloped viruses at room temperatures, although this difference is less evident at low or ultra low temperatures [49]. The effects of multiple freezing and thawing of serum specimens on TT virus and Hepatitis B virus stability has been evaluated by Durmaz et al. [50]; they showed, by using semi-nested PCR and a hybrid capture system, that viral DNAs continue to be positive until the seventh cycle of freezing and thawing, but these qualitative results do not allow any conclusion on the possible quantitative effects on the viral load of repeated freezing and thawing. More detailed quantitative studies on HIV RNA have demonstrated that RNA levels in plasma stored for up to one year at −70 °C are quite stable [51, 52]. In these studies, the stability of nucleic acids was ensured by the fact that the protocol for plasma handling included an additional centrifugation step to eliminate contaminating blood cells that contained RNases. Our quality control data on plasma EBV-DNA, however, suggest that a certain loss of viral DNA occurs after two to three years of cryopreservation (P. De Paoli, MT Bortolin, unpublished data).
Storage of genetically modified microorganisms
Very few studies have monitored viability and plasmid retention in frozen recombinant microorganisms. These cultures are widely used for industrial and research purposes, like production of enzymes, nucleic acids, controls, etc. Recently, Koenig [53] studied the long-term viability and plasmid retention of recombinant Escherichia coli strains. She demonstrated that frozen cultures showed stable viability and high plasmid retention rates over periods of up to 11 years; in few cases lower viability and/or plasmid retention were due to an inappropriate selection of initial colonies. Although this study did not address plasmid structural stability, it showed no changes in the restriction patterns of the strains, indicating the absence of gross structural instability of transfected sequences. Similar results were obtained by investigating storage stability in liquid nitrogen and post-thaw productivity of a recombinant strain of Saccharomyces cerevisiae expressing the human factor XIIIa protein [54]. Storage stability and optimal product expression after thawing are essential for the proper use of cryopreserved recombinant microorganisms.
Storage of human and animal cells or cell lines
Human or animal cells or cell lines may be stored for further detailed phenotypic, genomic or functional studies in living cells, to recover virions or viral nucleic acids and to enhance the availability of nucleic acids from the study subjects (i.e., cultured lymphoblastoid cell lines allow harvesting virtually unlimited amounts of DNA from single subjects). Cryopreservation for transfusion or for bone marrow transplantation is beyond the scope of this review and will not be included. The different response of human and animal cell components to freezing conditions and the existence of cell-to-cell contacts or mutual relationships between cells are major problems when storing these cell types. Human and animal peripheral blood mononuclear cells (PBMCs) can be easily obtained by density centrifugation through gradients and are the most frequently stored cell types. The generally used cryoprotectants include proteins (fetal bovine serum) and dimethylsulfoxide (DMSO). Factors influencing the quality of stored blood cells include the anticoagulant used and the storage temperature. For instance, disadvantages of heparin used as blood anticoagulant include its influence on T cell proliferation and problems in inhibiting the polymerase chain reaction assays [55, 56], which have not been confirmed in other studies [9]. Cryopreservation at −80 °C or in liquid nitrogen is the only established method to preserve PBMCs samples valuable for future experiments, and published guidelines establish that PBMCs may be stored for a minimum of 24–48 h at −80 °C and then transferred to liquid nitrogen for long-term storage [12]. One major problem of cryopreservation is the significant reduction in the number of viable cells after thawing [57, 58]. In PBMCs, cryoinjury appears to be mediated also by the specific activation of caspases and the induction of mitochondrial membrane permeability [57]. In HIV-infected subjects, the immune function, as measured by in vitro assays, improves significantly in response to antiretroviral therapies [59]. As a result, in vitro measurements of lymphocyte proliferation and cytokine production are being explored as a substitute for clinical endpoints in therapeutic clinical trials [60]. However, due to the damage induced by the HIV itself, by additional viral infections, and by antiretroviral therapies, lymphocytes from HIV infected subjects may pose special problems of cryopreservation. Therefore, extensive studies investigated the effects of cryopreservation on the immune functions of HIV-infected patients. The results of these studies indicated that the median of cells recovered after several years of preservation was around 50% of the initial number, and that viability greater than or equal to 70% was necessary to obtain reproducible and affordable results of the in vitro testing [61, 62]. These data imply that storing at least 107 PBMCs per vial yields sufficient viable cells for phenotypic and/or functional analysis. Phytohemoagglutinin assay and cytokine production are useful in vitro indicators of PBMC functions; both these assays demonstrated that cryopreserved cells maintain a consistent proportion of their functional activities even after ten years of storage [60, 62]. Granulocytes are seldom used in biological banks; these cells are particularly sensitive to storing procedures, although their yield after storage is >80%. Granulocytes are mainly used as a source of DNA, but simpler methods, using peripheral blood buffy coat or whole blood as a DNA source, have been recently employed [8, 9]. These methods require minimal sample manipulation; therefore they are less expensive and less time consuming.
It has been demonstrated that the presence of cell-cell and cell-matrix attachments may alter the cryobiological properties of cells [63], suggesting that special methods are necessary to store adherent cells or cell lines. Pegg [64] recently overcame these limitations developing a method for the cryopreservation of a human endothelial cell line as a single suspension or as a confluent layer on microcarrier beads. Furthermore, a recent report [65] described a simple method of freezing human or animal cells while still attached to the substratum of a multi-well plate; these authors demonstrated an effective storage of adherent mammalian cells up to three months at −80 °C. This system offers consistent savings of time and effort and may be very useful when screening large panels of cell clones.
Storage of serum/plasma
Plasma and serum can be used to measure antibodies against selected pathogens, to measure immune related substances like cytokines or vitamins and other metabolites. In addition, these blood products may permit to obtain nucleic acids from blood borne viruses or from bacteria during bacteriemic diseases [11, 12, 66]; finally, when a cellular source is not readily available, they can be also used to extract small quantities of DNA of human or animal origin [67]. Plasma can be obtained from anti-coagulated blood samples through separation of cellular components, while serum recovery can be optimized by gel separators and clot activators [68]. Plasma is more suitable for nucleic acid and cytokine detection, although the choice of the anticoagulant may hamper the possibility to subsequently test some specific analytes, while serum specimens are ideal to measure antibody levels. Quality control programs have shown that, due to the appreciable stability of immunoglobulins during time, serum/plasma specimens for antibody assays can be stored for years at −20 °C [68, 69]. Cytokines have short half-lives in vivo and may be further degraded in vitro. On the other hand ex vivo induction of cytokines can occur during blood processing. Therefore plasma or serum samples for cytokine assays must be collected and processed almost simultaneously. The stability of cytokines after storage has been studied by Kenis et al. [70]. They showed that serum samples for the determination of Interleukin-6, soluble IL-6 and cytokine inhibitors can be stored at −20 °C for several years, while for Interleukin-10 determinations storage at −70 °C is recommended.
The quality control program for storage of blood specimens in the Malmoe Diet and Cancer Study demonstrated, by the use of bioassays based on the measurement of endogenous oxygen metabolism, that plasma and serum can be preserved without any degradation for at least up to 140 weeks [71].
DNA and RNA extraction and storage
Sample processing may be quite simple, for example separation and freezing of serum or plasma or freezing of bacterial cells. More complex sample processing includes the extraction and purification of nucleic acids. The quantity and quality of nucleic acids obtained for biobanking purposes depends from several factors: (a) the quality of the original biological sample; (b) the extraction method used; and (c) the storage conditions [72]. DNA can be extracted from nucleated cells, including whole blood, buffy coats, mucosal swabs, and from fresh or formalin-fixed tissues. DNA and RNA can be also extracted from serum/plasma or other fluids, from which low yields can be obtained.
The quality of nucleic acids obtained from biological samples depends from the quality of the original sample, as mentioned in the chapter "Sample collection and processing". The quality of extraction and the conditions used for storage are the other factors of paramount importance when handling DNA or RNA. DNA can be extracted from biological samples using phenol-chloroform extraction [73], while RNA is extracted by the guanidinium isothiocyanate plus ethanol technique [74]. New methods, including commercially available kits (Quiagen, etc.) may include the use of resins or affinity gels [75–77]. As compared with new systems, traditional methods have generally a higher yield of nucleic acids, although the presence of toxic chemical reactants may discourage their use. The presence of bacterial and fungal integuments may require the use of stronger extraction systems, like those based on alkali wash and heat lysis [78].
DNA is the most stable component in biological samples. In fact, isolated DNA is generally stored at 4 °C for several weeks, at −20 °C for several months and at −80 °C for several years [8]. RNA is very sensitive to degradation by RNAses and its integrity is guaranteed with RNAse-free handling and by the addition of RNAse inhibitors. Furthermore, the integrity of RNA is maintained at very low temperature and must be stored at −80 °C [8]. The structural integrity of nucleic acids can be measured by the determination of the size of extracted DNA using agarose gel or SDS-PAGE electrophoresis, while functional integrity can be assessed by specific amplification procedures [72]. To address this issue, Jerome et al. [79] performed quantitative Real time PCR for herpes simplex virus DNA in a large series of fresh specimens and again after 16 months of storage. They demonstrated that viral DNA remains quantitatively stable for at least 16 months when frozen at −20 °C. Similarly, Walther et al. [80] revealed that closed circular plasmid forms, which are sought for efficient gene transfer procedures, are preserved after long-term storage. Although these conclusions may apply for other microbial DNAs, there are presently no additional experimental evidences. RNA integrity can be measured by reverse transcription PCR or by a commercial system using RNA chip that is able to determine purity, degradation and ribosomal RNA contamination of separated RNA preparations [81].
Filter paper represents an attractive alternative to freezing for the long-term storage of DNA. Filter paper methods require less equipment, less technical expertise and are less expensive. Samples stored with this method have been used to amplify human, viral and bacterial DNAs [72, 82, 83].
Documentation and data bases
Informatics can facilitate sample registration and unique identification, the cataloguing of sample properties (type of sample, type of specimen, presence of associated diseases and/or therapeutic protocols, environmental information, experimental conditions, etc.), sample tracking, quality assurance and specimen availability [6, 81]. The components of a banking inventory system must permit data entry, queries across groups to facilitate any combination of sample identification/retrieval per trial or project, and the preparation of periodic reports; furthermore, data must be available for a long period of time, maintained in a standardized format and combined from collaborative sources [81].
Studies that store identified or identifiable specimens must maintain confidentiality. Anonymous and anonymized biological materials are impossible to link to their sources, while identified and identifiable materials can be linked. In the latter cases, although national regulations may differ, coding of the biological materials and for the code key to be stored separately is usually recommended [85].
Software for biobanks have now been designed based on cooperative study networks [6, 81] or tailored on an individual basis [84]. Management software integrating sample preparation, processing and storage are also present on the market (CryoByo System, Paris, France). Biological banks storing microorganisms may require additional information that partially differ from those required for biological samples of human or animal origin; for instance, the software used in the laboratory of medical microbiology at the Centro di Riferimento Oncologico, Aviano, includes specific fields for each bacterial strain stored in the bank, indicating the pertaining microbial genus and species and the biological material from which the microorganism has been cultured. Specific queries for bacteria include the identification in the repository of microorganisms of the same species, or isolated from the same disease group, or having serum or DNA samples obtained from the same patient on the same date, etc.
Quality control
The Swedish Medical Research Council [85] recommends that all biobanks containing biological materials and associated information must have an organization with explicit procedures for quality assurance, including systems for storage, coding and registration. Biobanking facilities must adopt general laboratory standards, such as Good Laboratory Practices or ISO series and participate in an external proficiency program reflecting the specialty of the laboratory. In addition, there are four levels of quality control applying more specifically to biological bank facilities: (a) training and certification of personnel and assignment of bankers' responsibilities; (b) control of instrument variability; (c) verification of yield and purity of processed materials; (d) long-term control of stored samples. Briefly, the quality control of instrument or equipment performance includes the documentation of function checks, as specified by the manufacturers, the documentation of preventive maintenance, and the maintenance of records for the life of the instruments [86]. Yield and purity of the materials depend on general quality rules in microbiology [11, 12, 86], but the specific biological or molecular assays to test these parameters in samples containing microorganisms, animal, and human cells or nucleic acids have been mentioned in the previous sections dealing with each material. Storage quality control includes the periodic verification of alterations induced in the samples during storage and the documentation of freeze-thaw cycles of each sample. Quality control should also specify the limits of acceptability of recovery, purity, structural and/or functional integrity of the samples after storage, and the number of freeze-thawing cycles, which permit the distribution and further use of the samples for epidemiological, research or diagnostic purposes. Finally, an audit consisting in the control of the application of the quality system is recommended [87].
Ethical issues
Regulations pertaining to the collection and storage of samples of human origin, including tissues, cells and nucleic acids, and of the related information are at the beginning stage in most European countries and in the U.S.A. The first issue to be regulated regards the identification types that may be employed to store the samples, while a second issue includes the requests for banking: the American society of human genetics stated that "a DNA bank or a DNA diagnostic laboratory should accept samples only in response to requests from health-care professionals and not in response to requests from individuals …" [88]. National or international regulations must ideally also define the security and regulations of banking facilities. Extensive reviews of the existing professional guidelines, legal frameworks, ethical issues and other documents related to the data storage and DNA banking practices in public and private sectors have been recently published [85, 86]. Presently, ethical issues and regulations do not apply to microorganisms stored in biological banks.
Concluding remarks
This review presented the state of the art in staff and equipment necessary to set up a biobanking facility, in the problems related to sample collection and processing of various cell types or cellular components, and in data management and quality control. Microbiology laboratories rely even more frequently on biological bank specimens for diagnostics and research. To ensure quality, a biobanking facility must organize the space, equipment and personnel, adopt strict protocols for proper handling of the specimens, set up a quality program including periodic controls of the instrumentation, training of the laboratory staff, and quality control of stored specimens. Because biological banks constitute an invaluable resource for research and diagnostics, it is conceivable that they will require, in the near future, additional investments in terms of human and material resources. Single laboratories may be unable to support their own biobanking facility and institutional or multi-laboratory approaches will be the future of biobanking. The Commission of the European Community has already recognized the need to dedicate a financial contribution for the implementation of European quality standards for biobanking, aiming at supporting large scientific projects involving common diseases. It is conceivable that similar examples may allow the creation of reliable biobanking facilities providing materials that support research projects and thus improve our knowledge in biological sciences.
This work was supported by the Fifth Framework Programme of the European Community for Research 2002, Project "Evaluation of the Role of Infections in Cancer using Biological Specimens Banks" and by Progetto "Infezioni e Tumori", Fondazione per la Vita, Pordenone. Mrs. Luigina Mei is acknowledged for English language corrections in the manuscript.


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Hi all! For those with more experience in Luanda... How is the movement in the city on festive days like Christmas? Restaurants and bars are closed or opened?Does anyone know if there is any Christmas dinner (or party) on the 24th evening?Thank you all for any information.

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- Project Details
Title International Program for Certified Future Strategist
Project Number 2012-1-SE1-LEO05-11667
Product Inventory of future methodologies
Title Inventory of future methodologies
Product Type
Marketing Text Background report on similar training systems and course material.
Description We produced an Excel file with all existing training courses in the area of future studies. The inventory included methodologies and training offerings by private partners as well as universities and other public institutions.
Target group Partners in the consortium
Area of application
Product Languages English


text: Off all the bearings on a bike, the pulley wheels spins at the highest RPM. CeramicSpeed pulley wheels cut down on drag in your drivetrain compared to standard pulleys. The aluminum alloy is processed in Denmark and the Grade 3 ceramic bearing are installed at CeramicSpeed. CeramicSpeed has pulleys for Shimano and SRAM 9/10 speed road and mtb bikes and for Campagnolo 9/10 and 11 speed road. Feel the difference that high performance CeramicSpeed pulleys can do for your bike.


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Neat, respectful person. Happy to share a drink at the end of day.
I'm a musician, gardener and a great cook, with a good sense of humour. I love animals and art. Lefty politics, I make my own wine and sourdough bread. Pretty neat. Have 2 dogs that would visit one or two times a week but not stay full-time. I'm a community lawyer working for Legal Aid in Morwell. Looking to relocate permanently to the valley in 2018 depending on work. Need a place from the end of Feb but would consider paying rent from before that date for the right place.
Have a fun n outgoing bubbly personality, non smoker , easy to getalong with enjoy's a social drink. Im very clean n tidy (house proud) I work part time as a car detailer n substitute with centerlink payments. Im boarding in Trafalgar with friend but we need own space. I enjoy the outdoors tomboy @ heart.
Hi, I am University of Melbourne student, from India.
Hello My name is Mery Alvarez, I am from Colombia, I am 25 years old and I will be in Melbouerne to take an English course for 6 months. I am an open and cheerful person, I love to share and to know new cultures. I am clean and orderly and I would like to share a room with another woman. Anything you need to know about me you can write to me. Hugs Mery
My name is Ricky Fred I work full time at Rubber Tough installing rubber surfacing for schools parks and also kindergartens ect. I'm very tidy I'm easy to get along with I'm kind and consider others around me, I am very organised. And am a very happy person with a soft nature.
I'm a 30 year old male and until last year I worked full time at as an IT/AV technician where I worked for 10 years. I am seeking accomodation in Pakenham and the surrounding areas, as well as anywhere from Pakenham to Warragul including surrounding areas. I do not drive so it is essential the house be close to public transport, a train station is ideal but a nearby bus is good too. I will consider other areas if the house is a good match. I am hoping to have meals (just dinner really) included. Some storage space would also be required. Unfortunately throughout the last year things have been a difficult for me. I suffer from an anxiety based mental illness called obsessive compulsive disorder (OCD) which I started to really struggle with. Hopefully you won't judge me on that. As a result I ended up resigning from my job earlier this year. I am now coping a lot better now and I am currently working part-time (with almost full-time hours) but am looking for something different. I am a genuinely nice and caring person, I've just been through a lot lately and I'm just trying to get settled again. Unfortunately I have had issues with being screwed over by several housemates in the past, which certainly hasn't helped things. If you think you can help me or if you have any questions or advice at all please feel free to contact me - I have nothing to hide and I am happy to discuss my situation. Shaun
I generally keep to myself, like to stay fit and active, go to the gym often and eat healthy. I'm a clean and conscientious person, and pretty handy around the home with work or maintenance. I've been working full-time locally since late 2013 and occasionally get some tomatoes to bring home ;)
Adventuring out for a different share house. I enjoy jokes and having a laugh. I keep to myself mostly and find myself creative, currently working on an application for a tattoo studio. I am vegan and respect others life style choices. Currently a student studying part time as a beauty therapist in the CBD and currently working part time as a barista in Pakenham.
I have been travelling around Australia staying at various Buddhist Monasteries. I have decided to settle down for a while. I have a PhD, meditate, and like nature. I am a quiet person but also enjoy social interaction. I am an artist and am looking to start my practice again in the near future. I am a non drinker and non smoker. oh, and I like cats.
Lived independently since 17, house trained, quiet and keep to myself. Currently not working due to injury and surgeries, but about to commence post-grad psych studies, and (pre-time off) work in acute psychiatric care. Have a son who is currently only spending weekends with me.
We both work full time and are very respectable, looking for a place to call home. We are interested in team ups too. contact me to find out more!
Hi My name is Young. I am a quiet , neat and clean person. I have a long history of the house sharing, however I never had an issue with other people. I am very responsible and respect others.
Hi I'm Shannon, I am a student at Fed uni and in desperate need of accommodation. I have recently lives in a house with 8 people and only 1 bathroom so I won't take up the bathroom for very long! I am clean, I know how to cook and budget. I have my license and I have an addiction to Netflix so I will pay or help pay for internet!
Freindly, funny, tidy but not a clean freak and I understand personal spaces
Like my own space but also very friendly and respectful Clean and enjoy a few wines and out for dinner with friends Gym 3 to 4 days a week
easy going


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For the third time this season, Glasgow went down to defeat, missing enough kicks to have reversed the result against Munster. But this time Sean Lineen, the coach, says he could not have been more proud of the way his players performed in adversity at the reopening of Thomond Park.
"In the end, we thoroughly deserved a bonus point, but it was not to be," he said yesterday, after racing back from the Saturday night match in Limerick to experience life on the other side of the whistle as referee at a mini-rugby tournament in Edinburgh. "I'm proud of the


text:
The Cloister and the Hearth: A Tale of the Middle Ages.
READE, Charles.
Description: Illus., lettered tissue guards. 4 vols. 8vo, orig. embossed boards, d.w.; (chipped), in extra mylar wrs., t.e.g. N.Y., 1892.
Comments: One of 100 numbered copies on large paper.Binding: hardcover
Language: English
Type: Books
Book ID: 58114


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J. Varisco Events is a full service Wedding and Special Event planning company. Janie has been planning weddings and special events since 1997. She began her career helping her friends plan and execute their weddings. She then teamed up with Stage Call Entertainment to assist in the production of larger events and festivals such as Le Festival de Mardi Gras a Lafayette, Freedom Fest and The International Rice Festival. She has also worked with several major recording artists such as Joe Diffie, Los Lonely Boys, Kool and the Gang, Blake Shelton, Montgomery Gentry, John Connelly, Little Big Town, Percy Sledge, Evanescence and Kansas.
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Rusty, hi. I can't remember if anyone answered this request for you yet or not.
ORANGE TREAT (B. Hamner, 990) TB 37", M. Ruffled orange (RHS 26B);
tangerine beard; slight fragrance.
I couldn't find a ROYAL FLUSH, which doesn't mean it doesn't exist,
necessarily. I'm not perfect for one thing, and for another, I don't have
the '97 list. But I did find ROYAL BLUSH, and knowing of Mr. Keppel's
attitude toward easily confused names, I suspect this is your plant.
ROYAL BLUSH (H, Nichols '78) SDB, 14", E-L. S purple; F purple, washed
red-violet, heavily ruffled, electric blue beard.
Little Rock


text: Well, is it? Is there a such thing as bad, destructive diversity which should not be tolerated? If yes, how should we go about identifying good and bad diversity? And, what are some examples of good and bad diversity? Or, is all diversity good and should, therefore, not only be tolerated, but celebrated? Joseph Botwinick


text:
In Pirates Past Noon, it's a treasure trove of trouble when the Magic Tree House carries Jack and Annie back to the days of deserted islands, secret maps, and dangerous pirates! Will they discover the hidden buried treasure, or will the pirates discover them? Jack and Annie are put to the test when they are accidentally brought on board a pirate ship, and they are forced to think fast or face the possibility of walking the plank!
Don't miss any of Jack and Annie's adventures in the Magic Tree House series.
Mummies in the Morning ©1993 Mary Pope Osborne, Pirates Past Noon ©1994 Mary Pope Osborne; (P)2000 Random House, Inc., Imagination Studio, an imprint of the Random House Audio Publishing Group
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Are you ready for a Stress-Free Holiday Season?
By Catherine Cassidy
OK. That might sound like an oxy-moron, but bear with me … it's possible!!
Don't hold this against me, but I rarely stress about the holidays. I think it mostly has to do with the fact that I love every aspect of it – getting together with family, dressing up for all the parties, buying gifts for my favorite people, etc.
So, since you might not have the same love (or you just have SO much on your plate), I want to share mine with you!
Top 3 Holiday Buzzkills and how to Overcome them …
Gift Shopping … with the masses
I'm going to let you in on a little secret: I strongly dislike shopping. I'm not a shopper AT ALL! Well, I don't like it when I have to do it with everyone else and deal with the long lines, finding a parking spot and the crowds (not to mention here in LA at least, the traffic!).
So, I avoid all of that.
I only shop on a weekday or first thing in the morning on a weekend (IF I HAVE TO!). The optimal weekend time is 10-1. I know when stores and boutiques open and close and I take advantage of that. The chain stores usually open before the boutiques, so I plan accordingly.
(SIDENOTE: Thank you to my mom for instilling this shopping ethic. Though I fought getting up so early on a weekend when I was growing up, I thoroughly appreciate this awareness now.)
When I was at BCBG I was especially spoiled. I could get fantastic gifts for friends and family ridiculously discounted AND I never had to leave my office. I had knowledge of the entire line, how the sizing ran and could pick it up from the warehouse downstairs. Definitely a silver lining!
I shop online when possible. And I totally take advantage of those online sample sale sites. For a list and to sign up yourself all in one place, click here for my blog post on my favorites! Another resource I've used is gifts.com because you can shop by personality … which definitely helps narrow down the selection.
Personally, I love gift giving because it's an opportunity to give a thoughtful gift someone wouldn't buy for themselves. Of course, the thoughtful part is where it can get tricky. If you're really strapped for time, I'd recommend outsourcing it completely. U*styled can help with this as can your favorite salesperson at your preferred store. Call them ahead of your visit with a list of what you need and they can pull it all together for you BEFORE you get there. How cool is that?
Getting ready for the Bosses Holiday Party … with a closet full of clothes and nothing to wear!
Nothing is worse than building a pile of clothes that you don't want to wear when you've got negative 5 minutes to get ready.
Avoid that completely by being prepared. Plan your outfits for your events in advance.
It sounds simple, but it's not because that means setting aside an hour just to have fun playing with clothes. And if you're a busy woman, there is always something else to do with that hour.
So, think of it in terms how many minutes late you'll be to each event PLUS how many minutes it took you to get dressed. 20+20=40 … for ONE event! That's almost an hour right there! Not to mention how much fun you have when you're frustrated with NOTHING to wear … oh yea, that's NOT fun!
Break it down and make it simple …
First, take an inventory of your basics … black pumps, nude pumps, black skirt, LBD, black pants, white blouse, etc.
Now, take an inventory of what could pass for evening and/or holiday … a jewel tone blouse, your sequin tank, your little purple dress, your leopard heels, all of your accessories (an easy way to add holiday sparkle to an outfit!), etc.
Finally, play with mixing and matching. Think outside of the box. Put together your outfits and even take a picture if that's helpful for planning. Just have fun! Seriously, you will surprise yourself! And, I'd love for you to share a pic of your inspired masterpiece. :)
Or, enlist the help of an expert if you REALLY want to dress to impress this holiday season!
Family is FUN … or is it?
I love my family. I love my friends. They are incredibly important to me and I feel really lucky to have them all in my life. I know I'm pretty fortunate to have this. What family drama we might have is very little and more about us never really growing up (my brothers and I definitely still bicker and our dog, Aggie, still gets in the middle of it to break it up!).
This is mostly about stress relief and focusing on what you need.
Make sure you're exercising. Even if it's a 15 minute walk … something to relax and release stress. If you can fit in a hard run, a good weights session or some power yoga, even better!
Take some time for yourself. Indulge in a good book, meditate for 10-15 minutes or even just browse the internet. Whatever feels like a guilty pleasure that will recharge you quickly.
Don't take it personally. Whatever family stuff comes up, don't play into it. You're only responsible for yourself and your actions and what YOU want to do. Simply taking a deep breath can be really helpful to calm you down when temperatures are rising.
Play a game! Get the family together focused on something else – a game, a movie, making dinner, etc. Just enjoy each others company.
Any additional tips are more than welcome. I'd love to share them with the U*styled community. If you're an expert on any of these topics, please feel free to share and I'll post on Facebook for you all! Happy Holidays!! xo
Taking leave of the Corporate Fashion Industry, Catherine Cassidy founded U*styled in 2008 to support busy women everywhere. YOU are our mission. U*styled is dedicated to empowering women and giving you the tools to discover the lifestyle you deserve through personal style. Her unique approach to Personal Styling and Brand Image has had her work across a broad range of industries and career stages including Real Estate, Finance, Consulting, Marketing, Advertising, Entertainment, Entrepreneurs and Celebrity Entrepreneurs.
Additional SMW Stylish Resources:
6 Holiday Party Dresses to Help You Shine


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Hotfrog Canada provides information regarding Cintas-les Gens d'Uniformes in Longueuil QC. Cintas-les Gens d'Uniformes is located at 730 Blvd Nobert, Longueuil QC J4J 5K5 and provides Advertising And Marketing services. Contact them on (450) 674-1585 or by visiting them on their website http://www.sintas.com.
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Rue Saint LONGUEUIL QUEBEC J4H 2X5
bureau 102 LONGUEUIL QUEBEC J4K 5G5
1111 StCharles Street W East Tower - Suite 555 LONGUEUIL QUEBEC J4K 5G4
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text: Seller: decorativeantique (36) 95.2%, Location: Roorkee, UL, Ships to: Worldwide, Item: 332031459834 Home and Office Decor Fully Black Antique Reproduction Table Top Clock This is an Fully Antique Nautical Table Clock.Fully functional Item. Very interesting item!!!! You can take this as decoration piece for your home & office OR as GIFT purpose! Bid With confidence!!!!! You will get the same Clock as shown in pictures. Dimensions: Total Height :7.5 Inches Width is 3.5 Inches NOTE : All Custom Duty And Taxes Will Be Paid By Buyer In His Country. We Use to Deliver our product internationally BY We do Believe and Prefer PayPal for payment. BY Decorativeantique Condition: "BRAND NEW ITEM"


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The Dirtiest Bacteria That Spreads Disease: Caution!
Avoiding Colds, the Flu, and More
Kimberly Clark just did a survey that's pertinent to us all. They went to six, major U.S. cities to determine the "dirtiest" objects we encounter on our way to work and at the office. These are objects that we ALL touch that can spread colds and flu.
The study emphasizes the need for cleanliness and good personal hygiene to maintain good health. After all, the average company loses $1,685 per employee due to them being absent, or showing up at work sick.(1)
1. Stewart, W.F., Ricci, J. A., Chee, E., Morganstein, D.(2003). Lost productive work time costs from health conditions in the United States: results from the American Productivity Audit. J Occup Environ Med 45.12 1234-46.
Bacteria: What the Researchers Found!
Researchers measured the levels of Adenosine Triphosphate (ATP) on common objects we encounter. Any with an ATP reading of 300 or higher are considered to be at high risk to transmit illness.
Here's the "Dirtiest Seven" that they found, in order of the highest percentage levels of contamination:
- Gas Pump Handles
- Mailbox Handles
- Escalator Rails
- ATM Buttons
- Parking Meters/Kiosks
- Crosswalk Buttons
- Vending Machine Buttons
Don't Get Sick: An Easy Solution...
This study is dramatic because it proves that the most common objects, that many of us use on a daily basis, are highly contaminated with bacteria that spreads illness.
You get your gas tank filled on the way to work, stop at the ATM machine, grab the escalator rail on the way to your office... and then you touch your face, most particularly your eyes, nose or mouth... and Voila! You're on your way to getting a cold or flu and even worse, to contaminating those around you!
The Solution: Wash your hands! As soon as you get to work, do it! Then wash your hands throughout the day and the first thing when you get home from work! It's so simple to avoid getting the colds and flu, and to avoid passing it onto others...
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Recently, I wrote about the "Dirty Dozen" or 12, non-organic fruits and vegetables, which actually impart 47-67 servings of pesticides along with the fruit and/or vegetable you may be eating. These 12 include:...
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Anyone can suffer from a urinary tract infection or UTI. In fact, they are the most common type of bacterial infection. They strike children and the elderly, but most of all they strike women. Almost 50% of all...
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Very interesting, and SO TRUE! I'm a nurse assistant, and you are right on the money. Ya got a good hub on a good soothing lotion for my hands? With all the gel and washing, my poor dry hands say "Help us".


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By CALYPSO St. Barth -
Merge yacht hopping stripes with wear now cashmere in this tunic length cashmere pullover. Charmed with a functional button placket at left shoulder and side seam button plackets, this piece depicts nautical at its finest.
28" front & back body length (from shoulder seam)
17 ½" chest width (1" below armhole)
11 ½" neck width (edge to edge)
25 ¼" sleeve length (from shoulder seam)
measurements taken from size small
*Available in sizes XS-L
model is wearing a size small
model measurements are as follows:
height: 5'10"
bust: 34"
waist: 24"
hips: 35"
100% cashmere
dry clean only
made in China
FOR CUSTOMER SERVICE INQUIRIES:Phone
Monday - Friday: 9:00am-5:30pm ET
Saturday - Sunday: closed
JOIN LA LISTE: Be the first to know about new collections and special offers.


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For those of us in the IT service management (ITSM) space, DevOps appeared on our scene around five years ago. And at that time, to most in ITSM, DevOps was seen as an outsider pushing in to places it didn't know enough about. Specifically, it seemed to be an entirely development-driven concept that suggested IT operations wasn't actually needed – that getting development right meant NoOps was perfectly possible, where NoOps is "the concept that an IT environment can become so automated and abstracted from the underlying infrastructure that there is no need for a dedicated team to manage software in-house."
In the absolute sense, this is perhaps even correct. If you could build an IT system that was perfect, in that it never failed, and is so flexible that it's adaptable to every change in customer needs, environment, legislation, etc. – then surely all you need to do is build and test it, then let it go delivering the service without any need for tinkering, fixing, or updating.


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Events Assistant Internship
32.728107, -117.14682)
Cause Areas
WhenIt's flexible! We'll work with your schedule.
Where2215 Park Blvd Suite 2San Diego, CA 92101
The Japanese Friendship Garden is in need of an Events Assistant Intern for our Events Department. Students encouraged to apply!
Students are eligeble for internship.
Please send your resume and cover letter to firstname.lastname@example.com
- Provide on-site coordination assistance for JFG events (including but not limited to interaction with event participants, set-up and/or breakdown, and vendor coordination)
- Answer phone and web inquiries about weddings and private event venue rentals
- Schedule walkthroughs/wedding tours according to Event & Marketing Coordinator's calendar
- Maintain and organize vendor database
- Provide on-site coordination support to Garden staff at private events
- Assist with design, development, and distribution of promotional materials including: newsletters, brochures, flyers, posters, and invitations.
- Market events on community calendars and other marketing channels
- Develop wedding/special event related content for website and blog
- Conduct marketing surveys and research
Please send your resume and cover letter to firstname.lastname@example.com
- Networking
- People Skills
- Organization
- Problem Solving
- Cross-cultural Communication
- Multi-Tasking
Good Match For
Requirements & Commitment
- Background Check
- Must be at least 18
- Orientation or Training
- 5 months minimum committment
- Bilingual a plus!


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Among those of the ornithological persuasion, the St. Louis region is of prime interest because of our natural flyways. The Mississippi River underwrites that status; it's a superhighway for migrating birds. We have another feathered fact to boast about: While the renowned birdman John James Audubon was still alive, the St. Louis Mercantile Library acquired a rare reserved copy of his masterwork, Birds of America, from his family. This is tantamount to owning a Gutenberg Bible. Celebrate it with the exhibit Audubon and Beyond: Collecting Five Centuries of Natural History at the St. Louis Mercantile Library on the University of Missouri-St. Louis campus (1 University Drive at Natural Bridge Road; 314-516-7240 or www.umsl.edu/mercantile). The extensive exhibit incorporates sections relating to not only birds but also reptiles, mammals, fish, insects, humans, astronomy, geology, meteorology and more. Audubon and Beyond is open 7:30 a.m. to 10:30 p.m. Monday through Thursday, 7:30 a.m. to 5 p.m. Friday, 8 a.m. to 4 p.m. Saturday and noon to 8 p.m. Sunday (November 9 through June 2017). Admission is free. free admission
You could do worse things in your life than learn to ice skate. It's great exercise, sure, but skating's real value is that it's a solitary pursuit that gives you time to think. Once you become proficient you can turn your brain off and glide along it's the cheapest re-set button available. The restorative powers are even greater if you can do it outdoors, and that's exactly where Steinberg Skating Rink (400 Jefferson Drive; www.steinbergskatingrink.com) is located. The city's largest rink is a great place to stretch your legs, get some fresh air and realign your system, especially during the stressful holiday season. Steinberg is open from 10 a.m. to midnight December 16 to January 7 so you can burn off some steam late into the evening. Regular hours are 10 a.m. to 9 p.m. Sunday through Thursday and 10 a.m. to midnight Friday and Saturday through February 23. Admission is $7 for an all-day pass, and skate rental is $6 for hockey or figure skates. $7 admission, $6 skate rental
This full-career survey of playwright, novelist and visual artist Rosalyn Drexler offers a rare opportunity to see the breadth of the self-taught artist's work. Her paintings feature bright colors and figures appropriated from films and print media, which she cropped, enlarged and printed on her canvases and then painted over them. The effect is somewhere between photo-realism, pop art and the visual language of a dream. Chubby Checker depicts a large Chubby mid-twist against squared fields of scarlet and blue and yellow, with couples dancing in 45-sized circles to the left; a smaller Checker echoes the larger one to the right. Love And Violence is far more sharp, a suited man looming over a crumpled blonde woman, grabbing her chin. A triptych of blue windows beneath the tableau show the same man helping to assault a fellow in a trench coat. free admission
St. Louis was booming in the early twentieth century, growing beyond its frontier boundaries at a rapid rate. The St. Louis Street Department documented these growing pains, both to record the challenges it faced and show how much work was being done. Charles Clement Holt marshaled a force of photographers to shoot street work in progress, dilapidated areas needing improvement, finished municipal projects and -- quite by chance -- the daily life of a burgeoning city. At its peak, the project knocked out 6,000 photographs a year. Many of these were eventually thrown out, but a historian rescued some 300 prime images. A selection of these images comprise the exhibition Capturing the City: Photographs from the Streets of St. Louis, 1900-1930. These images show a St. Louis that is familiar but vastly different: Horses being hoisted out of holes in the street are a rarity these days, and Market Street never has musicians on flatbed trucks anymore urging us to keep the city clean. free admission
Before the interstate highway system was developed, Route 66 was the safest, fastest way to cross the western half of the country. Starting in Chicago and ending Santa Monica, the "Main Street of America" came right though St. Louis, but not in the mostly straight lines we're accustomed to now. At various points in time, Route 66 traversed Watson Road, Manchester Road, the Martin Luther King Bridge and the Poplar Street Bridge. That shifting route helped spur the growth of cities and businesses along the way, as travelers stopped overnight at the Coral Court Motel or grabbed a bit to eat at the Parkmoor Restaurant. Route 66: Main Street Through St. Louis, the new exhibition at the Missouri History Museum (Lindell Boulevard and DeBaliviere Avenue; 314-746-4599 or www.mohistory.org), tells the story of the byway through roadside signs and gas pumps, historic vehicles, bus tours and photographs. Route 66 opens Saturday, June 25, and remains open through July 16, 2017. Admission is free. free admission
Monday, February 20 and Tuesday, February 21 Story Hour – President's Day 10 – 10:45am, Free, Reservations not required With Diane Van Booven, Kids Club Coordinator In honor of President's Day, Diane will read "Arthur Meets The President" by Marc Brown. The kids will sing some patriotic songs and end story hour with a President's Day craft and a healthy snack. Free
The Chamber Music Society St. Louis bring a lovely collection of sonatas for harp, violin, piano and cello, by Rosetti, Bach and Beethoven. Concerts performed cabaret-style in the Sheldon Ballroom. 38http://chambermusicstl.org/
Vino van Gogh isn't just about painting; it's about creating balance and being healthy. Studies show the act of putting paint on canvas, regardless of skill, utilizes the creative side of your brain, giving you balance from the overuse of the productive, logic side of our brains. And our events are just fun – helping you relieve stress! $25 with online promo codehttp://vinogogh.com/StLouis/Calendar.aspx
The best comedy open mic south of I-40! Every Monday of each month the Heavy Anchor, 5226 Gravois, presents The Comedy Shipwreck open mic. Free to get in. See St. Louis' newest and brightest comedians before they get big! Also, bottle PBR's are $1 all night! comics sign up @ 9:30pm show @ 10pm Freehttps://www.facebook.com/TheComedyShipwreck/
What is the role of the visual arts in establishing and sustaining heroic status through which societies define and articulate their values? By encouraging viewers to contemplate the conception of the hero in the visual arts across the centuries—from early Greek coins and pottery to nineteenth-century paintings and prints—the exhibition invites critical reflection on the potential for images to shape the narratives of communal identity of which they are a part. 0
This first full-career retrospective highlights the groundbreaking work of Rosalyn Drexler, celebrating her unique contributions to the history of Pop art and her long and multifaceted practice as an artist, novelist, and award-winning playwright. The exhibition features major paintings and collages alongside rarely seen early sculptures as well as photographic and video documentation encompassing Drexler's wide-ranging and colorful career from the 1950s to the present. 0
Bringing together a broad selection of prints, posters, photographs, and film, this exhibition offers an overview of the thriving entertainment cultures of Paris in the last decades of the nineteenth century. The art of Pierre Bonnard, Edgar Degas, Édouard Manet, Henri de Toulouse-Lautrec, Pablo Picasso, and others addresses the pulsating life of the boulevards, the speed of the racetrack, and the performance worlds of the café-concert, ballet, opera, and the theater, both classical and popular. These diverse themes inspired traditional as well as innovative mediums, from fine art prints to commercial posters. 0
Noodles & Company kicked off a fundraising effort at its nine area locations to raise money for the Children's Miracle Network. During the month of February, all proceeds collected in donation boxes by the registers will be used to purchase hundreds of toy bears that will be delivered later this month to young patients of SSM Health Cardinal Glennon Children's Hospital and St. Louis Children's Hospital in St. Louis, and MU Children's Hospital in Columbia, Missouri. Proceeds collected after February will be used by the Children's Miracle Network Hospitals to expand existing programs and facilities of the three area hospitals. Freehttp://www.noodles.com
The start of spring is just one month away and if you're itching to resume your weekly rides, the Fat Tire Camp Fire will surely get you in the mood. This cycling expo gives you a sneak peek at the latest bikes and new gear, and also lets you meet with Pro Skills instructors from Roots Mountain Biking and Off-Road Cyclists representatives. You can also test your skills on the Fat Bike Log Ride Challenge and virtual indoor MTB rides. The Fat Tire Campfire starts at 6 p.m. tonight at Ballpark Village (601 Clark Avenue; www.stlballparkvillage.com). Admission is free. free admission
Graeme loves Game of Thrones so much that he decides to recreate it on stage with help from his best friend and former crush. But Graeme doesn't have the budget, cast or acting talent to pull it off. Regardless, he works to put on a show that will bowl over the influential producer who is rumored be in the audience. Graeme believes George R.R. Martin would approve. Would he? Who knows, but the critics certainly have. Game of Thrones fans will, too. Graeme of Thrones is performed at 8 p.m. Tuesday to Friday, 4 and 8 p.m. Saturday, and 2 and 6 p.m. on Sunday (February 21 to 26) at Playhouse at Westport Plaza (635 Westport Plaza; www.playhouseatwestport.com). Tickets are $60. $60
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Noa (Israel)
Genre: Rock
Catalog No: HBIM 5022970
Format: CD
Release Date: 2015-05-26
Number of Discs: 1
UPC Code: 888750853721
FreeFreight Is Available. Click for Details
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Keep checking BNN for the breaking news updates, and discuss it on the forums or our IRC channel #bulbapedia.Want to write an article? Go ahead! Have an opinion to share? An idea for a new column? Contact us!
96th Best Wishes episode title revealed
From Bulbanews, your community Pokémon newspaper.
'Meloetta and the Undersea Temple' to air September 27
The Digital TV-Guide has unveiled the title and airdate of the 96th episode of the Pocket Monsters Best Wishes series. 「メロエッタと海底の神殿!」Meloetta and the Undersea Temple! is scheduled to air on September 27, 2012.


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Panasonic Camcorder HC-MDH2 AVCHD(PAL)
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The Panasonic HC-MDH2 AVCHD Shoulder Mount Camcorder shoots SD and HD video that is compatible with PAL standards. It supports Full HD 1080p 50, using AVCHD version 2 compression at 28 Mb/s. It also shoots 1080i 50 recording at 24 Mb/s, as well as higher compression rates enabling longer recording times. The camera can also record SD footage, so it integrates into an SD or HD workflow. This makes it an option for those who are not yet ready to transition to HD but want to be able to use the same camera once they do.The camera features a 1/2.33" MOS sensor that has been designed with a thinner wiring layer, which allows the sensor to capture more light resulting in a better low light image with less noise than with a similar sensor with a thicker wiring layer. Panasonic's Crystal Engine Pro image processor converts the info from the sensor to a viewable image. It also handles the image processing of the 50X intelligent zoom, providing smooth looking video, and minimizing artifacts.The camera features built-in image stabilization that operates in 5-axes, compensating for vertical, horizontal, forward and back tilt, and panning motions that might otherwise be distracting to your audience. The HC-MHD2 features a level function, where the camera corrects shots that are not horizontally level. The camera's Intelligent auto mode allows the camera to select the appropriate settings to capture you image. However, you can choose to manually control, focus, zoom, iris, and white balance. The camera comes with an eyepiece viewfinder for a traditional ENG approach to shooting, but you can also use the built-in touch panel LCD instead. The camera records to available SD/SDHC/SDXC media, and features 2 x media card slots, allowing either relay or simultaneous recording to create an instant back up. Once you've captured your footage, you can connect an available USB drive to the camera and transfer directly to the drive without using a computer or other USB host device. An update on the little red recording light, the illuminated ring at the base of the lens glows to indicate the status of the camera, either blue for stand-by or red for recording.
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