[Construction and application of standard system for the preservation of pathogenic microorganism resources].

Standards are the technical support for economic activities and social development. The construction and standardization of the pathogenic microorganism preservation standard system is an important technical foundation for the high-quality development of preservation work. Establishing a pathogenic microorganism resource standard system is also important to the national biosafety standards. Through the standardization of pathogenic microbial resource preservation, we can ensure the effective management and sustainable utilization of pathogenic microbial resources, promote the transformation of resources, and serve as an important new element of new productivity to assist the innovative development of biosafety science and technology. This article elaborates and analyzes the establishment background, construction framework, standardization process, and application effects of the standard system for preserving pathogenic microbial resources, providing stronger support for further improving the standard system and promoting the standardization of pathogenic microbial resource preservation.

Deep silicification-assisted long-term preservation of structural and genomic information across biospecies: From micro to macro.

The concurrent preservation of morphological, structural, and genomic attributes within biological samples is paramount for comprehensive insights into biological phenomena and disease mechanisms. However, current preservation methodologies (e.g., cryopreservation, chemical reagent fixation, and bioplasticization) exhibit limitations in simultaneously achieving these critical combined goals. To address this gap, inspired by natural fossilization, here we propose "deep silicification," a room temperature technology that eliminates fixation requirements and overcomes the cold chain problem. By harnessing the synergy between ethanol and dimethyl sulfoxide, deep silicification significantly enhances silica penetration and accumulation within bioorganisms, thereby reinforcing structural integrity. This versatile and cost-effective approach demonstrates remarkable efficacy in preserving organismal morphology across various scales. Accelerated aging experiments underscore a 4,723-fold enhancement in genomic information storage over millennia, with whole-genome sequencing confirming nearly 100% fidelity. With its simplicity and reliability, "deep silicification" represents a paradigm shift in biological sample storage.

A simple culture medium for phenotypic characterization and long-term storage of medically relevant fusarioid fungi.

Fusarioid fungi, particularly Neocosmospora solani and Fusarium oxysporum, are emerging as significant human pathogens, causing infections ranging from localized mycoses to life-threatening systemic diseases. Accurate identification and preservation of these fungi in clinical laboratories remain challenging because of their diverse morphologies and specific growth requirements. This study evaluated a novel milk-honey and malt agar (MHM) against conventional media for cultivating and preserving 60 clinical fusarioid isolates, including Neocosmospora spp. (n = 47), Bisifusarium spp. (n = 5), and Fusarium spp. (n = 8). Compared with Sabouraud dextrose 2 % agar (SDA) and malt extract agar (ME2), MHM significantly increased conidia production (p < 0.0001, mean = 3.4 × 103, standard deviation (SD) = ±1.3 × 103), with results similar to those of carnation leaf agar (CLA). MHM facilitated superior preservation of fusarioid viability for up to one year at room temperature on slant cultures and over two years on swabs in Amies gel with charcoal, outperforming current methods such as Castellani (water) or cryopreservation. Morphological characterization of fusarioid fungi grown on MHM revealed distinct growth patterns and conidial structures for Neocosmospora, Bisifusarium, and Fusarium species, aiding in identifying these genera. The superior performance of MHM in stimulating conidiation, maintaining viability, and preserving morphology underscore its potential as a reference medium for medically relevant fusarioid fungi, with broad implications for clinical mycology laboratories and resource-limited settings.

Microbial DNA sample preservation and possible artifacts for field-based research in remote tropical peatlands.

Surveying bacterial and archaeal microbial communities in host and environmental studies requires the collection and storage of samples. Many studies are conducted in distant locations challenging these prerequisites. The use of preserving buffers is an important alternative when lacking access to cryopreservation, however, its effectivity for samples with challenging chemistry or samples that provide opportunities for fast bacterial or archaeal growth upon exposure to an aerobic environment, like peat samples, requires methodological assessment. Here, in combination with an identified optimal DNA extraction kit for peat soil samples, we test the application of several commercial and a homemade preservation buffer and make recommendations on the method that can most effectively preserve a microbiome reflective of the original state. In treatments with a non-optimal buffer or in the absence, we observed notable community shifts beginning as early as three days post-preservation lowering diversity and community evenness, with growth-driven artifacts from a few specific phyla. However other buffers retain a very close composition relative to the original state, and we described several metrics to understand some variation across them. Due to the chemical effects of preservation buffers, it is critical to test their compatibility and reliability to preserve the original bacterial and archaeal community in different environments.

Unlocking Antarctic molecular time-capsules - Recovering historical environmental DNA from museum-preserved sponges.

Marine sponges have recently emerged as efficient natural environmental DNA (eDNA) samplers. The ability of sponges to accumulate eDNA provides an exciting opportunity to reconstruct contemporary communities and ecosystems with high temporal and spatial precision. However, the use of historical eDNA, trapped within the vast number of specimens stored in scientific collections, opens up the opportunity to begin to reconstruct the communities and ecosystems of the past. Here, we define the term 'heDNA' to denote the historical environmental DNA that can be obtained from the recent past with high spatial and temporal accuracy. Using a variety of Antarctic sponge specimens stored in an extensive marine invertebrate collection, we were able to recover information on Antarctic fish biodiversity from specimens up to 20 years old. We successfully recovered 64 fish heDNA signals from 27 sponge specimens. Alpha diversity measures did not differ among preservation methods, but sponges stored frozen had a significantly different fish community composition compared to those stored dry or in ethanol. Our results show that we were consistently and reliably able to extract the heDNA trapped within marine sponge specimens, thereby enabling the reconstruction and investigation of communities and ecosystems of the recent past with a spatial and temporal resolution previously unattainable. Future research into heDNA extraction from other preservation methods, as well as the impact of specimen age and collection method, will strengthen and expand the opportunities for this novel resource to access new knowledge on ecological change during the last century.

Kato-Katz slide preservation technique: Extension of viability and the benefits for schistosomiasis control programs.

The World Health Organization recommends the use of the Kato-Katz method in the procedures of schistosomiasis control programs. Studies show the importance of a fast reading of the slides due to the decline of their viability, with the appearance of fungi or desiccation of the sample, which hinders diagnosis. It is necessary to establish a procedure to improve the long-term preservation of these Kato-Katz slides in order to accomplish the following: (1) preserve the slides for future quality control procedures and readings; (2) allow for the production of durable materials for training; and (3) train health professionals involved in diagnosing schistosomiasis. Therefore, this study aims to test a slide preservation methodology for these purposes. The results showed that the modifications made to the experimental slides demonstrated that egg loss was within the expected range and the limit accepted by quality control standards, as well as improved the diagnostic durability of the slides during the preservation times tested. We concluded that the application of the preservation technique to the slides promoted stabilization and permanence for long-term storage.

Novel buffer for long-term preservation of DNA in biological material at room temperature.

The collection and preservation of biological material before DNA analysis is critical for inter alia biomedical research, medical diagnostics, forensics and biodiversity conservation. In this study, we evaluate an in-house formulated buffer called the Forensic DNA Laboratory-buffer (FDL-buffer) for preservation of biological material for long term at room temperature. Human saliva stored in the buffer for 8 years, human blood stored for 3 years and delicate animal tissues from the jellyfish Pelagia noctiluca comb jelly Beroe sp., stored for 4 and 6 years respectively consistently produced high-quality DNA. FDL-buffer exhibited compatibility with standard organic, salting out and spin-column extraction methods, making it versatile and applicable to a wide range of applications, including automation.

DNA extractions were performed by Salting out, PCI, ZymoQuick and DNAeazy methods. DNA quantity and quality were assessed using qPCR, Qubit, gel electrophoresis, as well as Sanger sequencing, microsatellite profiling and SNPchip analysis.

Systematic review of literature to evaluate global distribution of species of the Sporothrix genus stored in culture collections.

Introduction: Sporotrichosis is a subcutaneous mycosis caused by fungi of the genus Sporothrix sp. Phenotypic and genotypic differences have been associated with their geographic distribution, virulence, or clinical manifestation of sporotrichosis. In the past decade, the interest in identifying species of the Sporothrix sp. has been increasing, due to its epidemiological importance and, in consequence, is important to know how to preserve them for future studies, in culture collection. Aims: The purposes of this study were to analyze the global distribution of environmental isolates and/or causal agents of sporotrichosis identified by polyphasic taxonomy, with mandatory use of molecular identification, and to evaluate the percentages and distribution of isolates stored in culture collections. Methods: A systematic review of articles on animal and human sporotrichosis and/or environmental isolation of the fungus, from 2007 to 2023, was done. Results: Our results demonstrated that, S. globosa, S. schenckii, and S. brasiliensis were the most identified species. With respect to the deposit and maintenance of species, we observed that only 17% of the strains of Sporothrix sp. isolated in the world are preserved in a culture collection. Conclusions: This systematic review confirmed a difficulty in obtaining the frequency of Sporothrix species stored in culture collection and insufficient data on the molecular identification mainly of animal sporotrichosis and isolation of Sporothrix sp. in environmental samples.

Nucleic acid degradation after long-term dried blood spot storage.

Collecting and preserving biological samples in the field, particularly in remote areas in tropical forests, prior to laboratory analysis is challenging. Blood samples in many cases are used for nucleic acid-based species determination, genomics or pathogen research. In most cases, maintaining a cold chain is impossible and samples remain at ambient temperature for extended periods of time before controlled storage conditions become available. Dried blood spot (DBS) storage, blood stored on cellulose-based paper, has been widely applied to facilitate sample collection and preservation in the field for decades. However, it is unclear how long-term storage on this substrate affects nucleic acid concentration and integrity. We analysed nucleic acid quality from DBS stored on Whatman filter paper no. 3 and FTA cards for up to 15 years in comparison to cold-chain stored samples using four nucleic acid extraction methods. We examined the ability to identify viral sequences from samples of 12 free-ranging primates in the Amazon forest, using targeted hybridization capture, and determined if mitochondrial genomes could be retrieved. The results suggest that even after extended periods of storage, DBS will be suitable for some genomic applications but may be of limited use for viral pathogen research, particularly RNA viruses.

Environmental DNA Isolation, Validation, and Preservation Methods.

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.

Methods for the optimal preservation of blow fly intra-puparial forms for morphological analysis in forensic casework.

Accurate minimum post-mortem interval (minPMI) estimations often rely on a precise age determination of insect developmental stages, which is significantly influenced by environmental temperature. An optimal preservation of the entomological samples collected at crime scenes is pivotal for a reliable aging of immature insect samples. For blow flies (Diptera: Calliphoridae), the most widely used insect indicators in forensic investigations, an appropriate preservation of tissues is particularly important in the case of puparial samples because aging methods for intra-puparial forms usually depend on morphological analyses; however, although informative soft tissues and structures could be discoloured and/or distorted if they are not properly fixed, there is a lack of studies to assess different methods for the optimal preservation of intra-puparial forms collected in forensic investigations. The present study compares three preservation methods for intra-puparial forms of the blow fly Calliphora vicina Robineau-Desvoidy, 1830: (i) direct immersion into 80% ethanol, (ii) puncturing of the puparium and hot water killing (HWK) prior to preservation in 80% ethanol, and (iii) HWK without puncturing before preservation in 80% ethanol. External and internal morphological analyses of intra-puparial forms of different ages were conducted to assess the quality of preservation. The results indicate that direct immersion in ethanol led to poor preservation, affecting both external and internal tissues. Both methods with HWK resulted in a better preservation, but puncturing resulted, in some cases, in physical damage of the specimens. HWK without puncturing emerged as the optimal preservation method, consistently yielding high preservation scores for both external and internal morphological analyses. These findings have practical implications for forensic practitioners and emphasise the need for updating some published guidelines and protocols in forensic entomology.

Methodological Proposal for the Adequate Use of the Osteotechnics Technique / Propuesta Metodológica para el uso Adecuado de la Técnica Osteotécnica

SUMMARY: Osteotechnics is one of the different anatomical preservation techniques and can be defined as the technique designed to prepare, clean, obtain and preserve bone structures that can be used in the teaching, museographic or research field. The osteotechnical technique procedure consists of the following phases: debulk and disjoint, maceration, cooking, cleaning, degreasing, bleaching, and labeling to obtain bone material. Seven phases will be explained in detail, as well as the materials, instruments, quantities of the substances used, and the time required to obtain human bone material. We consider that this article can serve as a guide, given that all the experimentation was carried out with human biological material. This methodological proposal could be consolidated and established based on the experience acquired during the creation of the contemporary skeletal collection of the department of innovation in human biological material (DIMBIH). Therefore, the purpose of our proposal is to provide tools that facilitate the work of those who carry out this work and fundamentally to avoid irreversible or irreparable damage to the osteological material, since it is of great value and difficult to acquire for disciplines as anatomy, veterinary, physical and forensic anthropology, medicine, dentistry and biology.

La osteotecnia es una de las técnicas diferentes de conservación anatómica y puede definirse como la técnica destinada a preparar, limpiar, obtener y conservar estructuras óseas que pueden ser utilizadas en el ámbito docente, museográfico o de investigación. El procedimiento de la técnica osteotécnica consta de las siguientes fases: descarnado y desarticulado, maceración, cocción, limpieza, desengrase, blanqueo y marcaje para la obtención de material óseo. Se explicarán en detalle siete fases, así como los materiales, instrumentos, cantidades de las sustancias utilizadas y el tiempo necesario para obtener material óseo humano. Consideramos que este artículo puede servir de guía, dado que toda la experimentación se realizó con material biológico humano. Esta propuesta metodológica pudo consolidarse y establecerse a partir de la experiencia adquirida durante la creación de la colección esquelética contemporánea del Departamento de Innovación en Material Biológico Humano (DIMBIH). Por lo tanto, el propósito de nuestra propuesta es brindar herramientas que faciliten el trabajo de quienes realizan este trabajo y fundamentalmente evitar daños irreversibles o irreparables en el material osteológico, ya que es de gran valor y difícil adquisición para las disciplinas como la anatomía, veterinaria, antropología física y forense, medicina, odontología y biología.

Light-Assisted Drying for the Thermal Stabilization of Nucleic Acid Nanoparticles and Other Biologics.

Cold-chain storage can be challenging and expensive for the transportation and storage of biologics, especially in low-resource settings. Nucleic acid nanoparticles (NANPs) are an example of new biological products that require refrigerated storage. Light-assisted drying (LAD) is a new processing technique to prepare biologics for anhydrous storage in a trehalose amorphous solid matrix at ambient temperatures. Small volume samples (10 µL) containing NANPs are irradiated with a 1064 nm laser to speed the evaporation of water and create an amorphous trehalose preservation matrix. In previous studies, samples were stored for 1 month at 4 °C or 20 °C without degradation. A FLIR SC655 mid-IR camera is used to record the temperature of samples during processing. The trehalose matrix was characterized using polarized light imaging to determine if crystallization occurred during processing or storage. Damage to LAD-processed NANPs was assessed after processing and storage using gel electrophoresis.

Methodological considerations for aqueous environmental RNA collection, preservation, and extraction.

Environmental RNA (eRNA) analysis is expected to infer species' physiological information (health status, developmental stage, and environmental stress response) and their distribution and composition more correctly than environmental DNA (eDNA) analysis. With the prospect of such eRNA applications, there is an increasing need for technological development for efficient eRNA detection because of its physicochemical instability. The present study conducted a series of aquarium experiments using zebrafish (Danio rerio) and validated the methodologies for capture, preservation, and extraction of eRNA in a water sample. In the eRNA extraction experiment, an approximately 1.5-fold increase in lysis buffer volume resulted in a more than sixfold increase in target eRNA concentration. In the eRNA capture experiment, although GF/F and GF/A filters yielded similar eRNA concentrations, a GF/A filter may be capable of passing through more volume of water samples and consequently collecting more eRNA particles, given the time required for water filtration. In the eRNA preservation experiment, the use of RNA stabilization reagent (RNAlater) allowed for stably preserving target eRNA on a filter sample at - 20 and even 4 °C for 6 days at least. Altogether, the findings enable the improvement of eRNA availability from the field and easily preserve eRNA samples without deep-freezing, which will contribute to the refinement of eRNA analysis for biological and physiological monitoring in aquatic ecosystems.

Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis.

Here, we aimed to compare the effects of different preservation methods on outcomes of fecal microbiota. We evaluated the effects of different preservation methods using stool sample preservation experiments for up to 1 year. The stool samples from feces of healthy volunteers were grouped based on whether absolute ethanol was added and whether they were hypothermically preserved. Besides, we performed a systematic review to combine current fecal microbiota preservation evidence. We found that Proteobacteria changed significantly and Veillonellaceae decreased significantly in the 12th month in the room temperature + absolute ethanol group. The four cryopreservation groups have more similarities with fresh sample in the 12 months; however, different cryopreservation methods have different effects on several phyla, families, and genera. A systematic review showed that the Shannon diversity and Simpson index of samples stored in RNAlater for 1 month were not statistically significant compared with those stored immediately at -80°C (P = 0.220 and P = 0.123, respectively). The -80°C refrigerator and liquid nitrogen cryopreservation with 10% glycerine can both maintain stable microbiota of stool samples for long-term preservation. The addition of absolute ethanol to cryopreserved samples had no significant difference in the effect of preserving fecal microbial characteristics. Our study provides empirical insights into preservation details for future studies of the long-term preservation of fecal microbiota. Systematic review and meta-analysis found that the gut microbiota structure, composition, and diversity of samples preserved by storage methods, such as preservation solution, are relatively stable, which were suitable for short-term storage at room temperature. IMPORTANCE The study of gut bacteria has become increasingly popular, and fecal sample preservation methods and times need to be standardized. Here, we detail a 12-month study of fecal sample preservation, and our study provides an empirical reference about experimental details for long-term high-quality storage of fecal samples in the field of gut microbiology research. The results showed that the combination of -80°C/liquid nitrogen deep cryopreservation and 10% glycerol was the most effective method for the preservation of stool samples, which is suitable for long-term storage for at least 12 months. The addition of anhydrous ethanol to the deep cryopreserved samples did not make a significant difference in the preservation of fecal microbiological characteristics. Combined with the results of systematic reviews and meta-analyses, we believe that, when researchers preserve fecal specimens, it is essential to select the proper preservation method and time period in accordance with the goal of the study.

[How to handle unkown fluids in fluid-preserved collections : Cloudy prospects?] / Zum Umgang mit Präparaten in unbekannten Konservierungslösungen in Feuchtpräparatesammlungen : Trübe Aussichten?

BACKGROUND: Fluid collections are gaining more importance for research and teaching, but they are facing preservation problems. In the case of historical collections, the methods of fixation and preservation are poorly documented. The liquid used is unknown. In order to ensure the preservation of such collections, it is essential to have available a preservation liquid that is compatible with the most common historical liquids and techniques. MATERIALS AND METHODS: A universal liquid based on historical recipes was developed for such problematic preparations. RESULTS: The use of distilled water, glycerin and ethanol (80%) in a ratio of 10:6:1 offers a good alternative that is harmless to the health of the user. It can be used for colour-preserving conserved preparations and for pure ethanol and formaldehyde preparations and is recommended as a universal solution for preparations in unknown preservation liquids.

Long-term whole blood DNA preservation by cost-efficient cryosilicification.

Deoxyribonucleic acid (DNA) is the blueprint of life, and cost-effective methods for its long-term storage could have many potential benefits to society. Here we present the method of in situ cryosilicification of whole blood cells, which allows long-term preservation of DNA. Importantly, our straightforward approach is inexpensive, reliable, and yields cryosilicified samples that fulfill the essential criteria for safe, long-term DNA preservation, namely robustness against external stressors, such as radical oxygen species or ultraviolet radiation, and long-term stability in humid conditions at elevated temperatures. Our approach could enable the room temperature storage of genomic information in book-size format for more than one thousand years (thermally equivalent), costing only 0.5 $/person. Additionally, our demonstration of 3D-printed DNA banking artefacts, could potentially allow 'artificial fossilization'.

Long-term preservation of germ cells and gonadal tissues at ambient temperatures.

Objective: To present an overview of different approaches and recent advances for long-term preservation of germ cells and gonadal tissues at ambient temperatures. Methods: Review of the existing literature. Results: Preserving viable spermatozoa, eggs, embryos, and gonadal tissues for the long term is critical in human fertility treatment and for the management of animal populations (livestock, biomedical models, and wild species). The need and number of banked germplasms are growing very fast in all disciplines, but current storage options at freezing temperatures are often constraining and not always sustainable. Recent research indicates that structures and functions of gametes or gonadal tissues can be preserved for the long term using different strategies based on dehydration and storage at supra-zero temperatures. However, more studies are needed in rehydration and reanimation of germplasms (including proper molecular and cellular evaluations). Conclusions: While a lot of research is still warranted to optimize drying and rehydration conditions for each sample type and each species, alternative preservation methods will change the paradigm in fertility preservation and biobanking. It will transform the way we maintain and manage precious biomaterials for the long term. Lay summary: Living sperm cells, eggs, embryos, and reproductive tissues can be preserved at freezing temperatures for human fertility treatments and used to manage breeding in livestock, laboratory animals, and wild species through assisted reproduction. These cells can be stored in cell banks and demand for them is growing fast. However, current long-term storage options at freezing temperatures are expensive. Instead of using low temperatures, recent research indicates that these cells can be dried and stored above freezing temperatures for an extended amount of time. While a lot of research is still needed to optimize how different samples are dried and rehydrated, alternative methods of preserving cells will make fertility preservation and cell banking easier. It will also transform the way we keep and manage samples for the long term.

Freeze-drying can replace cold-chains for transport and storage of fecal microbiome samples.

Background: The transport and storage of samples in temperatures of minus 80 °C is commonly considered as the gold standard for microbiome studies. However, studies conducting sample collection at remote sites without a reliable cold-chain would benefit from a sample preservation method that allows transport and storage at ambient temperature. Methods: In this study we compare alpha diversity and 16S microbiome composition of 20 fecal sample replicates from Damaraland mole-rats (Fukomys damarensis) preserved in a minus 80 °C freezer and transported on dry ice to freeze-dried samples that were stored and transported in ambient temperature until DNA extraction. Results: We found strong correlations between relative abundances of Amplicon Sequence Variants (ASVs) between preservation treatments of the sample, no differences in alpha diversity measures between the two preservation treatments and minor effects of the preservation treatment on beta diversity measures. Our results show that freeze-drying samples can be a useful method for cost-effective transportation and storage of microbiome samples that yields quantitatively almost indistinguishable results in 16S microbiome analyses as those stored in minus 80 °C.

Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics.

Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug-microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at -80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.