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.

Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA.

Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

A practical guide to sample preservation and pre-PCR processing of aquatic environmental DNA.

Environmental DNA (eDNA) is rapidly growing in popularity as a tool for community assessments and species detection. While eDNA approaches are now widely applied, there is not yet agreement on best practices for sample collection and processing. Investigators looking to integrate eDNA approaches into their research programme are required to examine a growing collection of disparate studies to make an often uncertain decision about which protocols best fit their needs. To promote the application of eDNA approaches and to encourage the generation of high-quality data, here we review the most common techniques for the collection, preservation and extraction of metazoan eDNA from water samples. Specifically, we focus on experimental studies that compare various methods and outline the numerous challenges associated with eDNA. While the diverse applications of eDNA do not lend themselves to a one-size-fits-all recommendation, in most cases, capture/concentration of eDNA on cellulose nitrate filters (with pore size determined by water turbidity), followed by storage of filters in Longmire's buffer and extraction with a DNeasy Blood & Tissue Kit (or similar) has been shown to provide sufficient, high-quality DNA. However, we also emphasize the importance of testing and optimizing protocols for the system of interest.

Influences of swab types and storage temperatures on isolation and molecular detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Routine diagnosis of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is performed by collecting oropharyngeal swabs, followed by isolation and/or detection by molecular methods. The storage temperature, storage duration and the type of swab could be critical factors for successful isolation or molecular detection. The aim of this study was to compare the influence of different types of cotton-tipped swab stored at different temperatures, on the detection of MG and MS. To achieve this, combined use of traditional culture analysis (both agar and broth), with modern molecular detection methods was utilized. Performances of wooden and plastic shaft swabs, both without transport medium, were compared. Successful culture of M. gallisepticum was significantly more efficient from plastic swabs when compared to wooden, whereas no difference was seen for the re-isolation of M. synoviae. Storage at 4°C compared to room temperature also increased the efficiency of culture detection for both Mycoplasma species. When stored at room temperature, PCR detection limits of both MG and MS were significantly lower for wooden compared to plastic swabs. The qPCR data showed similar detection limits for both swab types when stored at both temperatures. The results suggest that swabs with a plastic shaft are preferred for MG and MS detection by both culture and PCR. While a lower storage temperature (4°C) is optimal for culture recovery, it seems that both temperatures investigated here are adequate for molecular detection and it is the swab type which carries a greater influence.

Accuracy of body mass estimates of formalin-preserved fish - a review.

This paper highlights possible effects of physical and chemical mechanisms of formalin fixation and preservation on biological tissue and reviews the consequent potential inaccuracies on estimates of body mass of small fishes fixed and preserved in formalin. Twenty-six papers including 65 independent experiments with 35 species which examine effects of formalin on body mass estimates on small fishes are included. The effect of the formalin on the specimens depends on the salinity of the water used to dilute the commercial formalin (usually 1:9 formalin: water) before being used to fixate and preserve fish. Mean wet body mass of the specimens from the studies using seawater or fresh water diluted formalin deceases by 13% and increases by 7%, respectively, from before to after being immersed in formalin. The same trend is found with condition factor in the few papers that report this parameter. Body length decreases on average by c. 2% in fixated and preserved fish regardless of whether the formalin is diluted in seawater or fresh water.

Benefit by design: Determining the 'value' of donor human milk and medical products derived from human milk in NICU.

The use of donor human milk to provide therapeutic benefit to infants should only proceed where there is positive 'value'. This can be determined through an assessment of the benefit and the known risks. The emergence of new products derived from human milk requires new value assessments. The known hazards in human milk are modified by differences in the donor selection, processing methods and intended use and result in a unique risk assessment where any of these factors vary. The human source of the raw product requires high ethical standards in the design of these services with care taken to protect donors and recipients from harm. Any supplement to maternal milk should be provided cautiously to avoid displacement of maternal lactation.

Holding donated human bodies in conservation: A novel interactive safe-keeping system meeting high ethical and safety standards.

"Mortui vivos docent". Learning from donated bodies is widely considered a corner stone in pre-clinical education, advanced clinical training, and scientific progress in medicine. Making such use of dead human bodies must, of course, accord with high ethical standards and legal constraints. Piety and respect towards donors require using their remains (i) for valuable purposes, (ii) with what we call 'practical decency', (iii) in an efficient way, and (iv) with the utmost safety for all parties involved. With regard to these goals, practical aspects of preservation, safekeeping procedures (for up to several years), and complete documentation become of great importance, but have so far only been realized unsatisfactorily. Here, we describe the new Safe-Keeping System-Münster (SKS-Münster) that has been developed and implemented in the Anatomy Department of the University of Münster. Integrated components of the system include a paternoster transport system, a removal station with ventilation and an air barrier, RFID transponder technology, and an easy to use software package allowing the system together to provide all required functions in an unprecedented way.

Robust sampling and preservation of DNA for microbial community profiling in field experiments.

OBJECTIVE: Stabilising samples of microbial communities for DNA extraction without access to laboratory equipment can be a challenging task. In this paper we propose a method using filter paper disks for the preservation of DNA from diverse microbial communities which are found in a fermented milk product. RESULTS: Small adaptations to the DNA extraction method used for liquid fermented milk delivered DNA of sufficient amounts and quality to be used for later analyses, e.g. full community 16S amplicon sequencing. The microbial community structure obtained via the filter paper method showed sufficient resemblance to the structure obtain via the traditional DNA extraction from the liquid milk sample. This method can therefore successfully be used to analyse diverse microbial communities from fermented milk products from remote areas.

A Reusable Perfused Human Cadaver Model for Surgical Training: An Initial Proof of Concept Study.

OBJECTIVES: Today's surgical trainees have less exposure to open vascular and trauma procedures. Lightly embalmed cadavers may allow a reusable model that maximizes resources and allows for repeat surgical training over time. METHODS: This was a three-phased study that was conducted over several months. Segments of soft-embalmed cadaver vessels were harvested and perfused with tap water. To test durability, vessels were clamped, then an incision was made and repaired with 5-0 polypropylene. Tolerance to suturing and clamping was graded. In a second phase, both an arterial-synthetic graft and an arterial-venous anastomosis were performed and tested at 90 mmHg perfusion. In the final phase, lower extremity regional perfusion was performed and vascular control of a simulated injury was achieved. RESULTS: Seven arteries and six veins from four cadavers were explanted. All vessels accommodated suture repair over 6 weeks. There was minor leaking at all previous clamp sites. In the anastomotic phase, vessels tolerated grafting, clamping, and perfusion without tearing or leaking. Regional perfusion provided a life-like training scenario. CONCLUSIONS: Explanted vessels of soft-embalmed cadavers show adequate durability over time with realistic vascular surgery handling characteristics. This shows promise as initial proof of concept for a reusable perfused cadaver model. Further study with serial regional and whole-body perfusion is warranted.

Monitoring the microbial contamination of donor cornea during all preservation phases: A prospective study in the Eye Bank of Rome.

BACKGROUND: In most European eye banks, human donor corneas are microbiologically tested after storage in organ culture conditions, and the tissues that are free of contamination are distributed for transplantation. In this prospective study, 100 donor corneas were tested for microbial contamination after cold storage, corneal culture and corneal deswelling at the Eye Bank of Rome. METHODS: Samples of cold storage medium (EUSOL-C), corneal culture medium (TISSUE-C) and deswelling medium (CARRY-C) were tested after three, seven and one days of corneal storage, respectively. The CARRY-C medium, used to transport the cornea to the operation theatre, was retested 1 day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. RESULTS: We found 67% of the EUSOL-C samples were contaminated mainly by Staphylococcus spp, 14% of TISSUE-C media were contaminated by bacteria and fungi and 3% of CARRY-C media by Staphylococcus spp The analysis performed after removing the antimicrobial and antifungal agents showed growth in three additional TISSUE-C samples (S viridans, S haemolyticus and E faecalis) and one CARRY-C (S cerevisiae and P acnes). CONCLUSION: Tissue contamination was unexpectedly high on arrival to the eye bank, indicating the need to review and update decontamination procedures during tissue recovery, and renew training for the recovery teams. Storing donor corneas in organ culture conditions significantly reduced the microorganism burden. Using devices to remove antimicrobial and antifungal agents from samples before testing can increase the sensitivity of the standard microbiological method, and thus help further reduce the risk of microbial transmission.

Comparison of clinical performance of commercial urine growth stabilization products.

INTRODUCTION: Urine specimens for quantitative culture for the diagnosis of urinary tract infection may be unreliable due to bacterial overgrowth within 4 h after collection, at room temperature. Because specimen transportation may take longer than 4 h, urine preservatives may reduce overgrowth. Further evidence is needed to support a recommendation for use of preservative and to compare preservative products. METHODS: Consecutive midstream urine specimens submitted for culture were quantitatively cultured on receipt and then inoculated into 3 storage conditions [BD Urine Vacutainer (BD), Copan UriSwab (US), and refrigeration, with a room temperature control] for 72 h, with quantitative culture performed every 24 h. Odds ratio for significant growth interpretation was reported. RESULTS: Ninety-five of 501 (19.0%) urine specimens demonstrated significant growth. Within 24 h of storage, unpreserved urine at room temperature demonstrated a significantly increased odds ratio for significant growth as compared to preserved urine, and urine in refrigeration demonstrated similar odds ratio for significant growth as compared to preserved. There was no significant difference between the performance of US and BD. Over 48 and 72 h of storage, odds ratio for significant growth further increased. CONCLUSIONS: Preservation performed similarly to refrigeration. Preserved urine demonstrated a doubling in odds ratio for significant growth after 24 h. This increase may negatively impact antibiotic treatment decisions.

Effect of acids produced from carbohydrate metabolism in cryoprotectants on the viability of freeze-dried Lactobacillus and prediction of optimal initial cell concentration.

For the industrial production of probiotics powder, various sugars have been used as cryoprotectants to preserve probiotics during freeze-drying. Some of these sugars can be metabolized by Lactobacillus with the production of acids during the mix. In this study, we investigated the effect of acids on ATPase, ß-galactosidase, lactate dehydrogenase (LDH), integrity and fluidity of cell membrane and the survival rate of Lactobacillus during freeze-drying. In the presence of Lactobacillus, acids were produced from cryoprotectants containing fermentable sugars before freezing, resulting in a decrease in the pH of the bacterial suspension to below 5.0. During freeze-drying, the acids caused a loss of viability of Lactobacillus due to aggravated damage to ATPase, ß-galactosidase and cell membrane fluidity, but not LDH and cell membrane integrity. This finding implied that cryoprotectants that do not lead to the production of acids are effective in improving the survival rate of freeze-dried Lactobacillus. Here, a new formula was proposed for a protectant containing whey protein isolate (WPI) and rhamnose, which were not metabolized. In addition, linear-regression analyses were performed on the proportion of cryoprotectants (M) against cell paste (m), total cell count (N), total surface area (St) and total volume (Vt) of bacteria for 100% survival rate. The total surface areas of bacteria were found to be highly correlated with the amount of proposed cryoprotectant. The following prediction equation was established for the optimal initial cell concentration for a 100% survival rate of freeze-dried Lactobacillus: N (4πr2+2πl)=(0.66±0.03)M.

Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding.

BACKGROUND: Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. METHODS: We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. RESULTS: Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. CONCLUSIONS: Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center

Abstract Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.

Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center.

Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.

Improved outcomes of feeding low birth weight infants with predominantly raw human milk versus donor banked milk and formula.

OBJECTIVES: To investigate the benefits of treating low birth weight infants predominantly with mother's own raw milk and early initiation of breastfeeding (raw human milk/breast-fed infants), in comparison to feeding only with donor banked milk (until the third week of life) and afterwards a preterm formula until hospital discharge (donor banked/formula-fed infants). METHODS: One hundred and ninety-two predominantly raw human milk-fed infants (70% of raw and 30% of donor milk) were matched to 192 donor/formula-fed ones (on 1:1 ratio). Aggressive nutrition policy and targeted fortification of human milk were implemented in both groups. RESULTS: The two groups show similar demographic and perinatal characteristics. Predominantly raw milk-fed infants regained earlier their birth weight, suffered less episodes of feeding intolerance and presented a higher body length and head circumference at discharge (p < 0.001). Those treated mainly with their mothers' milk were able to initiate breastfeeding almost 2 weeks earlier compared to those fed with donor milk who achieved to be bottle-fed later on post-conceptual age (p < 0.001). Infants being breastfed until the 8th month of life conducted less visits for a viral infection to a pediatrician compared to those in the other group (p < 0.001). CONCLUSIONS: Feeding predominantly with mother's raw milk seems to result in optimal neonatal outcomes.

Standard operating procedures for standardized mass rearing of the dengue and chikungunya vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae) - II - Egg storage and hatching.

BACKGROUND: Management of large quantities of eggs will be a crucial aspect of the efficient and sustainable mass production of mosquitoes for programmes with a Sterile Insect Technique component. The efficiency of different hatching media and effectiveness of long term storage methods are presented here. METHODS: The effect on hatch rate of storage duration and three hatching media was analysed: deionized water, boiled deionized water and a bacterial broth, using Two-way ANOVA and Post hoc Tukey tests, and the Pearson correlation coefficient was used to find the effect on the proportion of collapsed eggs. Two long term storage methods were also tested: conventional storage (egg paper strips stored in zip lock bags within a sealed plastic box), and water storage (egg papers in a covered plastic cup with deionized water). Regression analyses were used to find the effect of water storage and storage duration on hatch rate. RESULTS: Both species hatched most efficiently in bacterial broth. Few eggs hatched in deionized water, and pre-boiling the water increased the hatch rate of Ae. aegypti, but not Ae. albopictus. A hatch rate greater than 80% was obtained after 10 weeks of conventional storage in Ae. aegypti and 11 weeks in Ae. albopictus. After this period, hatching decreased dramatically; no eggs hatched after 24 weeks. Storing eggs in water produced an 85% hatch rate after 5 months in both species. A small but significant proportion of eggs hatched in the water, probably due to combined effects of natural deoxygenation of the water over time and the natural instalment hatching typical of the species. CONCLUSIONS: The demonstrated efficiency of the bacterial broth hatching medium for both Ae. albopictus and Ae. aegypti facilitates mass production of these two important vector species in the same facility, with use of a common hatching medium reducing cost and operational complexity. Similarly the increased hatch rate of eggs stored in water would allow greater flexibility of egg management in a large programme over the medium term, particularly if oxygenation of the water by bubbling oxygen through the storage tray could be applied to prevent hatching during storage.