Ethanol Preservation Research Articles

Study of Cell Viability and Etiology of Contamination in Decalcified Bone Allograft: A Pilot Study.

Bone allografts can elicit immune responses which is correlated with thepresence of Human Leukocyte Antigen (HLA) and cellular DNA. It also has risk of causingoccult infection arising out ofcontaminationduring its processing and storage. The presence of immunogenic materials like cells, cellular remnants and DNA in a decalcified bone allograft during different phases of processing has never been studied. Present study was conducted to explore- thecell viability using routine Hematoxylin and Eosin, presence of DNA usingFeulgen staining and etiology of contamination in decalcified bone allograft during procurement, demineralization and ethanol preservation. The harvested bones from patients undergoing hemireplacement/THR/TKR were processed to prepare decalcified bone allografts. The samples during procurement (A), HCL treatment (B)and ethanol preservation (C) were sent forhistopathological analysis (number of osteocytes in the maximum density field under 40xand the cells demonstrating presence of DNA on feulgen stain) and microbiological assessment (aerobic/anaerobic/fungal cultures). Histopathological study demonstrated the presence of osteocytes and other cells like bone marrow, adipocytes, endothelial cells in the decal bone allograft. The average number of osteocytes gradually decreased from 55.47, 9.6, 0.86 in sample A, B, C, respectively. Feulgen staining confirmed the presence of DNA in osteocytes and other cells which decreased both qualitatively and quantitatively in subsequent stages of processing. Rate ofcontamination demonstrated at the procurement was 6.67% (Staphylococcus aureus). After treatment with HCl (demineralisation), 7.14% of non-contaminated allografts were found contaminated (Staphylococcus epidermidis). None of the remaining 13 non-contaminated allografts showed contamination after storage in ethanol. Overall 13% of the patients had positive cultures on microbiological assessment. The population of osteocytes in the harvested bone reduced significantly after processing with HCl and ethanolpreservation. Presence of DNA, demonstrated byusing Feulgen staining, was observed in bone marrow cells, adipocytes along with osteocytes which showed quantitative reduction on processing. Hence, antigenicity, conferred by cells and their DNA, reduced significantly after processing of decal bone. Contamination rate of banked decalcified allograft was 13%. Thus, culture and sensitivity tests should be carried out at each step of processing of decal bone allograft.

Microbiota identified from preserved Anopheles

BackgroundMosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution.MethodsMicrobial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks.ResultsElizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices.ConclusionsThis study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

Effects of Tissue Preservation on Carbon and Nitrogen Stable Isotope Signatures in Syngnathid Fishes and Prey.

Simple SummaryStable isotope analysis (SIA) was used to assess the influence of various preservation methods (freezing, ethanol and formaldehyde) on syngnathid (seahorses and pipefishes) fins, seahorse newborns (seahorses), and prey (copepods and Artemia). The first available conversion models for syngnathids are provided, enabling their application to isotopic studies in the field and in the laboratory.Isotopic stable analysis (SIA) is a powerful tool in the assessment of different types of ecological and physiological studies. For that, different preservation methods for sampled materials are commonly used prior to isotopic analysis. The effects of various preservation methods (freezing, ethanol and formaldehyde) were analyzed for C:N, and δ13C and δ15N signals on a variety of tissues including dorsal fins (three seahorse and two pipefish species), seahorse newborns (three seahorses species), and prey (copepods and different stages of Artemia) commonly used to feed the fishes under rearing conditions. The aims of the study were: (i) to evaluate isotopic effects of chemical preservation methods across different types of organisms and tissues, using frozen samples as controls, and (ii) to construct the first conversion models available in syngnathid fishes. The chemical preservation in ethanol and, to a lesser extent, in formaldehyde significantly affected δ13C values, whereas the effects on δ15N signatures were negligible. Due to their low lipid content, the isotopic signals in fish fins was almost unaffected, supporting the suitability of dorsal fins as the most convenient material in isotopic studies on vulnerable species such as syngnathids. The regression equations provided resulted convenient for the successful conversion of δ13C between preservation treatments. Our results indicate that the normalization of δ15N signatures in preserved samples is unnecessary. The conversion models should be applicable in isotopic field studies, laboratory experiments, and specimens of historical collections.

Fluid-preserved fishes are one solution for assessing historical change in fish trophic level.

There are few resources available for assessing historical change in fish trophic dynamics, but specimens held in natural history collections could serve as this resource. In contemporary trophic ecology studies, trophic and source information can be obtained from compound‐specific stable isotope analysis of amino acids of nitrogen (CSIA‐AA‐N).We subjected whole Sebastes ruberrimus and Clupea pallasii to formalin fixation and 70% ethanol preservation. We extracted tissue samples from each fish pre‐fixation, after each chemical change, and then in doubling time for 32–64 days once placed in the final preservative. All samples were subjected to CSIA‐AA‐N, and their glutamic acid and phenylalanine profiles and associated trophic position were examined for differences over time by species.Glutamic acid and phenylalanine values were inconsistent in direction and magnitude, particularly during formalin fixation, but stabilized similarly (in 70% ethanol) among conspecifics. In some cases, the amino acid values of our final samples were significantly different than our initial pre‐preservation samples. Nonetheless, significant differences in glutamic acid, phenylalanine, and estimated trophic position were not detected among samples that were in 70% ethanol for >24 hr.Our results suggest that the relative trophic position of fluid‐preserved specimens can be estimated using CSIA‐AA‐N, and CSIA‐AA‐N estimates for fluid‐preserved specimens should only be reported as relative differences. Timelines of trophic position change can be developed by comparing specimens collected at different points in time, revealing trophic information of the past and cryptic ecosystem responses.

Effects of ethanol preservation and formalin fixation on amino acid stable isotope analysis (δ13C and δ15N) and its ecological applications

AbstractCompound‐specific isotope analysis of amino acids (CSIA‐AA) is a promising nascent technique that alleviates many shortcomings of conventional bulk‐tissue stable isotope analysis (“bulk SIA”) in ecological studies involving the tracing/reconstruction of carbon and nitrogen pathways. While CSIA‐AA has been increasingly applied to preserved tissue samples (e.g., material in natural history collections), the effects of sample preservation on amino acid δ13C and δ15N profiles are poorly understood. It is therefore unclear if mathematical correction factors are necessary for interpreting isotopic profiles of preserved samples. In this study, we investigated effects of ethanol preservation and formalin fixation on amino acid δ13C and δ15N profiles. We also assess how these effects translate to two ecological applications of CSIA‐AA: quantification of organic carbon sources, and estimation of trophic positions. Results from an 8‐week controlled experiment on freshwater fish tissue show negligible preservation effects on most amino acid δ15N profiles, and results are similar for δ13C profiles of essential amino acids. Findings from mixing models using essential amino acid δ13C profiles similarly show that preserved samples can yield robust estimates of carbon source contributions. We also empirically demonstrate, for the first time, the use of amino acid δ13C profiles to enhance δ15N‐based estimates of trophic position in food webs with multiple producers, and show that these estimates are not compromised by preservation effects. Overall, our findings support the view that amino acid δ13C and δ15N profiles from ethanol‐ and formalin‐treated CSIA‐AA samples can be directly used for addressing ecological questions.

A self‐preserving, partially biodegradable eDNA filter

Abstract eDNA studies often rely on water filtration in the field and immediate sample preservation to prevent DNA degradation during sample transport. However, filter membrane transfer steps for preservation can increase risk of sample contamination and the reliance on typical single‐use filter housings produces significant plastic waste. We created a new eDNA filter housing (compatible with any suction pump) partially comprised of a biodegradable, hydrophilic material that functions to automatically preserve captured eDNA via desiccation—no filter membrane transfer steps, no chemical or cold storage required. We tested the self‐preservation capabilities of the desiccating filter housings by filtering replicate samples in an eDNA mesocosm study and compared with ethanol preservation. Self‐preserving filters were placed back into original packaging for storage, and samples for both preservation methods were kept at room temperature until extraction at prescribed time points (11 days, 18 days, 25 days, 32 days, 60 days, 88 days, 172 days) post‐filtration. Paired field samples were also collected from six pond locations targeting an additional species to demonstrate field performance. Quantitative PCR results from the mesocosm study indicated that both methods effectively preserved eDNA over a 6‐month storage period, with the self‐preserving filters yielding slightly more target DNA on average (SQ = 329 copies) than ethanol‐preserved samples (SQ = 288 copies) (F1,38 = 4.050, p = 0.051). Neither method showed signs of degradation after 172 days. Results from field sampling indicated a larger difference between preservation methods, with the self‐preserving filters containing approximately 2X the eDNA of ethanol‐preserved samples on average (paired t test, p = 0.020). These data suggest that self‐preserving eDNA filter housings are a viable alternative to standard ethanol preservation methods and may provide higher detection sensitivity in some circumstances. The new filter housings should also help in reducing the risk of sample contamination, minimize protocol steps and result in less plastic waste. Such innovations are important to assure eDNA data quality and to help in facilitating the inclusion of non‐expert sample collectors (e.g. citizen scientists) in research programs.

Effect of preservation on fish morphology over time: Implications for morphological studies.

It has long been recognized that the process of preserving biological specimens results in alterations of body shape, though detailed studies examining the degree to which morphological changes occur throughout the preservation process are lacking. We utilize geometric morphometric analyses, an increasingly common tool for examining shape variation in a wide variety of biological disciplines, to examine the effects of formalin and ethanol preservation on the body shape of 10 freshwater fish species over time: from fresh specimens to eight weeks after preservation. We found significant changes in body shape among fresh and formalin fixed specimens. Furthermore, changes in body shape continue to occur after subsequent ethanol preservation. Two fish species collected at multiple localities show significant morphological differences for a limited number of morphometric characters. However, the significance, or lack thereof, often changed inconsistently from one stage of preservation to another. We conclude that morphometric analyses would ideally be performed on fresh specimens. However, recognizing that this is not always feasible, it is important to be aware of the morphometric changes that can occur during preservation.

Impact of freezing and ethanol preservation techniques on the stable isotope analysis of humpback whale (Megaptera novaeangliae) skin.

RationaleDistinct techniques employed to preserve different types of tissues may affect stable isotope analyses conducted on samples, and this is critical when field work takes place in remote areas. To investigate this, the stable isotope ratios (δ13C and δ15N values) obtained using two methods commonly used to preserve humpback whale (and other cetaceans) skin samples were compared.MethodsA total of 54 pairs of skin samples of humpback whales from the southern Baja California Peninsula, Mexico, were preserved in ethanol (90%) and by freezing, between 2007 and 2009. The δ13C and δ15N values were determined using a PDZ Europa ANCA‐GSL elemental analyzer interfaced to a PDZ Europe 20‐20 isotope ratio mass spectrometer. Parametric and nonparametric tests were used to compare the isotopic results.ResultsA significant (t = 4.93; p = 0.000003) variation of −0.92‰ was found between the mean δ13C values in ethanol (from −19.38‰ to −16.07‰; mean = −17.86‰) and freezing (from −20.67‰ to −16.44‰; mean = −18.78‰) techniques. No significant (U = 1314, p = 0.38) differences were observed in the δ15N values. The δ13C values were compared between preservation methods for each of the three years under analysis. Significant differences were observed in 2007 (t = 3.45; p = 0.0012) and 2008 (t = 3.13; p = 0.0030), but not for 2009 (t = 1.66; p = 0.12).ConclusionsBased on the results of this study, the use of ethanol to preserve humpback whale skin samples collected for stable isotope analysis is not recommended, particularly regarding the analysis of δ13C values. This study serves as a point of reference for future research on humpback whales or other whales involving skin samples preserved by freezing or in ethanol.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a useful tool fortherapid identification of wild flea vectors preserved in alcohol.

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re-emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI-TOF MS profiling. The goal of the present work was to assess the performance of MALDI-TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI-TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C.felis and C.canis.

Correction of the isotopic composition (δ13C and δ15N) of preserved Baltic and North Sea macrozoobenthos and their trophic interactions

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 595:1-13 (2018) - DOI: https://doi.org/10.3354/meps12543 Correction of the isotopic composition (δ13C and δ15N) of preserved Baltic and North Sea macrozoobenthos and their trophic interactions Jacqueline Umbricht1,*, Joachim W. Dippner1, Brian Fry2, Ingrid Kröncke3, Iris Liskow1, Petra Nehmer3, Franziska Thoms1, Maren Voss1 1Leibniz Institute for Baltic Sea Research Warnemünde, Seestr. 15, 18119 Rostock, Germany 2Australian Rivers Institute, Griffith University Brisbane, Queensland, Australia 3Senckenberg am Meer, Dept. for Marine Research, 26382 Wilhelmshaven, Germany *Corresponding author:jacqueline.umbricht@io-warnemuende.de ABSTRACT: To better understand food webs and the trophic relationships of marine animals studied by means of preserved material requires knowledge of the effects of preservation on the organic materials of interest. We examined the effects of formalin and ethanol preservation on the stable isotope values of carbon and nitrogen in the bivalves Mya arenaria and Tellina fabula and in the polychaetes Magelona spp. and Hediste diversicolor. Samples of these organisms were collected in the southern North Sea and the southern Baltic Sea, and were preserved with formalin, ethanol, and by freezing. The stable isotope patterns of carbon and nitrogen in the chemically preserved samples were related to those in the frozen samples. The influence of different preservation methods on the stable isotope composition was analyzed using a correction model that is independent of the molar C:N ratio. For most samples from the North Sea, significant correction factors were obtained. In contrast, none of the samples from the Baltic Sea were impacted by the preservatives with respect to their δ15N values, and, for H. diversicolor, also with respect to the δ13C values. In these cases, corrections of the δ13C and the δ15N values were not necessary. Quantitative metrics using a phenotype-based approach were computed to characterize aspects of the trophic structure of the benthic community. Phenotypic clustering in isotopic diversity indicates no competition for food between the dominant species. Our results indicate that a representative dominant mollusk and polychaete species have a higher trophic position in the Baltic Sea than in the North Sea. KEY WORDS: Macrozoobenthos · Preservation effects · Stable isotopes · Phenotype model · North Sea · Baltic Sea Full text in pdf format Supplementary material NextCite this article as: Umbricht J, Dippner JW, Fry B, Kröncke I and others (2018) Correction of the isotopic composition (δ13C and δ15N) of preserved Baltic and North Sea macrozoobenthos and their trophic interactions. Mar Ecol Prog Ser 595:1-13. https://doi.org/10.3354/meps12543 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 595. Online publication date: May 14, 2018 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2018 Inter-Research.

Histological age estimation of the eggs of Calliphora vicina Robineau Desvoidy (Diptera: Calliphoridae)

ABSTRACTAging blow fly eggs can be critical to a forensic investigation, but there are currently no forensically useful timelines describing internal anatomical changes in embryological development. This is partly due to the lack of an economical, rapid and technically simple histological technique to allow mass production of slides for research and casework. We present a histological method that uses a slightly modified standard laboratory processing run with 1 h fixation in 10% formalin, 2 h softening in Molliflex and Haemotoxylin and Eosin (H&E) staining. We also present a summary of the internal anatomical changes that can be visualized using our technique in the developing eggs of Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). We examined eggs from at least three different females grown at 15 °C and sampled at 6 h intervals, and eggs grown at 20 °C and sampled at 3 h intervals. Blind aging trials demonstrated that it is possible to accurately age material grown at 20 °C to within 6 h (but attempts to further narrow this interval resulted in errors in one-third of cases). It was also possible to see sufficient anatomical detail to age eggs preserved for forensic casework 5, 8, 9, 10 and 11 years previously. Additionally, we determined that section quality was improved by 5 s fixation in hot water prior to preservation in ethanol. However, hot water fixation for longer than this increased the level of section artefact.

Validating fin tissue as a non-lethal proxy to liver and muscle tissue for stable isotope analysis of yellow perch (Perca flavescens)

ABSTRACTStable isotope ecology typically involves sacrificing the animal to obtain tissues. However, with threatened species or in long-term longitudinal studies, non-lethal sampling techniques should be used. The objectives of this study were to (1) determine if caudal fin tissue could be used as a non-lethal proxy to liver and muscle for stable isotope analysis, and (2) assess the effects of ethanol preservation on δ15N and δ13C in fin tissue of juvenile yellow perch Perca flavescens. The δ13C of caudal fin was not significantly different from liver (t23 = −0.58; p = 0.57), and was more correlated with δ15N in liver (r2 = 0.78) than muscle (r2 = 0.56). Ethanol preservation enriched 15N and 13C for caudal fins, but by using our developed regression models, these changes in δ15N and δ13C can now be corrected. Overall, caudal fin tissue is a more reliable proxy to liver than muscle for δ15N and δ13C in yellow perch.

Estimating cod egg developmental stage based on DNA concentration

Abstract Pelagic egg surveys are commonly used to map Atlantic cod spawning areas in the northeastern Atlantic. However, a sampling location may not necessarily indicate a spawning location, because more developed eggs may have drifted long distances in coastal currents. Newly spawned eggs have only a few embryonic cells, whereas eggs in later developmental stages have progressively larger numbers of cells and hence greater amounts of DNA. The progression through developmental stages largely depends on temperature, which influences cell division and growth. Preservation of the eggs for later analysis presents logistical dilemmas. Preservation in ethanol is suitable for DNA extraction but obscures developmental stage, as the eggs shrink and turn opaque. On the other hand, preservation in formaldehyde retains visual characteristics, but this method limits the ability to extract intact DNA. Here we report a method to estimate developmental stages in Atlantic cod eggs by assaying the amount of DNA from eggs preserved in ethanol. We used a correlation between the amount of DNA in an egg and its embryonic developmental stage in samples from western Norway and used this correlation to estimate developmental stages of eggs sampled in northern Norway.

Effect of conservation of fish in formalin and ethanol on length-weight relationships and condition factor in Tlaloc labialis (Günther, 1866)

The effect of the preserver on the length-weight relationship and condition factor were estimated using 50 specimens of Tlaloc labialis; a small stream fish from the Grijalva River basin, México. They were measured at different stages of preservation: freshly collected, fixed in 10% formalin and preserved in 70% ethanol for over a 2 years period. All fish decreased in weight and condition factor, with very significant differences compared to the fresh material. The value of the coefficient (b) increased and stabilized from the second month of preservation in ethanol.

Even with rehydration, preservation in ethanol influences the mechanical properties of bone and how bone responds to experimental manipulation

Typically, bones are harvested at the time of animal euthanasia and stored until mechanical testing. However, storage methods are not standardized, and differential effects on mechanical properties are possible between methods. The goal of this study was to investigate the effects that two common preservation methods (freezing wrapped in saline-soaked gauze and refrigerating ethanol fixed samples) have on bone mechanical properties in the context of an in vitro ribosylation treatment designed to modify mechanical integrity. It was hypothesized that there would be an interactive effect between ribose treatment and preservation method. Tibiae from twenty five 11week old female C57BL/6 mice were separated into 2 preservation groups. Micro-CT scans of contralateral pairs assessed differences in geometry prior to storage. After 7weeks of storage, bones in each pair of tibiae were soaked in a solution containing either 0M or 0.6M ribose for 1week prior to 4 point bending tests. There were no differences in any cortical geometric parameters between contralateral tibiae. There was a significant main effect of ethanol fixation on displacement to yield (−16.3%), stiffness (+24.5%), strain to yield (−13.9%), and elastic modulus (+18.5%) relative to frozen specimens. There was a significant main effect of ribose treatment for yield force (+13.9%), ultimate force (+9.2%), work to yield (+22.2%), yield stress (+14.1%), and resilience (+21.9%) relative to control-soaked bones. Postyield displacement, total displacement, postyield work, total work, total strain, and toughness were analyzed separately within each preservation method due to significant interactions. For samples stored frozen, all six properties were lower in the ribose-soaked group (49%–68%) while no significant effects of ribose were observed in ethanol fixed bones. Storage in ethanol likely caused changes to the collagen matrix which prevented or masked the embrittling effects of ribosylation that were seen in samples stored frozen wrapped in saline-soaked gauze. These data illustrate the clear importance of maintaining hydration if the eventual goal is to use bones for mechanical assessments and further show that storage in ethanol can alter potential to detect effects of experimental manipulation (in this case ribosylation).

Methodological issues affecting the study of fish parasites. III. Effect of fish preservation method.

The aim of this study was to evaluate the influence of preservation method on the results of parasite community studies. Two host species, European perch Perca fluviatilis and European bitterling Rhodeus amarus, were examined for parasites after having been subjected to 4 different storage treatments: freezing, preservation in 4% formaldehyde or 70% ethanol and transportation of live (fresh) fish as a control. Preservation prior to dissection resulted in a loss of information, leading to incomplete quantitative data (all preservation treatments), qualitative data (ethanol and formaldehyde preservation) and a lowered ability to determine parasites to species level based on morphology compared to dissecting fresh fish. Of the more abundant taxa, only crustaceans and acanthocephalans provided relatively even results between treatments. We conclude that preservation media, such as ethanol or formaldehyde, significantly affects the ability to obtain precise parasite community data; hence, we recommend the use of freshly sacrificed fish for parasite community studies whenever possible. Alternatively, freezing may prove acceptable for evaluating parasite community taxonomic composition.

Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter

Summary Aqueous environmental DNA (eDNA) is an emerging efficient non‐invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next‐generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex‐GP polyethersulfone, pore size 0·22 μm, hereafter called SX) with commonly used methods. Our experimental set‐up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish‐free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy® Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq‐values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq‐values than polycarbonate track‐etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq‐values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro‐organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector.

Using dedicated contrast agents high-quality X-ray imaging of soft tissue structures with isotropic micrometre resolution has become feasible. This technique is frequently titled as virtual histology as it allows production of slices of tissue without destroying the sample. The use of contrast agents is, however, often an irreversible time-consuming procedure and despite the non-destructive principle of X-ray imaging, the sample is usually no longer usable for other research methods. In this work we present the application of recently developed large-area photon counting detector for high resolution X-ray micro-radiography and micro-tomography of whole ex-vivo ethanol-preserved mouse organs. The photon counting detectors provide dark-current-free quantum-counting operation enabling acquisition of data with virtually unlimited contrast-to-noise ratio (CNR). Thanks to the very high CNR even ethanol-only preserved soft-tissue samples without addition of any contrast agent can be visualized in great detail. As ethanol preservation is one of the standard steps of tissue fixation for histology, the presented method can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft-tissue visualisation.

A paired comparison of scat-collecting versus scat-swabbing methods for noninvasive recovery of mesocarnivore DNA from an arid environment

Noninvasive DNA sampling from scats can provide powerful tools for wildlife research depending in large part on how scats are collected in the field. Preservation of scat in 95–100% ethanol can be highly effective, but not always practical. Use of swabs to sample the mucous layer from scats is a popular alternative, but has not been adequately tested in arid environments, where the mucous layer can rapidly desiccate. We conducted a paired comparison of methods, extraction from scats stored in ethanol (“direct method”) versus from swabs (“swab method”), for carnivore scats collected under typical field conditions during May–September 2013 and January–February 2015 in northern California, USA. Direct quantification of DNA indicated that the extracts from ethanol-preserved direct-method samples contained, on average, an order of magnitude (14×) more target DNA than extracts from swab method (P < 0.03). For mitochondrial sequencing, which is typically used for species-typing, we obtained slightly higher success for the direct (81%) than the swab (73%) extracts, but not significantly so (P = 0.25, n = 104). Among 62 canid scats, microsatellite genotyping (typically used for individual identification) was much more successful for the direct extracts (74%) than the swab extracts (35%; P < 0.001, n = 62). Together, our findings indicated a substantial advantage of ethanol preservation followed by extraction directly from scats over swabbing in our arid study area. Our results apply most specifically to canids in the arid study environment; swabbing is potentially more effective in humid environments or in other carnivore species. Nevertheless, our findings highlight the importance of conducting pilot studies before committing to swabbing as a means of fecal DNA collection. © 2015 The Wildlife Society.

Length–mass relationships for lake macroinvertebrates corrected for back-transformation and preservation effects

Length–mass relationships are widely used to estimate body mass from body dimensions for freshwater macroinvertebrates. The relationships are influenced by environmental conditions and should be applied within ecosystems and geographic regions similar to those for which they were estimated. However, very few relationships exist for littoral macroinvertebrates, and thus we provide length–mass relationships for macroinvertebrates from lakes of the Central European lowlands. We compared log-linear and nonlinear methods for fitting length–mass relationships and tested the smearing factor for removing bias in mass predictions from log-linear models. We also estimated conversion factors to correct for mass changes during ethanol preservation and assessed the transferability of our results to different geographical regions. We showed that the log-linear approach gave better results in fitting length–mass relationships, while residuals showed that nonlinear models over-predict the mass of small individuals. The smearing correction factor successfully removed bias introduced by log transformation, and relationships transferred well between lakes in the same and different geographical regions. In total, 52 bias-corrected length–mass relationships are provided for littoral macroinvertebrates that are applicable also to lakes in geographic regions with similar environmental conditions, such as the Central European lowlands or the temperate lowland zone of America.