Ethanol Fixation Research Articles

Simple, fast, cost-efficient, reliable, and highly automated DNA content analysis of cells in adherent cultures.

The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double-stranded DNA. This is a laborious and time-consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample-processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well-proven commercial tool-The BD Cycletest™ Plus DNA Reagent Kit-primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.

Gelatin Zymography Can Be Performed on Fixed Brain Tissue.

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).

Assessment of stained direct cytology smears of breast cancer for whole transcriptome and targeted messenger RNA sequencing.

Rather than surgical resection, cytologic specimens are often used as first-line clinical diagnostic procedures due to higher safety, speed, and cost-effectiveness. Archival diagnostic cytology slides containing cancer can be equivalent to tissue biopsies for DNA mutation testing, but the accuracy of transcriptomic profiling by RNA sequencing (RNA-seq) is less understood. This study compares the results from whole transcriptome RNA-seq and a targeted RNA-seq assay of stained cytology smears (CS) versus matched tumor tissue samples preserved fresh-frozen (FF) and processed as formalin-fixed paraffin-embedded (FFPE) sections. Cellular cytology scrapes from all 11 breast cancers were fixed and stained using three common protocols: Carnoy's (CS_C) or 95% ethanol (CS_E) fixation and then Papanicolaou stain or air-dried then methanol fixation and DiffQuik stain (CS_DQ). Agreement between samples was assessed using Lin's concordance correlation coefficient. Library yield for CS_DQ was too low, therefore it was not sequenced. The distributions of concordance correlation coefficient of gene expression levels in comparison to FF were comparable between CS_C and CS_E, but expression of genes enriched in stroma was lower in cytosmear samples than in FF or FFPE. Six signatures showed similar concordance to FF for all methods and two were slightly worse in CS_C and CS_E. Genomic signatures were highly concordant using targeted RNA-seq. The allele fraction of selected mutations calculated on cytosmear specimens was highly correlated with FF tissues using both RNA-seq methods. RNA can be reliably extracted from cytology smears and is suitable for transcriptome profiling or mutation detection, except for signatures of tumor stroma.

The Near-Infrared Spectroscopy of Ethanol-Fixed Tissues to Detect Illicit Treatments with Glucocorticoids in Bulls.

This study aimed to set up indirect, rapid methods involving near infrared (NIR) spectroscopy analysis, to detect illicit treatments with glucocorticoids in bull. The ethanol fixation method (EtOH) was applied to 7 different tissues obtained from 20 Friesian bulls, 12 of which were experimentally administered with dexamethasone as part of a growth-promoting protocol for 60 days and slaughtered 26 days after the end of the treatment. A perfect discrimination was obtained for the 7 sampled tissues, considering a full UV-Vis-NIR range (350 ÷ 2500 nm), for both false positive and negative animals. The validated true positive and negative errors were zero for the longissimus thoracis muscle, 10% for the skin-dermis, 15% for the fat, 25% for the thymus gland and the semitendinosus muscle, 30% for the sternomandibularis muscle and 35% for the skin-hair. A multiple test on the most accessible tissues, that is, the thymus gland, the sternomandibularis muscle and fat, can be used as an alternative to provide indications about animals that have been subjected to illicit treatments. In the short space of three days from the slaughter, NIR spectroscopy of ETOH fixed tissues, would allow at least cost the detection of a probable illicit which could eventually be reported to health authorities for specific investigation in the frame of official controls.

Effects of fixatives and storage duration on avian sperm morphology

Sperm cells are variable both within and among species. To be able to accurately measure sperm cells and understand their function, it is important that sperm cells are preserved in a manner that maintains their structural integrity. Formalin is a widely used fixative and storage medium for sperm cells, but few studies have examined the effect of fixation and long-term storage on their morphological integrity. Ethanol is also a common fixation and storage agent for tissue samples, and here we examine if fixation and storage in formalin or ethanol alters sperm cell size and structural integrity. We found no significant effects of the fixation process on fresh sperm cells fixed in formalin or ethanol. Further, there were no consistent length changes in sperm cells stored in formalin or ethanol over a period of 227 days, or in sperm cells stored in formalin for three years. A comparison across 13–14 years of storage time showed a small but significant reduction in sperm cell length of 0.93%. Furthermore, sperm cells initially fixed in formalin remained quite stable in dry storage on glass slides for a minimum of six months (we found a mean reduction in sperm cell length of 0.18% after 6 months). The proportion of sperm cells with head damage was, however, much higher for samples stored in ethanol than for those stored in formalin. Overall, 70% of sperm cells had acrosome damage in ethanol versus only 3% in formalin. Finding intact sperm cells for measuring length, therefore, required greater effort in ethanol samples than in formalin samples. Our findings indicate that use of sperm cells from long-term storage for the study of sperm morphometrics is justified for either fixative, although formalin clearly preserves the sperm cells better.

P05.03.B Application of rapid cytology in intraoperative diagnosis of primary central nervous system lymphoma

Abstract Background To assesment the value of rapid cytological diagnosis in primary central nervous system lymphoma (PCNSL) surgery. Material and Methods A total of 213 PCNSL specimens from the First Affiliated Hospital of Fujian Medical University between 2012 to 2022 were included, 1-2mm3 was taken from the tissues diagnosed for intraoperative pathology and made into cell smears, rapid cytological diagnosis was performed after 95% ethanol fixation and H&E staining, most of the remaining tissues were diagnosed by frozen section. With the results of paraffin section pathological diagnosis as the control standard, compared the accuracy of rapid cytology and frozen tissue section and their combined diagnosis. Results Cytological smears can reveal microscopic structures within the cells under a microscope, furthermore, it can avoid the formation of artifacts or deformation of cells when frozen due to excessive water in brain tissue. Compared with paraffin section pathological diagnosis, the diagnostic accuracy of rapid cytology was 151cases (70.89%) , that of frozen tissue section was 175 cases (82.16%) , and that of the combination of the two methods was 187 cases (87.79%) . There were 26 cases (12.21%) in which neither method could give a definite conclusion intraoperatively and only depended on the results of paraffin tissue sections. Conclusion Rapid cytology alone is less accurate in diagnosing PCNSL than frozen sections alone. However, the combination of the two diagnosis can improve the accuracy of intraoperative diagnosis of PCNSL and correct some errors that may occur when only relying on frozen section diagnosis. It is especially suitable for stereotactic biopsy surgery or cases with very little tissue, as well as primary medical units without frozen section machine and other equipment.

Neutrophil fixation protocols suitable for substrates to detect anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence

Anti-neutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that recognize neutrophil cytoplasmic antigens. The major ANCA antigens are myeloperoxidase and proteinase 3. Necrotizing small vessel vasculitis accompanied by ANCA production is called ANCA-associated vasculitis (AAV). In addition to AAV, ANCA is sometimes produced in patients with connective tissue diseases, such as systemic lupus erythematosus, and inflammatory bowel diseases. Indirect immunofluorescence (IIF) and enzyme immunoassay (EIA) have been used to detect ANCAs. Recently, the accuracy of EIA has improved and it has become the gold standard for ANCA detection. However, IIF does not lose its role in ANCA detection because EIA cannot detect ANCAs that recognize antigens other than those coated on the plate. For IIF, neutrophil substrates prepared with two different fixations, namely, ethanol fixation and formalin fixation, are used. There is a recommended protocol for ethanol fixation but not for formalin fixation. This study prepared neutrophil substrates according to the recommended protocol for ethanol fixation and protocols in the literature and original protocols for formalin fixation and then examined ANCA specificity and how storage period would influence the number of cells, antigen distribution, and antigenicity of the substrates. As a result, the number of cells and antigen distribution did not change after storage for up to 2 months regardless of fixation protocols, whereas a time-dependent decline in ANCA antigenicity and a fixation protocol-dependent difference in ANCA specificity were observed. How neutrophils are fixed on the glass slide needs to be checked upon evaluation of ANCAs by IIF.

The effects of formalin fixation and fluid storage on stable isotopes in rodent hair

Abstract Stable isotopes are increasingly being used to unlock the wealth of information contained in specimens preserved in museum collections. However, preservation methods that employ formalin may confound ecological interpretations. To quantify the effects of formalin fixation and subsequent fluid storage in ethanol on the isotopic signatures of small mammal hair, we analyzed δ 13C and δ 15N values from specimens of seven rodent species that were sampled repeatedly both before and after varying lengths of formalin fixation (1–11 days) and ethanol storage (1–6 years). We supplemented these data with a 2-week fixation experiment using deer mice (Peromyscus maniculatus) in which no ethanol storage was employed. As expected, preservation in formalin and ethanol had no discernable effect on δ 15N values. In contrast, specimen δ 13C values decreased in a saturating fashion during formalin fixation and over subsequent years of fluid storage in ethanol. On the basis of models that we fit to these time series, we estimate the long-term effect of fixation and storage on δ 13C values to be −0.92‰ after 4 years. This biologically relevant shift in δ 13C values should be accounted for when inferring the diets of species from fluid-stored museum collections and when comparing across specimens with different preservation histories.

Novel attempt at discrimination of a bullet-shaped siphonophore (Family Diphyidae) using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS)

One major difficulty in identifying the gelatinous bodied bullet-shaped Siphonophore, Diphyids, is that their shape is deformed following ethanol fixation. Ethanol often is preferred over other fixatives, since samples fixed in ethanol can be used for molecular studies that can supplement morphological findings. To overcome this problem, we obtained protein mass spectra of ten species of Diphyidae found in the waters of the Kuroshio Current (Northwest Pacific and South Coast of South Korea) to test whether MALDI-ToF MS could be used as a methodology for species identification. In addition, a number of morphological characteristics that can be used with ethanol-treated samples was summarized. Concatenated phylogenetic analysis was also performed to determine the phylogenetic relationship by obtaining partial sequences of four genes (mtCOI, 16S rRNA, 18S rRNA, and ITS regions). Based on our integrative analysis, MALDI-ToF MS was evaluated as a potentially fast, inexpensive, and accurate tool for species identification along with conventional morphological and DNA barcoding for Diphyidae.

Characterization of the gut microbiome in wild rocky mountainsnails (Oreohelix strigosa)

BackgroundThe Rocky Mountainsnail (Oreohelix strigosa) is a terrestrial gastropod of ecological importance in the Rocky Mountains of western United States and Canada. Across the animal kingdom, including in gastropods, gut microbiomes have profound effects on the health of the host. Current knowledge regarding snail gut microbiomes, particularly throughout various life history stages, is limited. Understanding snail gut microbiome composition and dynamics can provide an initial step toward better conservation and management of this species.ResultsIn this study, we employed 16S rRNA gene amplicon sequencing to examine gut bacteria communities in wild-caught O. strigosa populations from the Front Range of Colorado. These included three treatment groups: (1) adult and (2) fetal snails, as well as (3) sub-populations of adult snails that were starved prior to ethanol fixation. Overall, O. strigosa harbors a high diversity of bacteria. We sequenced the V4 region of the 16S rRNA gene on an Illumina MiSeq and obtained 2,714,330 total reads. We identified a total of 7056 unique operational taxonomic units (OTUs) belonging to 36 phyla. The core gut microbiome of four unique OTUs accounts for roughly half of all sequencing reads returned and may aid the snails’ digestive processes. Significant differences in microbial composition, as well as richness, evenness, and Shannon Indices were found across the three treatment groups.ConclusionsComparisons of gut microbiomes in O. strigosa adult, fetal, and starved samples provide evidence that the host internal environments influence bacterial community compositions, and that bacteria may be transmitted vertically from parent to offspring. This work provides the first comprehensive report on the structure and membership of bacterial populations in the gastropod family Oreohelicidae and reveals similarities and differences across varying life history metrics. Strong differentiation between these life history metrics demonstrates the need for wider sampling for studies of dynamics of the snail gut microbiome.

White matter imaging of ethanol-fixed rat brain by phase-contrast X-ray computed tomography.

Phase-contrast X-ray computed tomography imaging (PCI) based on crystal X-ray interferometry can detect minute density differences within biological soft tissues without contrast agents. Ethanol fixation yields increased tissue-background density differences due to the dehydrating and delipidifying effects of ethanol. To obtain high image contrast of cerebral white matter structures in PCI, tissue fixation using ethanol and routinely used formalin have been examined. Ethanol-fixed (EF) (n = 4) and formalin-fixed (FF) (n = 4) rat brains were imaged by crystal X-ray interferometry-based PCI. Tissue staining/microscopy was also performed for histological comparison and myelin density evaluation. Three-dimensional white matter tract images were reconstructed. Superior image contrast was obtained in the images of EF brains (EF images) compared to those of formalin-fixed brains (FF images), particularly for white matter structures. Significant density differences between the white matter structures and hippocampus (P < 0.01)/thalamus (P < 0.001) were observed in the EF, but not FF, images. Ethanol fixation enhanced the image contrast of white matter tracts by approximately sixfold compared to formalin fixation, and close agreement (r2 = 0.97; P < 0.05) between the density values on the CT images and the myelin density values in histological images was observed for the EF brains. Three-dimensional reconstruction of the white matter tracts was possible from the EF images, but not FF images. Ethanol fixation resulted in marked contrast enhancement of cerebral white matter structures in PCI. Thus, high-resolution PCI using ethanol for tissue fixation could be valuable for experimental neurological studies and postmortem neuropathology evaluation.

The Combination of Paraformaldehyde and Glutaraldehyde Is a Potential Fixative for Mitochondria.

Mitochondria are highly dynamic organelles, constantly undergoing shape changes, which are controlled by mitochondrial movement, fusion, and fission. Mitochondria play a pivotal role in various cellular processes under physiological and pathological conditions, including metabolism, superoxide generation, calcium homeostasis, and apoptosis. Abnormal mitochondrial morphology and mitochondrial protein expression are always closely related to the health status of cells. Analysis of mitochondrial morphology and mitochondrial protein expression in situ is widely used to reflect the abnormality of cell function in the chemical fixed sample. Paraformaldehyde (PFA), the most commonly used fixative in cellular immunostaining, still has disadvantages, including loss of antigenicity and disruption of morphology during fixation. We tested the effect of ethanol (ETHO), PFA, and glutaraldehyde (GA) fixation on cellular mitochondria. The results showed that 3% PFA and 1.5% GA (PFA-GA) combination reserved mitochondrial morphology better than them alone in situ in cells. Mitochondrial network and protein antigenicity were well maintained, indicated by preserved MitoTracker and mitochondrial immunostaining after PFA-GA fixation. Our results suggest that the PFA-GA combination is a valuable fixative for the study of mitochondria in situ.

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers

We designed an experiment to analyze the gut content of Rotifera based on DNA barcoding and tested it on Asplanchna sp. in order to ensure that the DNA extracted from the rotifer species is from the food sources within the gut. We selected ethanol fixation (60%) to minimize the inflow effects of treated chemicals, and commercial bleach (the final concentration of 2.5%, for 210 s) to eliminate the extracellular DNA without damage to the lorica. Rotifers have different lorica structures and thicknesses. Therefore, we chose a pretreatment method based on Asplanchna sp., which is known to have weak durability. When we used the determined method on a reservoir water sample, we confirmed that the DNA fragments of Chlorophyceae, Diatomea, Cyanobacteria, and Ciliophora were removed. Given this result, Diatomea and cyanobacteria, detected from Asplanchna, can be considered as gut contents. However, bacteria were not removed by bleach, thus there was still insufficient information. Since the results of applying commercial bleach to rotifer species confirmed that pretreatment worked effectively for some species of rotifers food sources, in further studies, it is believed to be applicable to the gut contents analysis of more diverse rotifers species and better DNA analysis techniques by supplementing more rigorous limitations.

Low Temperature Enzymatic Hydrolysis (LTEH) and Fermentation for Bioethanol Generation from Suweg (Amorphophallus campanulatus B) Starch

The ethanol generation through low temperature enzymatic hydrolysis (LTEH) of Suweg (Amorphophallus campanulatus B) starch followed by anaerobic fermentation using Saccharomyces cerevisiaehas been studied. Granular starch hydrolyzing enzyme (GSHE) as Stargen TM 002 was used in the hydrolysis to degrade starch into reducing sugar at 30°C and pH 4. The concentration of Suweg starch was 200 g/L, concentration of enzyme were 1; 1.5 and 2% (w/w), respectively. The fermentation was carried out at pH 4.5 and 30 °C for 72 h employing yeast concentration of 1gL−1. The ideal state of the procedure was fermentation utilizing concentration of Suweg starch 200 g L−1, concentration enzyme 1.5% (w/w), pH 4.5 and 30°C, for 60 h, which came about reducing sugar grouping of 31.32 g/L and further used for ethanol generation. It was discovered that most extreme ethanol fixation and profitability 13.12 g L−1 and 0.3727 gL−1h−1, separately. Although the result shows that Suweg starch is a potential raw material for ethanol generation, further investigations are required in both generation sustainability and techno-economical considerations.

Good Laboratory Practices for the Use of Ethanol Fixation of Fine Needle Aspiration Smears in the Era of Molecular Testing

Good Laboratory Practices for the Use of Ethanol Fixation of Fine Needle Aspiration Smears in the Era of Molecular Testing

Imaging of Mouse Brain Fixated in Ethanol in Micro-CT.

Micro-CT imaging is a well-established morphological method for the visualization of animal models. We used ethanol fixation of the mouse brains to perform high-resolution micro-CT scans showing in great details brain grey and white matters. It was possible to identify more than 50 neuroanatomical structures on the 5 selected coronal sections. Among white matter structures, we identified fornix, medial lemniscus, crossed tectospinal pathway, mammillothalamic tract, and the sensory root of the trigeminal ganglion. Among grey matter structures, we identified basal nuclei, habenular complex, thalamic nuclei, amygdala, subparts of hippocampal formation, superior colliculi, Edinger–Westphal nucleus, and others. We suggest that micro-CT of the mouse brain could be used for neurohistological lesions evaluation as an alternative to classical neurohistology because it does not destroy brain tissue.

Morphometric convergence among European sand gobies in freshwater (Gobiiformes: Gobionellidae).

The five genera of sand gobies inhabit the seas and freshwaters of Europe and western Asia and occupy habitats ranging from fully marine to exclusively freshwater. In this study, we use geometric morphometrics to quantify body shape among sand gobies, in order to investigate how shape has evolved and how it is related to habitat. We also compare body shape between preserved museum specimens and fresh specimens, to determine whether or not fixation and storage in ethanol introduce detectable bias. We confirm that the fixed specimens exhibit significant shape changes as compared to fresh specimens, and so, we perform the bulk of our analyses exclusively on fixed specimens. We find that Economidichthys, Orsinigobius, and Pomatoschistus occupy distinct regions of morphospace. Knipowitschia and Ninnigobius have intermediate forms that overlap with Pomatoschistus and Orsinigobius, but not Economidichthys. This pattern is also in rough accordance with their habitats: Pomatoschistus is fully marine, Economidichthys fully freshwater, and the others fresh with some brackish tolerance. We augment a recent phylogeny of sand gobies with data for P. quagga and interpret morphometric shape change on that tree. We then evaluate convergence in form among disparate lineages of freshwater species by constructing a phylomorphospace and applying pattern‐based (convevol) measures of convergence. We find that freshwater taxa occupy a mostly separate region of morphospace from marine taxa and exhibit significant convergence in form. Freshwater taxa are characterized by relatively larger heads and stockier bodies than their marine relatives, potentially due to a common pattern of heterochronic size reduction.

An improved method to identify osmium-stained organic matter within soil aggregate structure by electron microscopy and synchrotron X-ray micro-computed tomography

Organic matter (OM) in surface soil is largely present within porous mineral-dominant clusters called aggregates. Recent studies have shown the localization of OM within large macroaggregate structure based on synchrotron X-ray micro-computed tomography (μCT) coupled with a vapor-phase, osmium (Os)-staining pretreatment. Here we developed a new approach to identify OM locations within submillimeter-sized aggregates at higher spatial resolution and assessed its applicability using a well-characterized surface volcanic soil, an allophanic Andisol, under no-tillage with leaf compost addition. Water-stable aggregates (500–800 μm in diameter) were embedded in agarose, chemically fixed, and repeatedly stained by osmium tetroxide and thiocarbohydrazide, followed by gradual dehydration with ethanol and resin fixation. Scanning electron microscopy and μCT observation showed no signs of physical alteration during sample preparation. Energy-dispersive X-ray spectroscopy analysis of the microtome-cut surface of the aggregate identified plant detritus highly enriched in Os and much smaller amorphous OM (<10 μm) that were high in Fe, Al, and Si as well as Os. Synchrotron μCT imaging by photon energies above and below Os L3 X-ray absorption edge (10.87 keV) and subsequent image calculation showed the distribution of Os-stained voxels down to micrometer scale. The improved resolution compared to previous studies was partly attributable to liquid-state staining and thiocarbohydrazide bridging. Other advantages and some cautions of the proposed method were discussed.

Ethanol fixation method for heart and lung imaging in micro-CT.

The soft tissue imaging in micro-CT remains challenging due to its low intrinsic contrast. The aim of this study was to create a simple staining method omitting the usage of contrast agents for ex vivo soft tissue imaging in micro-CT. Hearts and lungs from 30 mice were used. Twenty-seven organs were either fixed in 97% or 50% ethanol solution or in a series of ascending ethanol concentrations. Images were acquired after 72, 168 and 336h on a custom-built micro-CT machine and compared to scans of three native samples. Ethanol provided contrast enhancement in all evaluated fixations. Fixation in 97% ethanol resulted in contrast enhancement after 72h; however, it caused hardening of the samples. Fixation in 50% ethanol provided contrast enhancement after 336h, with milder hardening, compared to the 97% ethanol fixation, but the visualization of details was worse. The fixation in a series of ascending ethanol concentrations provided the most satisfactory results; all organs were visualized in great detail without tissue damage. Simple ethanol fixation improves the tissue contrast enhancement in micro-CT. The best results can be obtained with fixation of the soft tissue samples in a series of ascending ethanol concentrations.