Preservation In Formalin Research Articles

Effects of preserving juvenile rainbow trout (Oncorhynchus mykiss) in formalin and alcohol on body length, mass, and condition factor estimates

Abstract Body length and weight are key parameters measured to provide information about the growth and condition of larval, juvenile, and adult fishes from both the wild and aquaculture. The goal of this study was to evaluate the effects of preservation in formalin and alcohol on juvenile rainbow trout, Oncorhynchus mykiss (Walbaum) standard body length (SL) and weight changes. Standard length (19.43–32.98 mm SL) decreased after 60 days of preservation by an average of 2.98% in 96% alcohol and 2.26% in 10% formalin. In contrast, fish body weight (0.094–0.63 g) decreased in alcohol (31.22%) but increased in formalin (11.65%). These percentage values can be used as correction factors, with the exception of fish body weight in alcohol since the size of individuals affected the magnitude of change. Accordingly, a correction formula accounting for fish weight must be applied (fresh weight (g) = -0.0363 + 1.667 × preserved length). Changes in fish length and body weight in different preservatives resulted in significant differences in the Fulton condition factor, with underestimation in alcohol (fresh F = 1.58; preserved F = 1.20) and overestimation in formalin (fresh F = 1.60; preserved F = 1.92).

Abundance and Distribution of Benthic Macro-Invertebrates of Luhu Dam, Michika, Adamawa State, Nigeria

The study uses benthic macroinvertebrates as indicators to assess water quality in the Luhu Mini Dam, Michika, Adamawa State. The study involves the collection of benthic macroinvertebrate samples over six months, utilizing the Van Veen grab method for sampling and preservation in formalin. Upon transportation to the laboratory, the samples were identified and subjected to statistical analysis, including the Shannon and Weiner Index, Margalef’s Index, Simpson’s diversity Index, and Pielou’s Evenness. The study aims to provide fundamental information on the health of the Luhu Mini Dam and to understand the water quality and pollution status, thereby informing necessary management measures. The distribution and diversity of species across three different stations. A total of 462 individuals representing 20 taxa from 3 phyla were collected. Station 1 yielded 16 taxa and 162 individuals, station 2 had 17 taxa and 201 individuals, and station 3 had 17 taxa and 99 individuals. The diversity indices revealed variation in the number of species across stations, with stations 3 and 2 having the highest number of species, while station 1 had the lowest. The general diversity (H') was highest at station 3, followed by stations 1 and 2, whereas station 3 also exhibited the highest Evenness index (E). Moreover, the taxa richness index, as calculated by Margalef's Index (d), peaked at station 3. Simpson's dominance index showed that Station 2 exhibited the highest dominance, followed by Station 1 and Station 3. These findings provide valuable insights into the distribution and diversity of species across different biotopes.

Plastination - A method for preservation of oral hard and soft tissue biopsy specimen v/s the conventional method of preservation with formalin.

Plastination is one of the most advanced method for preserving perishable biological specimen as well as tissue samples as for a longer period of time using polymers. To evaluate the changes in dimension of hard and soft tissue specimens after plastination procedure and compare it with the conventional method of preservation in formalin. Institution-based retrospective study. The study included 20 formalin-fixed soft tissue and 10 formalin-fixed hard tissue specimens. All the specimens were plastinated which involved four basic steps of fixation, dehydration and defatting, impregnation with polymer and curing of polymer followed by finishing and storage. The specimens were analysed for shrinkage and dimensional changes and changes in colour and consistency between formalin-fixed specimen and plastinated soft tissue and hard tissue specimen. Descriptive statistics were used. After plastination, soft tissues showed average shrinkage of 3.49%with a range of 0.80-7.90% in comparison to the original size. In case of teeth and hard tissue specimen, there was no evidence of dimensional changes or shrinkage before and after plastination. Changes in colour and consistency of the soft tissue specimens were also noted before plastination and after plastination. Although the plastinated specimens in the current study showed minimal shrinkage rate, they have proved to be an excellent alternative to formalin-fixed specimens as they are easy to handle and maintain, non-infectious, non-toxic, user-friendly.

Genetic and morphological identification of formalin fixed, preserved larval fishes; can we have the best of both worlds?

Surveys of larval fishes require accurate identifications of larvae, which are essential to understand early life history of fish, fish ecology and fisheries. However, the identification of larval fishes requires microscopic examination that is substantially more difficult than that of juvenile and adult fishes, as many larval stages remain undescribed. Furthermore, the traditional, formalin fixation of larval fishes were previously thought to prevent genetic sequencing compared to ethanol preserved larvae. In this study, we used an integrative taxonomic approach based on morphology, imaging and DNA barcoding of the mitochondrial (mtDNA) cytochrome c oxidase subunit (COI) gene. We used this approach in both cultured yellowtail kingfish, Seriola lalandi and wild sourced fish larvae that had been fixed in 5% formalin. Based on controlled and in-field formalin treatments, DNA barcoding and genetic species identification was 100% successful in cultured yellowtail kingfish fixed in formalin for up to 6 months, while barcoding of wild caught fish larvae enabled species identification of 93% of up to 8-weeks formalin fixed specimens. Furthermore, we demonstrated the viability of using either whole larval individuals or a single eyeball (<1 mm diameter, thus retaining the specimen intact) from formalin fixed larval fish for genetic species identification. While COI genetic identifications from the in-field experiments were patchier than the controlled experiments, our study highlights the possibility of recovering suitable DNA from larvae that have been fixed in formalin for up to six months. This was achieved by applying DNA extraction methods that use de-cross-linking steps and species identification based on both full-length reference and mini-barcodes. Our study provides the larval fish research community with a practical framework for undertaking both morphological and genetic identifications of larval fish assemblages, particularly when geographic relevant reference sequence databases (based on vouchered adult fishes) are available for interrogation. It also simplifies field-based collection of samples allowing their preservation in formalin without compromising the genetic identification of species.

Shrinkage of Rio Grande Silvery Minnow after Preservation in Formalin and Storage in Ethanol

Understanding how the preservation process affects fish morphology is important for studies that use museum collections as voucher specimens. Sixty-nine Rio Grande silvery minnows (standard length [SL] 32.14–81.65 mm) were observed over 545 days during a formalin-to-water-to-ethanol preservation procedure. Median standard length decreased by the end of each preservation step; specimens shrank 1.06 mm in formalin solution, 1.97 mm in 35% ethanol, 2.17 mm in 50% ethanol, and 2.48 mm in 70% ethanol. Peak shrinkage occurred at 365 d, with a median decrease of 3.86 mm (SL 29.57–75.98 mm). After 545 days, Rio Grande silvery minnows began to increase in length, exhibiting a median shrinkage of 2.04 mm from live length. Research on museum specimens that includes morphological measurements should consider that changes in length or body shape may influence or hinder the ability to detect changes in morphology over time.

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.

Effect of Preservation in Formalin and Alcohol on the Growth Rate Estimates of Larval Northern Pike

Abstract Body length measurements and growth rate (GR) analysis are important steps in research on the ecology of early development stages of fish. In order to obtain accurate results, the lengths of field-collected and preserved larvae must be corrected for shrinkage. Northern Pike Esox lucius larvae were kept in the laboratory in freshwater conditions (300-L tank; temperature = 16°C), where they were fed six times a day ad libitum with aquaculture feed. Every 3 d over a period of 30 d, a subsample of approximately 30 larvae was collected, measured, and placed in separate vials in 96% alcohol and 4% formalin. All of the larvae (8.8–34 mm SL; n = 276) were measured again after 90 d of preservation. The shrinkage ranged from 0% to 11.6%, averaging 3.42% in alcohol (n = 134) and 3.74% in formalin (n = 139). The difference was not statistically significant. In both preservatives, the relative shrinkage (%) was higher for small fish than for large fish. The length correction for 8–34-mm SL fish in both alcohol and formalin should be done by adding 0.7 mm to the length measured after preservation. Shrinkage did not affect fish GRs estimated based on linear regression fitted to size-at-age data (0.645 mm/d for unpreserved larvae and 0.650 mm/d for preserved larvae). In the opposite case, when growth was estimated as mean GR between hatching and sampling, the shrinkage effect was significant: the mean GR was 0.604 mm/d for unpreserved larvae and 0.558 mm/d for preserved larvae. The presented shrinkage correction formula allows for obtaining accurate length and GR data for larval Northern Pike that should lead to conclusions that credibly support undertaking appropriate management strategies.

Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.

No evidence for effects of formalin storage duration or solvent medium exposure on avian sperm morphology

Morphometric analysis of avian spermatozoa from sperm samples preserved in formalin is a frequently adopted procedure in basic science (e.g. evolutionary ecology) and applied disciplines (e.g. animal breeding). Many research questions such as individual-based longitudinal studies of sperm traits require comparisons of formalin-stored sperm samples collected across multiple sampling events, which may be separated by years. Such analyses presuppose that prolonged storage in formalin does not affect sperm morphology, an assumption often implicitly made in the analysis of avian sperm morphology. This assumption, however, has never been tested, although for many study designs a potential effect of sperm storage duration may well confound the focal analysis. Based on pairwise comparisons of 22 experimental ejaculates from three passerine bird species, we found no evidence that differential storage duration of more than 1 year had affected the total length of spermatozoa stored in a 5 % formaldehyde solution. This suggests that formalin-stored sperm samples from long-term studies or museum collections can be merged in combined analyses without confounding differential storage duration with natural between-year variation in sperm dimensions or age effects in longitudinal studies. Based on pairwise comparisons of 29 split ejaculates, we also found no evidence that spermatozoa differed in length when solved initially in either phosphate buffered saline or Dulbecco’s Modified Eagle Medium, a standard medium for videotaping live sperm, prior to preservation and storage in formalin. Sperm samples treated differently in this respect may thus be merged into combined analyses, too.

Forensic toxicological analyses of drugs in tissues in formalin solutions and in fixatives

Forensic toxicological analyses of drugs in tissues in formalin solutions and in fixatives

Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies

Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies

A comparative analytical assessment of iodides in healthy and pathological human thyroids based on IC-PAD method preceded by microwave digestion

A comparative analytical assessment of iodides in healthy and pathological human thyroids based on IC-PAD method preceded by microwave digestion

Comparison of the DNA Preservation in Neutral-Buffered Formalin Fixed Paraffin-Embedded Tissue and in Non-Buffered Formalin Fixed Paraffin-Embedded Tissue

Background: The preservation of optimized DNA and its extraction from formalin-fixed, paraffinembedded (FFPE) tissues are important issues. There has been some doubt over whether 10% neutral-buffered formalin is an ideal fixation solution for DNA preservation over non-buffered for malin, as conventionally recommended. In this study, the correlation between the efficiency of DNA extraction from FFPE tissues and buffered formalin was evaluated. Methods: Several tissues with same conditions except fixatives were fixed in four different formalin solution groups and were routinely processed as paraffin-embedding protocols. DNAs were extracted from four different FFPE tissues that were stored for over 3 months and over 9 months. The quantity and quality of the DNAs were assessed with a NanoDrop ND-1000 spectrophotometer, and the polymerase chain reaction (PCR) amplification and degradation were analyzed via microchip electro phoresis. KRAS mutation analysis and microsatellite instability (BAT25) PCR were performed with each sample. Results: The results showed no remarkable difference in the four groups. Conclusions: The study findings demonstrate that DNA preservation is fairly unaffected by a neutral buf fer where there is short formalin manufacture period and an adequate formalin fixation time be fore embedding in paraffin.

Molecular identification of Taenia specimens after long-term preservation in formalin

Molecular identification of Taenia specimens after long-term preservation in formalin

The Critical Role of Permanent Voucher Specimens of Hosts and Vectors in Public Health and Epidemiology

The Critical Role of Permanent Voucher Specimens of Hosts and Vectors in Public Health and Epidemiology

Length correction for early-juvenile Brazilian herring Sardinella janeiro (Eigenmann, 1894) after preservation in formalin, ethanol and freezing

This work aims to quantify the variation in total length and body mass for the early-juvenile Brazilian herring Sardinella janeiro and to determine total length and body mass correction equation to allow fresh measures to be calculated from preserved ones. Fishes were randomly assigned to one of five preservation methods (freezing at - 20º C, 2.5% and 5% formalin, 70% and 95% ethanol), and measured for total length (TL) and body mass (W) before preservation, and on days 5, 15, 30, and 60 after storage. Significant reductions in total length and body mass occurred during the first 5 days after preservation and continued to contract significantly at a lesser rate through 30 days in most methods. Exceptions were shown for body mass in freezing and 5% formalin, where the greatest losses occurred after 30 days of preservation. The degree of shrinkage for total length and body mass was very much dependent on fish size, with smaller specimens shrinking more than larger ones. The fresh total length and body mass can be back-calculated using equations that describe the relationship between fresh and preserved individuals after 60 days storage for all methods except for body mass in freezing.

Effect of Different Preservative Methods on Fracture Behavior of Bovine Cortical Bone

Bone specimens used for evaluating the mechanical properties of bone may not have been necessarily preserved by the same method before they become available. To this end, the mechanical properties of bone may be affected by freezing, a common preservation method, as well as by formalin and other preservation solutions. In this study, bone specimens were preserved by different methods (i.e., freezing, preservation in saline, preservation in ethanol, and preservation in formalin) to examine the effects of each preservation method on the fracture characteristics of bovine femoral cortical bone. Regarding the crack extension behavior in the fracture toughness test, microcracks accumulated at the top of the slit in the low-load region before the maximum load was reached. When such accumulated microcracks grew to a visible size, a crack was formed from the top of the slit, and then grew with subsequent expansion of the processing zone. The fracture toughness values of the bone groups preserved in formalin and neutral buffered formalin were significantly lower than those of the bone groups preserved by other methods. As the fracture surface by scanning electron microscopic observations was smoother with a loading rate of 20 mm/min than with 1 mm/min, cracks were considered to develop easily, resulting in a lower fracture toughness value. Scanning electron microscopic observations of a test section subjected to a low loading rate showed that the fracture surface of the bone groups preserved in formalin or neutral buffered formalin was flat and smooth. In the other preservation groups, the lamellae adjacent to the top of the slit had undulating contours with plastic deformation.

Shrinkage of Gobiocypris rarus, Procypris rabaudi, and Sinilabeo rendahli preserved in formalin

Length measurements of preserved fishes are necessary in many types of fish surveys because logistics often do not allow for fish measurement immediately after catch. If the fixative causes significant shrinkage, then the preserved lengths cannot be directly used to indicate accurate live lengths. The objective of this study was to determine how preservation in formalin affects standard length of Gobiocypris rarus larvae (24-day-old and newly hatched), larval Procypris rabaudi (4-day-old), and larval Sinilabeo rendahli (12-day-old). Fishes were measured (to nearest 0.01 mm) and individually fixed in the appropriate formalin solution (2.5% or 5.0% formalin), then re-measured at 0.5, 1, 3, 7, 14, 30, 45 and 75 days after preservation to follow the time course of shrinkage. Most of the shrinkage occurred within the first half day after preservation. The 5.0% formalin caused a higher relative shrinkage rate than did the 2.5% solution; however, the difference was not statistically significant. In G. rarus, initial shrinkage of newly hatched larvae was higher than that of 24-day-old larvae.

Combined morphological and molecular analysis of individual nematodes through short‐term preservation in formalin

AbstractSmall metazoans such as marine nematodes are increasingly identified using both molecular and morphological techniques. Formalin is the preferred fixative for morphological analysis but specimens become unsuitable for molecular study due to formalin‐induced modification of DNA. Nematodes fixed in ethanol work well for molecular studies but become unsuitable for taxonomy due to shrinkage. Here we show for the first time that formalin can be used as a short‐term fixative (≤ 7 days) for marine nematodes, allowing both morphological and molecular work to be conducted on the same individual. No sequence ambiguities were detected in polymerase chain reaction (PCR) amplifications of 18S ribosomal DNA (rDNA) following short‐term formalin preservation.

The quality of DNA recovered from the archival tissues of atomic bomb survivors is good enough for the single nucleotide polymorphism analysis in spite of the decade-long preservation in formalin.

It is well known that the yield of DNA recovered form tissues preserved in formalin is inversely proportional to the stored duration. How is the quality? We tested the quality of DNA from archival tissues of atomic-bomb survivors stored in formalin for decades with the parameters of gene amplification efficiency by a polymerase chain reaction. All of the DNA extracted from the tissues preserved in formalin for 30 years amplified the 54- and 61-base pairs of the DNA fragments successfully. The direct sequencing of the PCR products confirmed the accurate amplification of the target sequence. A further trial to amplify the longer sequence of 111 base pairs succeeded in 20% of the samples tested. From these results, we propose a new utility of archival samples for the analysis of single nucleotide sequence polymorphism of genes, no matter how long the samples have been preserved in formalin.