Museum fluid collections preserve important biological specimens for study. Tissues are often fixed in 10% buffered formalin to halt metabolic activities and transferred to a solution of ethanol for long‐term storage. This process, however, forces water from the tissues and has been shown to alter the morphology of preserved specimens in ways that may influence the biological interpretation of results. The degree to which fluid preservation alters morphology is linked to multiple biological factors, such as tissue size and composition, and should therefore be examined prior to functional analysis. This study is undertaken as part of a more inclusive examination of mammalian volar morphology. A sample of five adult male and five adult female rats (
Gelatinous zooplankton can be difficult to preserve morphologically due to unique physical properties of their cellular and acellular components. The relatively large volume of mesoglea leads to distortion of the delicate morphology and poor sample integrity in specimens prepared with standard aldehyde or alcohol fixation techniques. Similar challenges have made it difficult to extend standard laboratory methods such as in situ hybridization to larger juvenile ctenophores, hampering studies of late development.
We have found that a household water repellant glass treatment product commonly used in laboratories, Rain-X®, alone or in combination with standard aldehyde fixatives, greatly improves morphological preservation of such delicate samples. We present detailed methods for preservation of ctenophores of diverse sizes compatible with long-term storage or detection and localization of target molecules such as with immunohistochemistry and in situ hybridization and show that this fixation might be broadly useful for preservation of other delicate marine specimens.
This new method will enable superior preservation of morphology in gelatinous specimens for a variety of downstream goals. Extending this method may improve the morphological fidelity and durability of museum and laboratory specimens for other delicate sample types.
- NSF-PAR ID:
- 10249322
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Frontiers in Zoology
- Volume:
- 18
- Issue:
- 1
- ISSN:
- 1742-9994
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract Rattus norvegicus ) was utilized to evaluate longitudinal changes in the dimensions of the volar pads across fixation in 10% buffered formalin and preservation in 70% ethanol for 1 year. No significant changes to the measured dimensions of the rat volar pads were present across stages of fixation and preservation, and no significant interactions of specimen size or sex were noted. These findings indicate that small mammalian volar pads that have been fixed in 10% buffered formalin and stored in 70% ethanol are appropriate for morphological study using the measurements described here without corrective algorithms. This finding is rare among preservation studies but highlights the variability of tissue behavior during chemical preservation and the necessity of preliminary investigations of preservation artifacts. Concurrence here between the preserved and unpreserved samples is likely related to the anhydrous nature of the volar pads and the supporting skeletal structure, and their confined position between major joints of the hands and feet. -
more » « less
Abstract Studies of morphology and developmental patterning in adult stages of many invertebrates are hindered by opaque structures, such as shells, skeletal elements, and pigment granules that block or refract light and necessitate sectioning for observation of internal features. An inherent challenge in studies relying on surgical approaches is that cutting tissue is semi-destructive, and delicate structures, such as axonal processes within neural networks, are computationally challenging to reconstruct once disrupted. To address this problem, we developed See-Star, a hydrogel-based tissue clearing protocol to render the bodies of opaque and calcified invertebrates optically transparent while preserving their anatomy in an unperturbed state, facilitating molecular labeling and observation of intact organ systems. The resulting protocol can clear large (> 1 cm3) specimens to enable deep-tissue imaging, and is compatible with molecular techniques, such as immunohistochemistry and in situ hybridization to visualize protein and mRNA localization. To test the utility of this method, we performed a whole-mount imaging study of intact nervous systems in juvenile echinoderms and molluscs and demonstrate that See-Star allows for comparative studies to be extended far into development, facilitating insights into the anatomy of juveniles and adults that are usually not amenable to whole-mount imaging.
-
Behavioral measurements of fragile aquatic organisms require specialized in situ techniques.We developed an in situ brightfield camera set-up for use during SCUBA diving in aquatic ecosystems.The system uses brightfield illumination with collimated light and an underwater camera to highlight morphological details, body motion and interactions between organisms with high spatial (4K: 3840x2160 pixels) and temporal resolution (up to 120 fps).This technique is particularly useful for gelatinous organisms because of their large (centimeters in length), transparent bodies.Further, the measurements are not subject to experimental artifacts produced in laboratory studies.This method is useful for anyone seeking detailed brightfield images of organisms or nonliving material (e.g. marine snow) in the natural environment.more » « less
-
more » « less
Abstract Muscle excursion and force potential can be estimated from architectural variables, including mass, volume, fascicle length, and density. These have been collected from fresh specimens, preserved specimens, and sometimes mixed samples of both. However, preservation alters the gross morphology of muscles. This study aims to quantify the effects of long‐term storage on myological properties across a sample of fresh and ethanol preserved
Mus musculus specimens ranging in storage time from 16 to 130 years. Masses, volumes, and densities of biceps femoris, quadriceps femoris, and triceps surae were measured, and histological cross‐sections of some specimens were used to evaluate the microscale effects of long‐term fluid preservation. For the remainder of the sample, chemically dissected fascicle lengths were measured to evaluate the fixation effects on the linear dimensions of muscle architecture. Relative muscle mass, volume, fascicle length, average fiber area, and density, and percent fiber area were regressed against years stored in ethanol. Muscle size dropped steeply between fresh and stored samples, ultimately decreasing by 62 and 60%, respectively. These losses correlate with histologically measured shrinking of average muscle fiber area. Density of stored specimens plateaued 5% below that of fresh ones. Although muscles lost mass and volume during ethanol storage, fascicle lengths did not shorten significantly (presumably because they were preserved attached on either end to bone). This study demonstrates that muscle mass, volume, and density of specimens stored long‐term in ethanol should be corrected by factors of 2.64, 2.49, and 1.054 respectively for comparability to fresh specimens. -
more » « less
Abstract Objectives The cervical spine is the junction between the head and trunk, and it therefore facilitates head mobility and stability. The goal of this study is to test several predictions regarding cervical morphology and intervertebral ranges of motion.
Materials and Methods Intervertebral ranges of motion for 12 primate species were collected via radiographs or taken from the literature. Morphometric data describing functionally relevant aspects of cervical vertebral morphology were obtained from museum specimens representing these species. We tested for correlations between intervertebral movement and vertebral form using phylogenetic generalized least‐squares regression.
Results Results demonstrate limited support for the hypothesis that range of motion (ROM) is influenced by cervical vertebral morphology. Few morphological variables correlate with ROM and no relationship is consistently significant across cervical joints.
Discussion These results indicate that the relationship between vertebral morphology and joint ranges of motion is, at most, weak, providing little support the use of bony morphology to reconstruct axial mobility in fossil specimens. Future work should investigate the role of soft tissues in vertebral joint stability.
