Phenoxyethanol as a nontoxic preservative in the dissection laboratory

In an effort to rid the dissection room of irritating and potentially health-threatening toxic chemicals, we have modified the phenoxyethanol technique for long-term preservation of embalmed cadavers. The new methods employ faster, less toxic embalming and reduced or eliminated phenoxyethanol immersions. Our results are comparable with or improved over those previously described and demonstrate that phenoxyethanol is an excellent, easily manageable alternative preservative to standard formaldehyde/phenol-based embalming fluids.     

Application and Evaluation of the Lead-Adenosine Triphosphatase Method in Skeletal Tissue

Application and evaluation of the lead-ATPase histochemical method in skeletal tissue has demonstrated an intracellular localization of enzyme activity. The skeletal tissue was demineralized for 72 hr in cold 10% aqueous EDTA adjusted to pH 7.2. Frozen sections were cut and placed on cold albumenized slides, oriented, thawed, dried in a cool air stream, and fixed for 10 min in cold (-2 to -3 C) 10% formalin buffered with Na-acetate and adjusted to pH 7.2. The sections were washed, treated with 10% EDTA for 20 min at room temperature, rewashed, and incubated for an optimal period of 30 min at 37 C. in the lead-ATP medium of Wachstein and Meisel. Following incubation the sections were washed, treated for 1 min with 1% (NH₄)₂S, rewashed, immersed for 30 min in 10% buffered formalin, dehydrated, cleared, and mounted. Evaluation of the substrate specificity suggests that other phosphatases associated with skeletal tissue do not complicate the ATPase reaction.     

Tissue Quality Assessment Using a Novel Direct Elasticity Assessment Device (The E-Finger): A Cadaveric Study of Prostatectomy Dissection

Introduction

Minimally invasive radical prostatectomy (RP) (robotic and laparoscopic), have brought improvements in the outcomes of RP due to improved views and increased degrees of freedom of surgical devices. Robotic and laparoscopic surgeries do not incorporate haptic feedback, which may result in complications secondary to inadequate tissue dissection (causing positive surgical margins, rhabdosphincter damage, etc). We developed a micro-engineered device (6 mm² sized) [E-finger]) capable of quantitative elasticity assessment, with amplitude ratio, mean ratio and phase lag representing this. The aim was to assess the utility of the device in differentiating peri-prostatic tissue types in order to guide prostate dissection.

Material and Methods

Two embalmed and 2 fresh frozen cadavers were used in the study. Baseline elasticity values were assessed in bladder, prostate and rhabdosphincter of pre-dissected embalmed cadavers using the micro-engineered device. A measurement grid was created to span from the bladder, across the prostate and onto the rhabdosphincter of fresh frozen cadavers to enable a systematic quantitative elasticity assessment of the entire area by 2 independent assessors. Tissue was sectioned along each row of elasticity measurement points, and stained with haematoxylin and eosin (H&E). Image analysis was performed with Image Pro Premier to determine the histology at each measurement point.

Results

Statistically significant differences in elasticity were identified between bladder, prostate and sphincter in both embalmed and fresh frozen cadavers (p = <0.001). Intra-class correlation (ICC) reliability tests showed good reliability (average ICC = 0.851). Sensitivity and specificity for tissue identification was 77% and 70% respectively to a resolution of 6 mm².

Conclusions

This cadaveric study has evaluated the ability of our elasticity assessment device to differentiate bladder, prostate and rhabdosphincter to a resolution of 6 mm². The results provide useful data for which to continue to examine the use of elasticity assessment devices for tissue quality assessment with the aim of giving haptic feedback to surgeons performing complex surgery.     

Senile changes in human lymph nodes

BACKGROUND: The degenerative process of lymph nodes is poorly documented. METHODS: 161 lymph nodes of seven fresh and one embalmed human cadavers in the head and neck were studied. We used 6% hydrogen peroxide, lead oxide injectant, and radiographs to demonstrate lymphatic vessels, and found both solidified and transparent lymph nodes. They were removed, fixed in 10% formalin and sent for histopathology cross section. RESULTS: Thirty-eight solidified and 123 transparent lymph nodes were found. A series of histopathological sections show the degenerative process is variable and continuous. Senile involution affects all elements of the lymph node including the cortex, the medulla, and the architecture. CONCLUSION: This study provides actual anatomical and histopathological images of lymph nodes in different degenerative stages in the head and neck region. It may help explain some clinical conditions in the elderly, especially their diminished immunological response to infection and cancer metastasis.     

Fixation-dependent immunolocalization shift and immunoreactivity of intracellular growth factors in cartilage

The effects of fixation on immunolocalization and immunoreactivity in cartilage tissues were studied using monoclonal antibodies against peptides that can effectively stimulate chondrocytes in vitro and have been shown to play a role in musculoskeletal tissue regeneration: transforming growth factor 1, transforming growth factor 3, insulin-like growth factor I, insulin-like growth factor II and fibroblast growth factor 2. Paraffin sections fixed in buffered formalin, buffered paraformaldehyde, Carnoy and methacarn, as well as cryosections, were tested. A strong immunoreaction was observed in tissue fixed in formaldehyde-based fixatives, with a resemblance to that in cryopreserved tissues. Immunoreactivity was reduced in alcohol-fixed tissues. Furthermore, a striking intracellular immunolocalization shift from cytoplasm to nucleus was observed using alcohol-based fixatives as compared to cryopreserved or formaldehyde-based fixatives. We concluded that, for the detection and localization of growth factors in cartilage tissues, fixation in buffered formalin or paraformaldehyde is optimal.     

Formic Acid Corrosion for the Demonstration of Pulmonary Elastic Tissue

By a revised technique, human pulmonary elastic tissue can be isolated in a form suitable for examination under the stereoscopic microscope. Fresh human lungs from autopsy are fixed by intrabronchial infusion with 10% formalin for 24 hr. Slabs 1.5 cm thick are cut and the formalin removed in running water. One such slab is embedded under intermittent vacuum in an aqueous mixture containing 15% gelatin, 10% glycerol, and 1% phenol; then allowed to gel. Frozen sections 2 mm thick are cut on a large-section MSE sledge microtome. Squares 3 × 3 cm from such a section are corroded for 4-5 days in 88% formic acid at 45 C, washed once with distilled water, and mounted in glychrogel containing 6% gelatin. The elastic tissue network of the lung will have been freed from surrounding elements. The preparation should be stored in a refrigerator. Blocks for thin sections and large thick un-corroded sections can be prepared from the same lung as part of an over-all procedure.     

Periodic Acid-Schiff-Toluidine Blue-Aurantia; A Stain for the Gland Cells of the Stomach

By a revised technique, human pulmonary elastic tissue can be isolated in a form suitable for examination under the stereoscopic microscope. Fresh human lungs from autopsy are fixed by intrabronchial infusion with 10% formalin for 24 hr. Slabs 1.5 cm thick are cut and the formalin removed in running water. One such slab is embedded under intermittent vacuum in an aqueous mixture containing 15% gelatin, 10% glycerol, and 1% phenol; then allowed to gel. Frozen sections 2 mm thick are cut on a large-section MSE sledge microtome. Squares 3 × 3 cm from such a section are corroded for 4-5 days in 88% formic acid at 45 C, washed once with distilled water, and mounted in glychrogel containing 6% gelatin. The elastic tissue network of the lung will have been freed from surrounding elements. The preparation should be stored in a refrigerator. Blocks for thin sections and large thick un-corroded sections can be prepared from the same lung as part of an over-all procedure.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.     

Variation in soft-tissue thicknesses on the human face and their relation to craniometric dimensions

The average thickness of soft tissues on parts of the face is known, but its variation has not been related to cranial morphology. To investigate this relationship, measurements of facial soft-tissue depths and craniometric dimensions were taken on adult, white Australian cadavers (17 male and 23 female). Significant correlations between many soft-tissue depths and craniometric dimensions were found, suggesting a relationship between the amount of soft tissue present on the face and the size of the underlying bony skeleton. Soft-tissue depths were highly positively correlated with each other; craniometric dimensions were correlated but to a lesser extent. Males had thicker soft tissues and larger craniometric dimensions than females; considerable overlap of ranges was also noted. Multiple regression analysis was used to produce equations predicting the soft-tissue depth at specified areas of the face from craniometric dimensions. A subsample of nine cadavers was examined for the effects of tissue embalming. Embalming caused significant initial increases in facial soft-tissue depths. Cadavers embalmed for less than 6 months had soft-tissue depths significantly greater than for fully embalmed cadavers. The evidence that facial soft-tissue thicknesses vary with craniofacial dimensions has implications for forensic identification, facial aesthetic surgery, and approximation of the facial features of extinct individuals.     

The Value of Alcohol for Fixation of Skin

Stained sections of skin fixed in 70% alcohol were compared with others from pieces fixed in 4% formaldehyde-saline. The sections of alcohol-fixed material were much more susceptible to the action of deoxyribonuclease and lipase than those from formalin-fixed, as demonstrated by a standardized hematoxylin staining method and by fluorescence microscopy. After formalin, cytoplasmic basophilia was increased, presumably because formalin fixation caused ribonucleic acid to diffuse from nuclei to cytoplasm. Both types of fixation damaged collagen, as seen in fluorescence induced by 5-anvmo-2-chloro-7-methoxyacridine, but alcohol caused less distortion than formalin. Probably fluorochroming of fresh tissue is the only satisfactory method for studying collagen in pathological conditions.     

Density of fresh and embalmed human compact and cancellous bone

This investigation was undertaken to determine the densities of fresh human compact and cancellous bone, with its marrow constituents intact, and compact and cancellous bone taken from embalmed cadavers. The density of 107 bone specimens was determined on the basis of the weight and volume of each specimen. Mean density results were: (1) fresh compact bone, 1.85; (2) fresh cancellous bone, 1.08; (3) embalmed compact bone, 1.85; and (4) embalmed cancellous bone, 1.09. Embalming did not appear to have altered the density of either compact or cancellous bone. It was observed also that the densities of several bone specimens taken from the same area of one bone varied greatly.     

Immunofluorescent Staining of Epon Embedded Kidney Sections Through Simultaneous use of Two Different Fluorochrome-Conjugated Antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.     

Immunofluorescent staining of Epon embedded kidney sections through simultaneous use of two different fluorochrome-conjugated antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.     

Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.     

Restoration of the softness and flexibility of cadavers preserved in formalin

Submerging parts of cadavers in a 5% aqueous solution of glacial acetic acid eliminates the undesirable properties of formalin and formalin-containing fixatives such as the irritating and unpleasant smell of formalin and phenol. It also considerably softens the tissues, renders the articulations flexible and makes dissection much easier. No significant bone softening occurs if the process is stopped at a suitable time. Parts of the bodies softened in this way can be kept for years in 70% alcohol without any signs of a loss of flexibility or decomposition.     

Impregnation of Oligodendroglia in Nervous Tissue Kept in Formalin for Many Years

A method for impregnating oligodendroglia in nervous tissue (monkey) fixed and preserved in formalin for many years is described. This tissue is reconditioned by placing 12 to 30μ frozen sections of it in concentrated ammonia (sp. gr. 0.90) and by washing them slowly for 24 hours with a 1 mm. stream of water. The fluid is then poured off the sections; the jar is refilled with concentrated ammonia; and washing is repeated for another 24 hours. The sections are then plunged into concentrated ammonia for 7 minutes.

After treatment in ammonia, the sections are incubated for one hour at 38^oC. in Globus' 5% hydrobromic acid solution. They are washed again, in distilled water, and then impregnated in a “medium” strength ammoniacal silver carbonate solution (5 ml. of 10% AgNO₃ added to 15 ml. of 5% Na₂CO₃. The precipitate is dissolved in concentrated ammonia and diluted to SO ml. with distilled water). Impregnation is followed by reduction in 1% formalin without agitation; fixation in 5% Na₂S₂O₃; dehydration, and mounting in clarite.

Typical oligodendroglia (Fig. 1) were made visible by use of the method outlined in this paper.     

Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium

A strictly anaerobic gram-positive, rod-shaped bacterium, strain LuPhet1, was isolated from sewage sludge with phenoxyethanol as sole carbon and energy source, and was assigned to the genus Acetobacterium. The new isolate fermented the alkylaryl ether compound phenoxyethanol stoichiometrically to phenol and acetate, whereas phenoxyacetic acid was not degraded. In cell-free extracts of strain LuPhet1, cleavage of the ether linkage was shown, and acetaldehyde was detected as reaction product. Coenzyme A-dependent acetaldehyde: acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results indicate that the ether linkage of phenoxyethanol is cleaved by a shift of the hydroxyl group to the subterminal carbon atom, analogous to a corrinoid-dependent diol dehydratase reaction, to form an unstable hemiacetal that releases phenol and acetaldehyde. Obviously, phenoxyethanol is degraded by the same strategy as in anaerobic degradation of the alkyl ether polyethylene glycol.     

A Modified One-Step Trichrome Stain for Demonstration of Fine Connective Tissue Fibers

Gomori's one-step trichrome procedure was modified to improve coloration of fine connective tissue fibers. Paraffin sections from tissues fixed in alcohol, acetone, Zenkerformol, 10% formalin, Kaiserling's or Carnoy's fluid were mordanted 1 hr at 56 C in Bouin's solution, stained 1 min in a trichrome solution (chromotrope 2R-phosphomolybdic acidaniline blue WS) adjusted to pH 1.3 with HCl, rinsed in 1% aqueous acetic acid, dehydrated and covered. Collagen, reticulum fibers, basement membranes, ring fibers around splenic sinuses, intercalated discs in cardiac muscle and cartilage were colored blue. Nuclei, cytoplasm, fibrin, muscle fibers and elastic fibers were stained red. Pretreatment of sections with Bouin's solution enhanced the affinity of tissues for chromotrope 2R and was found essential for satisfactory coloration of material fixed in alcohol, acetone, formalin or Carnoy's fluid. Because this method does not require differentiation, it gave uniform results even in the hands of inexperienced laboratory trainees. No fading was observed in sections stored for more than 8 yr.     

Enzyme histochemical investigation of glycol methacrylate embedded chick embryonic tissue

Synopsis The advantages of the water-soluble glycol methacrylate (GMA) embedding procedure make it highly applicable for use with fragile early embryonic material. Not only can one obtain tissue sections containing excellent histological detail, but numerous enzymes are retained for subsequent histochemical localization. For the purpose of establishing a methodology whereby concomitant histology and histochemistry could be obtainable, various fixatives and fixation times have been evaluated on GMA embedded chick embryonic mesonephros and gonad. It was found that fixing the tissues for 1 h in a solution of 95% ethanol, 5% acetic acid and 10% neutralbuffered formalin resulted in the retention of not only excellent histology but also alkaline and acid phosphatase. Thus, with this procedure, more specific investigations of early embryonic tissue can be performed.     

The Histological Examination of Mummified Material

Tissue from Egyptian mummy material is extremely brittle; hence it was handled in perforated glass tubes during processing. The first (softening) fluid consisted of 96% ethyl alcohol, 30 vol; 1% aqueous formalin, 50 vol; 5% aqueous Na₂CO₃, 20 vol. It was used in a fluid to tissue volume ratio of 100:1 and allowed to act overnight. A special dehydrating sequence: 80% alcohol, 3-6 hr; 8% phenol in 96% alcohol, and absolute alcohol followed by 3 changes of amyl acetate, 6-18 hr each; 3 changes of 1 % celloidin in methyl benzoate, 24 hr each; then through benzene and embedding in paraffin completed the special technic. This allowed regular sectioning and staining to be done successfully.