Safranin O reduces loss of glycosaminoglycans from bovine articular cartilage during histological specimen preparation

Summary The ability of Safranin O, added to fixation and decalcification solutions, to prevent the escape of glycosaminoglycans (GAGs) from small cartilage tissue blocks during histological processing of cartilage has been studied. GAGs in the fixatives and decalcifying solutions used and those remaining in the 1 mm³ cubes of cartilage were assayed biochemically. The quantity of GAGs remaining in the cartilage cubes were determined from Safranin O-stained sectins using videomicroscopy or microspectrophotometry. A quantity (10.6%) of GAGs were lost during a conventional 4% buffered formaldehyde fixation (48 h) and a subsequent decalcification in 10% EDTA (12 days) at 4°C. Rougly one-quarter of the total GAG loss occurred during the 48 h fixation, and three-quarters during the 12c days of decalcification. Inclusion of 4% formaldehyde in the decalcification fluid decreased the loss of GAGs to 6.2%. The presence of 0.5% Safranin O in the fixative reduced this loss to 3.4%. When 0.5% Safranin O was included in the fixative and 4% formaldehyde in the decalcification solution, Safranin O staining of the histological sections increased on average by 13.5%. After fixation in the presence of 0.5% Safranin O, there was no difference in the staining intensities when decalcification was carried out in the presence of either Safranin O or formaldehyde, or both. It took 24 h for Safranin O to penetrate into the deep zone of articular cartilage, warranting a fixation period of at least this long. In conclusion, the addition of Safranin O to the fixative and either Safranin O or formaldehyde in the following decalcification fluid, markedly reduces the loss of GAGs from small articular cartilage explants during histological processing. However, for immunohistochemical studies, Safranin O cannot be included in the processing solutions, because it may interfere.     

Fixation, Counting, and Manipulation of Heterotrophic Nanoflagellates

Quantitative effects of several fixatives on heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were investigated by hemacytometer and epifluorescence counting techniques. Counts of Monas sp. cultures before and after fixation with unbuffered 0.3% glutaraldehyde and 5% formaldehyde showed no loss of cells during fixation, and cell concentrations remained constant for several weeks after fixation. Buffering of fixatives with borax caused severe losses, up to 100% within 2 h. Field samples from Lake Vechten showed no decline of HNAN and total nanoflagellate concentrations for at least 1 week after fixation with 5% formaldehyde and with 1% glutaraldehyde. With 1% glutaraldehyde, the chlorophyll autofluorescence of PNAN was much brighter than with 5% formaldehyde, although it was lost after a few days and thus limited the storage time of samples. However, when primulin-stained slides were prepared soon after fixation and stored at −30°C, the loss of autofluorescence was prevented and PNAN and HNAN concentrations were stable for at least 16 weeks. Effects of filtration and centrifugation on HNAN were also studied. Filtration vacuum could not exceed 3 kPa since 10 kPa already caused losses of 15 to 20%. Similar losses were caused by centrifugation, even at low speed (500 × g).     

Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.     

PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Behaviour of DNA-RNase A complex in the presence of formaldehyde]

A formaldehyde-produced fixation of defects caused by a despiralizing action of a protein was studied in the case of DNA-RNAase A complex. The concentration of the defects fixed was measured by kinetic formaldehyde method (KF-method). It was shown that following processes take place in the complex in the presence of formaldehyde: (a) fixation of defects; (b) unwinding of DNA; (c) inactivation of the protein. The rates of all these processes depend on the concentration of formaldehyde, phi. At formaldehyde concentrations above some critical value phic the protein is inactivated before the defects are fixed. At phi less than phic the protein inactivation proceeds more slowly than the fixation of defects; at sufficiently low formaldehyde concentration no inactivation of protein occurs practically during the fixation time (20 min). The number of new defects formed during the time of fixation is linear with the formaldehyde concentration in the region where no inactivation of the protein occurs. Therefore the initial concentration of defects can be determined through an extrapolation to zero concentration of formaldehyde. On the basis of the data obtained a method is proposed for the evaluation of the number of defects in DNA caused by the despiralizing action of proteins. A model is proposed describing the behaviour of the complexes of DNA with despiralizing proteins in the presence of formaldehyde.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.     

The effects of mercaptoethanol-formaldehyde on tissue fixation and protein retention

Summary The study compared the effects of mercaptoethanol-formaldehyde and formaldehyde alone, on tissue fixation and protein retention in human and mouse tissues. Shrinkage of tissues and the penetration rate of the fixatives were assessed. The cross-linking ability of the fixatives was determined by viscometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and spectrophotometry, using bovine serum albumin and human haemoglobin. Tissues fixed in buffered 0.0025% mercaptoethanol-4% formaldehyde showed good nuclear and cytoplasmic detail, better than those fixed in buffered 4% formaldehyde. There was no significant difference in shrinkage. A mixture of 0.0025% mercaptoethanol-4% formaldehyde penetrated faster into adult liver than 4% formaldehyde. The mean penetration rate (±SE) or coefficient of diffusibility of 0.0025% mercaptoethanol-4% formaldehyde into adult liver was 1.32±0.01 and that of 4% formaldehyde was 1.12±0.06 (p<0.04). Both fixatives diffused more rapidly into mouse liver than into human liver. The cross-linking ability of mercaptoethanol-formaldehyde depends on the concentration of the fixative and the time of fixation. Bovine serum albumin (15%) and 0.1% mercaptoethanol alone formed a gel, whilst electrophoresis showed monomers in the supernatant. Mercaptoethanol (0.1%) also rapidly decreased the absorption at 420 nm, suggesting denaturation. It seems that mercaptoethanol increases the number of thiol groups available to form cross-links with formaldehyde. This study demonstrated that mercaptoethanol-formaldehyde fixed and cross-linked tissues better than formaldehyde at 3 h and 4 h, but not at 1 h and 2 h. The most effective concentration of mercaptoethanol for tissue fixation in 4% formaldehyde is 0.0025%.     

Formaldehyde fixation of cells does not greatly reduce the ability to amplify cellular DNA

Formaldehyde fixation of cells is routinely used to study DNA-protein interactions in vivo. In these studies, DNA is often analyzed using a polymerase chain reaction technique. Although it is known that formaldehyde can damage DNA, no studies have been performed so far to compare the efficiency of DNA amplification between normal and fixed cells. Here we show that formaldehyde fixation results in a 15% to 20% reduction in the ability to amplify cellular DNA. The loss of amplifiability is independent of the length of the amplification region and the degree to which DNA is compacted on packaging into chromatin.     

Protein fixation and antigen retrieval: chemical studies

Abstract

Fixation with formaldehyde is the first process to which most biopsy and necropsy specimens are exposed prior to dehydration and embedding in paraffin wax. Tissue specimens that have been fixed in formaldehyde have architectural characteristics that are familiar to virtually every pathologist and these facilitate routine diagnosis. Nevertheless, formaldehyde fixation has some deleterious effects including reduction in immunoreactivity and degradation of nucleic acids. Development of methods to counteract these deleterious effects requires an understanding of the chemical events that occur during tissue fixation and subsequent tissue processing. This short review illustrates some of the chemical consequences of formaldehyde fixation and ethanol dehydration. It also provides some insight into the molecular events accompanying heat-induced antigen retrieval.     

Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit.Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at −20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%.Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.Key words: fixation, GM1 ganglioside, cholera toxin, anhydrous acetone, 4% formaldehyde.     

MORPHOLOGICAL AND PHARMACOLOGICAL EFFECTS OF RESERPINE, GIVEN ALONE OR AFTER IPRONIAZID, ON THE CATECHOL AMINES OF THE ADRENAL GLANDS OF THE RAT

Adrenomedullary cells, after fixation with OsO₄, are filled with well formed granules which are considered to represent their catechol amine content. The submicroscopic appearance of these cells was studied in reserpine-treated rats during the late phase of catechol amine depletion and during the period of its restoration. At 3 days after the beginning of reserpine treatment, the granules appeared to be emptied of their content and small vesicles containing scattered, dense deposits of, presumably, catechol amines began to be seen. At 9 days after the beginning of treatment, these deposits had already become granules and the cells had attained a completely normal appearance. The submicroscopic structure of the adrenomedullary cells of rats pretreated with iproniazid (before reserpine), in which a complete inhibition of monoamine oxidase activity had thus been obtained, was similar to that seen in non-treated animals. In numerous cases, however, some characteristic features were noted: the sacs which usually contained a dense granule of catechol amines appeared swollen and many fine granules could be seen around them; the latter were dispersed in a way suggesting that they may represent a partial breakdown of the large granules which, under the inhibitory action of iproniazid, do not release the catechol amines contained within them.     

Relationship ofAzotobacter chrooccum siderophores with nitrogen fixation

In iron-limited medium, a siderophore producing soil isolate ofAzotobacter chroococcum showed a high level of hydroxamate with relatively low level of nitrogen fixation. Inclusion of iron in the medium resulted in increased nitrogen fixation with decreased hydroxamate production. Under shake culture conditions, the level of both hydroxamate and catechol type of siderophores decreased after 2 d of incubation in iron-deficient medium. However, under iron-sufficient conditions, both siderophore production and nitrogen fixation increased with time although the level of siderophore was quite low. A number of soil isolates and mutants ofA. chrococcum were tested for nitrogen fixation, hydroxamate and catechol type of siderophore production. Wide variation was observed in the siderophore level and nitrogen fixation in the cultures tested. Nitrogen fixation was higher in the iron-sufficient medium than in iron-limited one while hydroxamate yield was higher in iron-limited medium than in the iron-sufficient one in all the cultures. Inclusion of ammonium acetate in the medium induced catechol synthesis in more than 60% of the cultures.     

Effects of Aldehyde Fixation on Adenosine Triphosphatase and Peroxidase Activities in Maize Root Tips

The effects of aldehyde fixation of excised roots and subcellularfractions of Zea mays have been studied in relation to the preservationof ATP-ase and peroxidase activities. Total peroxidase was littleaffected by either formaldehyde or glutaraldehyde whereas ATP-aseshowed considerable loss of activity, particularly with glutaraldehyde.The activity remaining after fixation was dependent on boththe concentration of fixative and the pH of fixation. Subcellularfractions differed in their response to fixation with the cellwall fraction generally showing higher retention of activitythan the mitochondrial or microsomal fractions.     

Laccase-assisted Dyeing of Cotton

Cotton cellulose was dyed “in situ” with a polymeric dye generated by oxidative coupling of colourless 2,5-diaminobenzenesulfonic acid and 1-hydroxyphenol (catechol) with laccase. Up to 70% dye fixation was obtained increasing the concentration of catechol less soluble upon oxidation from 1 to 10 mmol, while 1 mmol of diamine was used. Dye fixation was not achieved using equal molar concentrations of the reagents.     

甲醛固定对GFP发光特性的影响

绿色荧光蛋白(GFP)能够作为报告分子对活体细胞中特定基因的时空表达进行实时追踪,因此广泛应用于生物学研究领域。在用GFP对细胞活动进行追踪的实验中,常有无法在取样后及时对样品进行荧光观察的情况,此时需要先将样品进行固定以便对其进行观测。然而,不恰当的细胞固定方法会导致胞内GFP荧光信号强度减弱、位置改变等后果。甲醛是最常用的细胞固定剂,也常被用于固定表达GFP蛋白的细胞样品。但对甲醛固定GFP样品的报道多是针对于真核细胞,且固定效果也存在较大差异。文章系统地探索了甲醛浓度、固定时间、固定缓冲液种类对两种细菌(E.coli及鱼腥蓝细菌Anabaena PCC7120)胞内GFP信号的影响。结果显示,较低浓度(≤1%)的甲醛处理2h后,细胞的荧光强度在1d后仍可保持80%以上,胞内荧光点无弥散现象发生。具有相近pH的几种常见缓冲液对荧光强度的影响无显著差异。随着甲醛浓度的增加、固定时间的延长、溶液pH的增加(中性至偏碱性),细胞中的荧光强度会逐渐降低。     

Effect of Fixation on Demonstration of Phosphatases of Eimeria tenella Grown in Chick Kidney Cell Cultures

SYNOPSIS. The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase > thiamine pyrophosphatase > 5'-nucleotidase > adenosine triphosphatase > acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase. TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase.
Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations.

BACKGROUND: Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho-Thr 202/Tyr 204-p44/42 extracellular-regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho-epitopes in leukocyte populations in whole blood. METHODS: Normal blood samples were activated with PMA followed by formaldehyde fixation and subsequent treatments with detergents and protein denaturants. The effects of each treatment were monitored by light scatter, selected CD expression intensity, and phosphorylated ERK (pERK) expression. RESULTS: Red cells could be lysed using 0.1% Triton X-100 after brief fixation of whole blood with 2% or 4% formaldehyde. Light scatter improved as a function of formaldehyde concentration and inversely with MeOH concentration. CD3 signal intensity increased when MeOH concentration was reduced. The ratio of pERK immunofluorescence in PMA-stimulated versus nonstimulated (control) samples was highest with high MeOH (90%) and lowest without MeOH treatment. This pattern is consistent with epitope unmasking by alcohol. The pERK epitope could also be unmasked by treatment with high salt, urea, acid, or heat, but none of these produced the level of unmasking of MeOH and each of these was associated with degradation of light scatter and CD3 staining intensity. The final procedure employed 4% formaldehyde, 0.1% Triton X-100, followed by 50% methanol denaturation. Samples prepared in this way demonstrated good preservation of light scatter and surface immunophenotypic patterns, similar to those obtained using a commercial whole blood/red blood cell lysing system (Q-Prep) and an acceptable PMA-stimulated pERK signal (essentially 100% of CD3+ cells that are pERK positive). CONCLUSIONS: Brief fixation of whole blood in 4% formaldehyde followed by treatment with Triton X-100 results in erythrocyte lysis and leukocyte light scatter and immunophenotypic features equivalent to those of other commercial lysis reagents. Intracellular pERK staining is significantly improved by treatment with methanol, but levels of MeOH above 50% degrade light scatter and CD3 expression. This protocol (formaldehyde/Triton X-100/MeOH) circumvents potential artifactual changes in phospho-epitopes due to removal of erythrocytes or erythrocyte lysis followed by fixation, and results in a pERK signal that resolves positive from negative cell populations.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Formaldehyde prepared from paraformaldehyde is stable

For critical histological investigations, tissue fixation is sometimes carried out in formaldehyde freshly prepared from paraformaldehyde by heating. The purity of formaldehyde produced in this way is superior to that of commercial stock solutions. We studied the stability of freshly prepared formaldehyde solutions by determination of pH and titration of acid, which reflect the formation of formic acid. It was found that very small amounts of acid are produced during the heating of paraformaldehyde. Prolonged heating or storage of freshly prepared formaldehyde for up to 8 days did not significantly increase the amount of acid. It was also found that heating of the paraformaldehyde is not necessary, since depolymerization may take place at room temperature.

We conclude that formaldehyde prepared from paraformaldehyde remains stable for considerable periods of time, and it is therefore unnecessary to prepare it immediately prior to fixation. Also, in many cases, buffering of the fixative may be omitted, since only minor changes in the pH occur during fixation.     

Implications of mechanical deformation and formaldehyde preservation for the identification of stage-specific characteristics of Baltic cod eggs

The identification of developmental stages in fish eggs from plankton samples is often complicated by deformation of the embryos due to mechanical stress during the sampling procedure and by dehydration during formaldehyde fixation. The effects of formaldehyde fixation and mechanical stress on Baltic cod eggs ( Gadus morhua callarias L.) were examined separately by visually comparing the morphological features of treated vs. live eggs of identical ontogenetic age. Microphotographs were made concurrently for documentation. In stage IA eggs, mechanical treatment resulted in scattered blastodiscs surrounded by single cells, while in further advanced stages the yolk membrane collapsed entirely, the yolk coagulated and the embryo extending over the yolk shrank. Formaldehyde fixation caused the yolk and the blastodisc or embryo to darken, and in some cases crystalline enclosures occurred. Eggs mechanically deformed during handling were clearly distinguishable from those that died prior to catching; however, staging was generally less accurate for formaldehyde-preserved eggs when compared with living specimens.