Zenker's Fixative Research Articles

Improved Methods for Staining Fibrin

Two methods used to demonstrate fibrin are the Fraser-Lendrum and Mallory's phosphotungstic acid-hematoxylin. One problem with the Fraser-Lendrum is that it requires the use of saturated alcoholic picric acid. To prepare this solution, a considerable amount of picric acid is required, which is an explosive. Federal and state regulations prohibit the storage of this chemical in the laboratory unless it is stored under water. We found that saturated aqueous picric acid could be substituted for the saturated alcoholic solution. Other changes were made to simplify the method. Mallory's method for fibrin requires treatment of tissue sections with potassium permanganate and oxalic prior to staining them with phosphotungstic acid-hematoxylin. We found that better staining results are obtained if the tissues are also treated with Zenker's fixative. However, Zenker's fixative contains mercuric chloride, a hazardous chemical that many laboratories have discontinued using. We found that zinc acetate can be substituted for mercuric chloride in preparing Zenker's fixative with excellent staining results when used in the Fraser-Lendrum and Mallory methods. (The J Histotechnol 26:127, 2003)Submitted April 10, 2003; Accepted April 22, 2003

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.

Identification of Spinal Cord Lesions through the Use of Zenker's Fixation and Radiography

Identification of Spinal Cord Lesions through the Use of Zenker's Fixation and Radiography

Observations on Fixation and Staining of Bone Marrow Biopsies

AbstractTen bone marrow (BM) biopsies were taken from the iliac crest of six autopsy cases. The tissues were fured in Zenker's fixative for intervals between one and 18 hours with and without decalcification. Ultrasonic bombardment was used for one and two hours. The same procedures were also used following 18 hours fixation in 10% neutral buffered formalin. Coded slides were double blind evaluated for section quality and cytologic detail after staining with H & E, three Giemsa variants, and Gomori's iron stain.Paraffin processed BM biopsies were optimally prepared by fixation in Zenker's fluid for 4-18 hours without decalcification. Prior formalin fixation did not influence the consistent brilliance of Giemsa staining with the preferred Sheehan Wright Giemsa stain. Overnight Zenker fixation with or without prior formalin exposure and the absence of decalcification is recommended for the routine preparation of BM biopsies for paraffin processing. (The J of Histotechnol 11:145, 1988.)

Perceiving mitosis in eukaryotic cells.

A sensitive method has been developed for visualizing eukaryotic cells in mitosis (M) phase. It employs Zenker's fixative, which makes the plasma membrane but not the nuclear envelope permeable to immunoglobulins. Zenker's-fixed cells are exposed to an antibody which recognizes a major constituent of chromatin. In this case the antibody is a monoclonal (MC 21) which recognizes histone H2b. Because cells in M phase do not have an intact nuclear envelope, the antibody has access to and interacts with their chromatin. The presence of a nuclear envelope in Zenker's-fixed interphase cells precludes access of the antibody to the nuclear chromatin. Consequently, this indirect immunofluorescence procedure selectively labels M-phase cells. At high enough magnification some details of the chromatin figures are revealed. MC 21 recognizes the chromatin of cells of many different species. With appropriate fixation it can be used effectively on cells in culture. With some procedural modifications it can also be used with more complex tissue systems. Detailed mitotic patterns for chick embryos up to Day 3 of development have been obtained by this method.

The Qualitative Use of Cason's One-step Triple Stain on <i>Hydra</i>

Cason's one-step triple stain for connective tissue yields a wide range of coloration when adapted to Hydra. Previous staining methods were either time-consuming or did not adequately contrast the mesoglea, cell types, or cell inclusions. Following Bouin's or Zenker's fixative, one observes blue mesoglea apposed to red muscle fibers. Immature cnidoblasts are a light blue color; mature ones have a dark blue capsule with red coils or barb. Epithelio-muscular droplets are blue, whereas glandulomuscular droplets as well as digestive cell inclusions are orange. Nerve cells are grey. Nuclei are pink with red nucleoli, although interstitial cells and gland cells have more purplish nuclei. Mucous cells are blue after Bouin's and light lavender after Zenker's; gland cell droplets are magneta and stain well only after Zenker's.For photography on black and white film, the use of filters is demonstrated to enhance the density of desired cell types.

Lesions of Hemorrhagic Enteritis

A 1:5 suspension of frozen (-40?C) gut contents of an experimental turkey with HE was prepared in sterile tryptose broth (Difco) at 4?C. The suspension was centrifuged at 8,000 X G for 15 min at 4?C. The supernatant was filtered through a sterile 0.22-/A Millipore filter (Millipore Filter Corp., Bedford, Mass.) under 5 psi of vacuum. No bacteria could be grown when the filtrate was cultured aerobically or anaerobically. The turkeys used in these experiments were from the specific pathogen-free Beltsville White turkey flock maintained in isolation at the Veterinary Science Department. All experimental birds were maintained in isolation rooms. Three turkeys were destroyed before inoculation as uninoculated controls, while additional groups of 3 birds each were killed 2, 3, 4, 5, 6, 7, 8, and 10 days after inoculation. Within 3 minutes of death, portions of the anterior small intestines of the birds were placed in Zenker's fixative. This is essential in order to prevent artifacts in the histology of the intestine. After fixation for 18 hr the tissues were washed, processed by standard techniques, and stained with hemotoxylin and eosin. Additional specimens from similar experimental groups were also examined, particularly during the 5th and 6th days after inoculation.

The Patterns of Development and Distribution of Collagen, Reticulin, and Elastin in the Placentae of the Golden Hamster (Mesocricetus auratus Waterhouse)

The patterns of development and distribution of collagen, reticulin, and elastin in the placentae and adnexa of the golden hamster, representing day 6 to term, were studied along with non-pregnant and post-partum uteri for comparisons. Collagen becomes widely distributed in the membranes of the gestation sac by mid-term, but diminishes in the endometrium thereafter. The distribution of reticulin follows that of collagen in large measure, and diminishes in the labyrinth in later gestation. Elastin is limited to the myometrium, to walls of umbilical vessels, and to the internal elastic membranes of arteries. Several papers have appeared in recent years concerning a variety of aspects in the development of the hamster placentae and adnexa. These include the studies of Graves (1945), Venable (1946), Ward (1948), Adams & Hillemann (1950), Orsini (1954, 1956, 1957, 1962), and Enders (1965). 1 Supported by Grant GM-10611 from the U. S. Public Health Service and by a supplemental grant-in-aid from the Graduate Council of Oregon State University. TRANS. AMER. MICROSC. Soc. 86(3): 294-303. 1967. , R. M. 1946. Cyt logie des globules c vitaires actifs et dormants chez le t rdigrade obiotus hufelandi Schultze. Arch. Anat. Mi rosc., 8: 136-150. This content downloaded from 157.55.39.27 on Mon, 05 Sep 2016 04:47:20 UTC All use subject to http://about.jstor.org/terms RITSCHARD AND HILLEMANN-HAMSTER PLACENTAL FIBROARCHITECTURE 295 In 1961, Hillemann & Gaynor included some details on the pattern of distribution of collagen, reticulin, and elastin, in their paper on the term placenta of nutria. Two abstracts by Hillemann & Ritschard (1963, 1964) served as preliminary accounts of these three fibers in chinchilla, nutria, and baboon term placentae. In 1967 these two authors published an extended account on the comparative fibroarchitecture of the term placentae of these three animals. The connective tissue fibers of the hamster placentae and adnexa have not been investigated, save for a brief reference in the Hillemann & Ritschard abstract of 1963. The paragraphs below include not only an account of the three above-mentioned connective tissue fibers in the term hamster placentae and adnexa, but also the content of these fibers in the developing hamster placentae and adnexa, including time of appearance, distribution, arrangement, and quantitative changes, beginning with the sixth day of gestation and extending to term on day 16. Additionally, both the non-pregnant and post-partum (day one) uteri were included for comparisons. The synopsis (Table I) in the paper by Hillemann & Ritschard (1967) containing information historically pertinent to the subject at hand, may be economically brought up to date by including references not only to this presentation, but also to Hillemann & Ritschard (1967) and Tenzer (1963). No previous work so far as the authors can ascertain, has given attention to the day by day developing patterns of these fibers in any placenta or associated membranes. MATERIALS AND METIODS The gestation ages of placentae taken from 15 hamsters were determined on the basis of the time which elapsed from observed coitus to time of autopsy. One non-pregnant, and one post-partum (day 1) uterus was included for comparison. A 16-day pregnancy period was used; in Table I, day 17 post-partum is the first day following the day of parturition. The smaller loculi were fixed whole, but the larger ones were injected through the anti-mesometrial aspect of the uterine wall and into the yolk sac. As Bouin's fluid, or Zenker's fixative was injected, a nearly equivalent amount of amniotic fluid was allowed to escape through an adjacent puncture. Following dehydration in a graded series of alcohol (for Bouin-fixed material) or dioxane (for Zenker-fixed material), clearing in xylene, embedding in paraffin (56-58 C), the tissues were sectioned at 7-12 ft. Infiltration of the larger loculi was facilitated with a vacuum pump. Harris' hematoxylin, and eosin were used as stains for general histology, Foot's method (Lillie, 1954) for reticulin (argyrophilic fibers), MacCallum's modification of Verhoeff's stain (Krajian & Gradwohl, 1952) or the orcinol new fuchsin method (Fullmer & Lillie, 1956) for elastin, and a modification of Masson's trichrome (Davenport, 1960) for collagen. The latter (an improvised collagen stain) consisted of a substitution of Biebrich's scarlet for ponceau 2R (staining time 1-2 min). Rapid dehydration prevented the loss of both the aniline blue and scarlet stains.

Acceleration of Mallory's phosphotungstic acid-hematoxylin staining for skeletal muscle fixed in formalin.

The staining time for mammalian skeletal muscle fixed in neutral phosphate-buffered formalin was shortened from 12-24 hr to 10-30 min. The permanganate-oxalate sequence was omitted although oxidation by periodic acid or with iodine was found to be necessary. The material was embedded in paraffin and cut 6 μ or less. Deparaffinized sections were treated with 1% alcoholic iodine for 10 rain followed by 5% Na2S2O3 for 2 min and placed in an oven at 60 C for 10-30 min to stain in a preheated mixture of 50 ml of ripened Mallory's phosphotungstic acid-hematoxylin and 1 ml of 2% phosphomolybdic acid. Experiments with fixation showed that the staining procedure followed Zenker's fluid successfully but not Bouin's fluid. Oxidation by KMnO4 was effective only after Zenker fixation; oxidation by CrO3 was unsuccessful.

Observations on the Structure of Cysticerci of Taenia solium and Taenia saginata (Cestoda: Taeniidae)

The author reports a detailed histological study of the cysticerci of Taenia solium and T. saginata. In spite of minor apparent differences, the structure of the two species is very similar and their specific differentiation cannot be guaranteed when the scolex is not available. The cysticerci of Taenia solium and T. saginata have been described and illustrated by many different investigators. Early workers such as Moniez (1880) studied the gross morphology and some of the histology of both species. Numerous illustrations of sections of cysticerci are to be found in various medical or veterinary texts. In recent years human cysticercosis of the brain caused by T. solium has been studied extensively by Trelles (1952), who also described in considerable detail some of the tissues of the cysticercus. McIntosh and Miller (1960) recorded the gross morphological changes during early cysticercus development of Taenia saginata. Silverman and Hulland (1961) studied the histology of Cysticercus bovis with special reference to growth rates and host responses. In spite of these and many other careful investigations, the detailed histologic structure of cysticerci of T. solium or T. saginata has not been described, and studies comparable to those by Crusz (1948) on Taenia taeniaeformis have not been published. The purpose of the present investigation is to describe the morphology of fully developed cysticerci of T. solium and T. saginata and to determine whether specific identification of the two species can be made on the basis of tissues other than the scolex. Appreciation is expressed to Dr. J. O. Trelles, San Marcos University, Lima, Peru, and to Dr. K. Nasr-alla Soliman, Veterinary Research Institute, Cairo, Egypt, for supplying some of the materials used in this study; to Mr. Zane Price for preparing the photographs included here; and to Mrs. Nora Liu for technical assistance. MATERIALS AND METHODS Cysticerci of Taenia solium were obtained from human brains, Lima, Peru, and from the muscle of pigs, Los Angeles, California. Cysticerci of T. saginata were collected from cattle in Cairo, Egypt, in the Sudan, and in Los Angeles, California. The material was fixed in 10% formalin or in Zenker's fixative, embedded in paraffin, and sections cut at 7 p. Stains used were Haidenhain's iron hematoxylin, Mallory's aniline blue stain, Mayer's hemalum, Gomori's trichrome, and Davenport's protargol stain. All these were used as described by Voge (1960). Rinehart's modification of Hale's stain (Rinehart and Abul-Hay, 1951) was used for the demonstration of collagen, and Himes' triple stain (Himes and Moriber, 1956) for polysaccharide and protein. All observations were made on fully differentiated, undamaged specimens.

Phloxine-methylene blue staining of formalin-fixed tissue.

It is at present difficult to obtain a good phloxine-metbylene blue stain on formalin-fixed tissue. When phloxine has been used, it is washed out in the process of staining with methylene blue and differentiating with colophony (rosin). In the original technic of Mallory, Zenker's fixation is used. The tissue is first stained with a 2.5% aqueous solution of phloxine, then with a solution of 1% methylene blue plus 1% azure II and differentiated in colophony.1

A Cytochemical Study of the "Volutin Granules" in Protozoa

Purple staining are commonly found with the Romanowsky method in the cytoplasm of certain SPOROZOA, RHIZOPODA, CILIOPHORA and MASTIGOPHORA. They have been called chromatoid grains by Minchin (1909) and are generally referred to now as granules on the assumption that they are constituted of the same substance which Meyer (1904) discovered in molds, yeast cells, and algae, and called volutin. Meyer also concluded from certain microchemical reactions that volutin is related to the nucleic acids. Some protozoologists, considering the staining properties of the volutin granules, related the substance to chromatin and even considered it to be a precursor of that nuclear material. Others, on a less morphological basis, considered the volutin of protozoa as a cytoplasmic reserve substance. The latter opinion received much credit for it interpreted satisfactorily the fact that the appear, increase in size, or disappear in the life cycle of certain protozoa and in certain stages of trypanosomes in culture. The recent progress in quantitative histochemical methods and qualitative cytochemical methods has made it possible to define more precisely the nature of cellular constituents. Caspersson and Brandt (1941), using the ultraviolet absorption method, concluded that the volutin of the yeast cells was nucleic acid. Several authors, Brachet (1942) and others, used ribonucleases extensively to detect ribonucleic acid. Ribonuclease, a ferment extracted from the pancreas either by the method of Dubos and MacLeod (1937, 1938) or after successive precipitations by ammonium sulfate, specifically splits ribonucleic acid. When a cellular structure stains red with pyronine, or blue with toluidin blue, or purple with Romanowsky stains, and when it does not stain by the same methods after a preliminary digestion with ribonuclease, one can positively conclude that the staining of the structure was due to the presence of ribonucleic acid. The method is easily applicable to blood smears after Zenker's fixation (van den Berghe, 1942a). When very active, the ferment digests the stainable elements completely in less than an hour at 37? C. The digestion should be done at pH 6.8. Control slides in physiological saline must be run under the same conditions.

Some Histopathological Changes Caused by Hypoderma Larvae in the Esophagus of Cattle

Material for this investigation was obtained from cattle slaughtered at an abattoir in Des Moines, Iowa. As soon after slaughter as possible tissue from infested esophagi, with larvae intact, was placed in Zenker's fixative and prepared for sectioning and staining. Paraffin was used exclusively for imbedding purposes. Several staining techniques were used to bring out the desired effects. Eosin-methylene blue staining proved very effective for demonstrating infiltrating leucocytes. Best results were obtained by staining in a 4 percent aqueous solution of eosin Y for 20 minutes, counter-staining with Goldhorn's polychrome methylene blue for 15 minutes, and then diffenentiating in 95 percent alcohol containing a few drops of rosin solution (I). Picrofuchsin (Van Gieson) was an excellent counter stain to hematoxylin for differentiating connective tissue, which, owing to pathological conditions, did not always appear as expected. The ordinary Delafield hematoxylin-eosin staining was employed in some instances. In estimating the number of eosinophils per cubic millimeter of tissue, counts were made of all the cells falling in the area of a crossline ocular micrometer. The square area of the ocular micrometer was computed for each power of the microscope, and the thickness of the tissue was known. In computing densities per cubic millimeter of tissue, the following formula was used:

THE USE OF COLOPHONIUM IN DIFFERENTIATING THE EOSIN-METHYLENEBLUE AND OTHER STAINS

One of the most important of the regressive staining methods used for general histological work is the eosinmethylene-blue method, hitherto only applicable to tissues fixed in Zenker's fixative. A successful stain by this method yields better results for cell-differentiation and the study of fine structures than any other single staining method. Its use has been limited because of the difficulty of securing constant results. Inquiries made in various laboratories, and the results of personal experience in a number of laboratories, have shown that the one uncertain step in the staining method is that of differentiation. It has been generally known that alcohols from different sources do not act alike when used for differentiation. The result of an attempt to determine the variable ingredient in the commercial 95 per cent, alcohol has yielded a method for differentiation which has given uniform results over a period of one year with a large