A method for cytogenetic and cytological examination of small shelled larvae of bivalves and other zooplankton

Studies on chromosomes and nuclei of very small bivalve larvae have been impeded by the veliger shell. It has been determined that the alcohol:acetic acid fixative commonly used in cytogenetic techniques can be made to act as a decalcifying agent upon repeated heating. In addition, transfer of formalin fixed shelled specimens, routinely used as marine bioassay organisms, into ethyl alcohol:acetic acid (3:1) fixative also yields clear cells for cytological examination of decalcified but otherwise intact oyster larvae and other zooplankton. Identification of cell type, such as germ-line primordia, in, for example, reproductive and ploidy level studies, and observations on the presence of bacteria can be made from the preparations. Material can be examined up to at least a year after preservation. The method is evaluated and its modifications are discussed.     

A Chrome-Alum Preparation for Delicate and Difficult Fixations

A chrome-alum fixative is recommended as a reagent for general use. The basic formula is: C.P. chrome-alum, 3 g.; 40% formaldehyde, 30 ml.; glacial acetic acid, 2 ml.; distilled water, 238 ml. This fixative permits easy sectioning of yolk-rich amphibian embryos. It can be used to make permanent slides of Euglena showing the flagellum. It is a satisfactory fixative for insect larvae and fixes sharply the slime droplets of Planarians. Fixation should not exceed two hours or the material being fixed will swell. Rinses of 70% alcohol or water may follow the fluid. The fluid keeps well, does not harden tissues and gives good cytological detail.     

Diagnostic morphology of four larval ascaridoid nematodes that may cause visceral larva migrans: Toxascaris leonina, Baylisascaris procyonis, Lagochilascaris sprenti, and Hexametra leidyi

The gross and histological morphology of the larvae of 4 ascaridoid nematodes, Toxascaris leonina, Baylisascaris procyonis, Lagochilascaris sprenti, and Hexametra leidyi, are described. The larvae of T. leonina, B. procyonis, and L. sprenti were recovered from experimentally infected mice at 32, 14, and 75 days of infection, respectively. Hexametra leidyi larvae used for morphological study were collected on day 159 postinfection from a rhesus monkey, Macaca mulatta, while H. leidyi larvae used for histological study were collected from mice when they had reached the same size as those found in the monkey, i.e., at 23 days postinfection. Larvae for morphological study were collected by pepsin digestion, fixed in glacial acetic acid, and cleared in glycerin. Tissues for histological study were fixed in 10% formalin or Bouin's fixative. All sections were stained with hematoxylin and eosin. The differences in the morphology of the larvae of these 4 species were found to be length of the body, shape of the anterior end, and shape of the tail. The major differences in the histological anatomy of these larvae were found to be the body diameter, form of lateral alae when present, presence or absence of internal cuticular bars, shape and internal patterns of the excretory columns, and size and number of intestinal cells. These 4 larvae are differentiated from other described species of ascaridoid larvae that may cause visceral larva migrans, and keys have been devised to aid in the making of a diagnosis.     

A procedure for obtaining erythrocytes from larval fish for cytological study and a description of larval blood of red hake, Urophycis chuss (Walbaum) and Atlantic mackerel, Scomber scombrus (Linnaeus)

A means of obtaining erythrocytes from larval fish and the blood of larvae of two fish species are described. Erythrocytes were obtained for cytological preparations from larvae of Urophycis chuss (Walbaum) and Scomber scombrus (Linnaeus) as small as 2.0 mm. Heart and gills were dissected from the larvae, stained, and flattened into a monolayer on a microscope slide. Mature erythrocytes were the only blood cell type observed in either heart or gill preparations, and these appeared to be the same as in the adult fish. Mature erythrocytes can be isolated from formalinfixed larvae, even as sorted from plankton, and prepared for cytological and cytogenetic study and for blood cell counts. Knowledge gained from the study of erythrocytes from larval teleosts can be important in assessing the normality and health of these early-life stages of fish in either laboratory assays or field studies. The method described is applicable to other fish species, even after years of storage in formalin fixative.     

Acetic Acid as a Diluent and Dehydrant in the Preparation of Whole, Stained Helminths

Aqueous 45% acetic acid can be used successfully as a diluent for Ehrlich's haematoxylin and for Horen's trichrome stain (chromotrope 2 R, 0.6 gm; phosphotungstic acid, 0.7 gm; glacial acetic acid, 1.0 ml; water, 100 ml). Glacial acetic acid is used for dehydration of the stained helminths, and followed by a glacial acetic acid-methyl salicylate series for clearing. The whole process can be completed within 1 hr, from fixation to the cleared specimen, with helminths up to 5 mm in length. A satisfactory fixative for Monogenea, Digenea and Acanthocephala is: 85% ethanol, 85; formalin (40% HCHO), 10; and glacial acetic acid, 5—parts by volume. For Cestoda, 5% aqueous formalin is preferable because they are hardened excessively by the alcoholic fixative.     

A PCR detection method for rapid identification of Paenibacillus larvae

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.     

Cytogenetic Analysis of Bone Marrow and Peripheral Blood Samples Stored in Fixative for Several Years

We have developed a method which improves the spreading of chromosomes and permits banding analysis of cytogenetic samples of bone marrow and unstimulated peripheral blood which have been stored in fixative for up to 15 years. Metaphase cells had been harvested as usual and stored in fixative (acetic acid:methanol 1:3) at -15 C. The procedure includes 4-5 changes of fixative (acetic acid:ethanol 1:1). Next, cells are dropped onto a chilled, wet slide. The back of the slide is then rinsed with 70% ethanol and dried by ignition. C-, G-, Q-, or R-banding patterns can now be obtained with these specimens. The procedure is useful for reinvestigation of cytogenetic samples that were obtained prior to the development of banding techniques.     

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

An Acetic Acid Dissociation, Air-Drying Technique for Insect Chromosomes, With Aceto-Lactic Orcein Staining

The method differs from mammalian techniques for somatic chromosomes in that it uses very small amounts of material. Drosophila melanogaster and an ant, Dorymyrmex sp., are used as examples. Pretreatment with 0.05% Colcemid in insect Ringer solution is applied to mature Drosophila larvae for 5 hr, by feeding, but Dorymyrmex prepupae require puncture and a 15 hr exposure of the puncture to the solution. Organs are removed under 1% sodium citrate, tansferred to fresh citrate for 10-20 min, than fixed in acetic-methanol, 1:3, for 30 min. Transfer to a drop of 60% acetic acid on a clean warmed slide dissociates the cells, which are spread by adding a small drop of fixative and tilting the slide in all directions. After immersion in acetic ethanol, 1:3, for 4 hr, rinsing in the stain solvent and draining the slides then have 2-3 drops of aceto-lactic orcein placed on each, coverslips added, and warmed (at about 50 C) for about 12 hr or until staining is sufficient. They can then either be treated as semipermanent or made permanent by allowing the coverslips to slide off in acetic-ethanol, dehydrating, and mounting in Euparal, or a synthetic resin.     

家蝇幼虫营养保健果冻的研制

家蝇Musca domestica L幼虫营养丰富,体内含有多种具有特异生物活性物质,有很好的开发利用价值。以家蝇幼虫为原料,运用正交试验设计对家蝇幼虫营养保健果冻的加工工艺进行研究。结果表明:35%的家蝇幼虫粗提液,15%的白砂糖,0.18%的柠檬酸和1.4%的卡拉胶的工艺参数,可生产具有良好感官和食用品质的家蝇幼虫营养保健果冻;家蝇幼虫粗提液的护色剂使用0.02%抗坏血酸与0.02%柠檬酸,果冻防腐剂使用0.04%山梨酸钾。     

An ethanol-acetic acid-formol saline fixative for routine use with special application to the fixation of non-perfused rat lung

An ethanol-acetic acid-formol saline fixative (40 : 5 : 10 : 45 v/v) has been developed which gives good results with non-perfused rat lung and which may be used routinely for the fixation of a wide range of rat tissues. The special qualities of the fixative include good penetration, good fixation of nuclei and mitotic chromosomes, and little shrinkage after paraffin embedding. The fixative is also easy to use and has a flexible fixation period (nominally 48 h). Although several fixative mixtures containing alcohol, acetic acid and formalin have previously been reported, none are identical to the present mixture, which was developed independently and systematically in accordance with specific listed requirements.     

Direct and Repeatable Bone Marrow Chromosome Preparations from Living Mice

Using a 27 gauge hypodermic needle, bone marrow is aspirated from a lumbar vertebra into 0.1 ml of Hanks' salt solution. The aspirate is kept well mixed in 1% sodium citrate for 15 min, centrifuged, and the cell pellet fixed for 30 min in Clarke's 3:1 ethanol-acetic fixative. After removal of the fixative the cells are suspended in 0.05-0.1 ml of 60% acetic acid, centrifuged and resuspended in 0.03 ml of this fixative. Chromosome preparations are made by spreading the suspension on a slide heated to 60 C.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

A Rapid Field Technique for Preparing Ant Chromosomes for Karyotypic Analysis

This technique for chromosomal preparation of ant tissues for karyotypic analysis is advantageous under field conditions because it reduces processing time and can be used under humid conditions. The cerebral ganglia from prepupae or early pupae are incubated 20 minutes in a hypotonic citrate solution, minced in a fixative solution of 3:3:4 glacial acetic acid: absolute methanol: distilled HOH, rinsed in a fixative solution of 1:1 glacial acetic acid: methanol followed by Carnoy's fixative, then immediately flame dried. The resulting metaphase chromosomes are well spaced and usually show banding characteristics.     

Honey as a substitute for formalin?

Formalin has long been the standard fixative for clinical routines worldwide. After the Formaldehyde Standard became law in the US in 1987, as a result of increasing concerns about the potential carcinogenicity of formaldehyde, attempts have been made to find safer alternatives. Alcoholic formalin is a useful fixative, because in addition to fixation, dehydration also is begun. For centuries, honey has been known to be an antibacterial agent with the potential to preserve compounds without harmful effects on its users. We compared the effects of honey fixation with other routine fixatives using conventional histochemical and immunohistochemical staining methods. Our results demonstrated that tissues fixed in either honey or alcoholic formalin and 10% neutral buffered formalin (NBF) have similar histomorphology. Honey fixation showed minor histomorphological differences among the various tissues; however, it did not influence affect correct diagnostic conclusions. Our results suggested that honey can be used as a safe alternative to formalin in histopathology.     

Technic for Studying Chromosome Structure

The methods described are modifications of various technics for the study of spiral structure in chromosomes. They enable permanent preparations to be made with better fixation and allow the use of stains which give clear and more critical definition. The first method described involves the use of ammonium, hydroxide (880 vols.) fumes for the treatment of pollen mother cells before fixation. Anthers of Tradescantia are smeared on a slide and wet in a 3% cane sugar solution. The preparation is then immediately placed in a dish of fixative where it remains for two hours. The slide can then be washed, bleached and stained with gentian violet or hematoxylin. It was found that fumes of nitric acid, hydrochloric acid and glacial acetic acid gave similar results. For the second method, boiling water is used for pre-treatment. A smear is made on a slide and immersed in boiling water for five to ten seconds. The smear is then fixed and treated in the usual manner.     

An Air-Drying Procedure for Mammalian Male Meiotic Chromosomes, Following Softening in Gluconic Acid and Cell Separation by An Ethanol-Acetic Mixture

A I cm³ sample of tubules from testes is placed in 5 ml of 0.7% Na-citrate for 20-30 min, then 5 ml of glacial acetic acid is added, mixed well, and allowed to stand for 30 min. The mixture is centrifuged, the supernatant removed, and 3 ml of 3 M gluconic acid is mixed with the tissue and allowed to act for 3 hr. The gluconic acid is removed with a pipette and the tissue is suspended in 5 ml of a freshly made 1:1 absolute ethanol-glacial acetic acid mixture. The tissue is drawn into and discharged from a syringe several times through an 18, a 20, and finally a 22 gauge needle to separate and suspend the cells. The cells are centrifuged and resuspended several times in fresh fixative to remove the gluconic acid. Finally, the cells are suspended in sufficient fixative to give a smear of suitable density, and air-dried preparations are made, or the suspension may be stored at 0-5 C for several days. The cells can be stained by any of the usual stains for chromosomes. This technique results in the improved spreading produced by the air-drying technique and permits recovery of all stages of meiosis and mitosis present.     

Theory and practice of combining coagulant fixation and microwave histoprocessing

The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples. A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realize that formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol.     

A Combined Hcl-Acetic-Alcohol Fixation and Hydrolysis Followed by Cresyl Violet Staining for Mosquito Chromosome Spreads

Mosquito tissues of cytogenetical importance were dissected out on a slide in 0.65% NaCl, under a dissecting microscope, and treated about 30 sec in a drop of 1:3 Carnoy's fixative diluted 1:19 with distilled water. Fixing and hydrolysis was done by a single step in a mixture consisting of: glacial acetic acid, 1; ethanol 96%, 3; HCl conc., 2; and distilled water, 2 (v/v) for 2-6 min at 20-25 C. The specimen was then rinsed with the acetic-alcohol fixative and covered in a drop of 1% cresyl violet in 50% acetic acid under a coverslip coated with Mayer's albumen. Washing was performed immediately by adding water dropwise to one side of the coverslip and drawing the fluid from the other side with absorbent paper. The preparation could be used either as a temporary slide or made into a durable mount. The DNA-containing bands of the giant polytenic chromosomes stained dark violet; interband regions, weakly stained or colourless against a clear background. Mitotic and meiotic figures in gonadal cells stained selectively dark violet or violet with a practically unstained cytoplasm.