A Colchicine, Hypotonic Citrate, Squash Sequence for Mammalian Chromosomes

The method was developed for bone marrow of mice but is applicable to other tissues and other species of small mammals. Mice are injected intraperitoneally with 0.5 ml of 0.025% colchicine solution and killed 1 hr afterwards. The femurs are dissected out rapidly, the epiphyses are removed, and the marrow is washed out of the shafts by warm hypotonic sodium citrate solution from a hypodermic syringe. Gentle aspiration of the marrow into and out of the syringe converts it into a fine suspension. The suspension is kept in the citrate solution at 37°C for 10 min. Connective tissue and bony spicules are removed by centrifuging through Nylon bolting cloth in a bacterial filtration tube, before fixing in acetic-alcohol (1:3) and staining by the standard Feulgen procedure. The cells are concentrated for each change of reagent by centrifuging slowly. The advantages of colchicine pretreatment and of working with cell suspensions are emphasized.     

Chromosome Preparatons of Bone Marrow Cells without Prior In Vitro Culture or In Vivo Colchicine Administration

Bone marrow (about 0.5 ml) from au erythropoietic region is freed of blood clots by washing 1-3 min in 1 μg/ml colchicine solution (2-3 ml) and then soaking 1-2 hr at 20-30° C in a second change. For mammalian or avian marrows, the colchicine is made up in phosphate-buffered (pH 7) physiological NaCl solution; for amphibian, Ringer's A solution. Next the specimens are soaked about 20 min in a hypotonic solution as follows: for mammalian, 1% Na-citrate; for avian, a 1:4 dilution of the buffered NaCl solution by distilled water; and for amphibian, Ringer's A-distilled water, 1:1. Then they are heated in a mixture of 2% orcein in 45% acetic acid and 1 N HCl, 9:1. Immediately after heating, squash preparations are made with 2% acetic-orcein in the usual manner. An alternative method is to dissociate the marrow cells by agitating after colchicine treatment. Then, recovering the cells between changes by low-speed centrifugation, to carry out the hypotonic treatment and subsequent fixation in Carnoy's solution I (alcohol acetic, 3:1) before drying the cells onto slides from the fixative. After thorough drying the slides may be stained 10-20 min in acetic orcein, or by other suitable technics.     

Male chromosomes of sea urchin hybrid andromerogones created with cryopreserved sperm

We developed a method for preparing male chromosomes from sea urchin hybrid andromerogones created with cryopreserved sperm. We obtained hybrid andromerogones by heterospermic insemination of Hemicentrotus pulcherrimus non-nucleate egg fragments produced by centrifuging unfertilized eggs in a stepwise saccharose density gradient. The hybrid andromerogones showed cleavage rates of 1%-93%, cleaved successively into two- and four- blastomeres and developed to early blastulae. The morulae or early blastulae were treated with colchicine (0.1-1.0 mg/ml), dissociated into single blastomeres by pippeting, swollen with 7%-10% sodium citrate for 10 min and fixed with methanol:acetic acid (3:1). The fixed cells were dropped on slides and air-dried. The andromerogones for 5 sperm species showed a half of their respective diploid chromosome numbers without chromosome elimination. This method is applicable for analysis of the haploid male chromosome complement in sea urchin species for which only sperm can be obtained.     

Chromosome preparations in the field from mammals long after death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01% colchicine solution to 5 ml of medium-cell suspension. After 1/2-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KCl. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanol:acetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Giemsa, if required after pretreatment of the preparations for banding, e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Chromosome Preparations in the Field from Mammals Long After Death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01 % colchicine solution to 5 ml of medium-cell suspension. After Vi-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KC1. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanokacetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Gietnsa, if required after prclieatment of the preparations for banding; e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Bone Marrow Response to Damage Induced By Hydroxyurea Or Colchicine

The extent of bone marrow damage caused by the administration of single or repeated doses of either hydroxyurea (1000 mg/kg b.w.) or colchicine (1 mg/kg b.w.) are comparable. This conclusion is based on serial studies of bone marrow cellularity and of the CFUc numbers in the bone marrow. the proliferation response of the pluripotential haemopoietic stem cells, determined by the cells forming colonies in the spleen of lethally irradiated mice (CFUs) markedly differs if the bone marrow damage is caused by hydroxyurea or colchicine. While hydroxyurea administration stimulates a large proportion of the resting G₀ cells into the cell cycle, the damage induced by colchicine is followed by only a mild increase in the CFUs proliferation rate. The seeding efficiency of the spleen colony technique has been determined after both hydroxyurea and colchicine administration. This parameter, important for the estimation of the number of the pluripotential haemopoietic stem cells in blood forming organs, is significantly affected by hydroxyurea administration, but also by repeated injections of colchicine. Following a single dose of hydroxyurea, the time-course of the CFUs numbers, which were corrected for the change in the seeding efficiency, shows an overshoot occurring after 18–20 hr. At the other time periods, the number of pluripotential haemopoietic stem cells is little affected by a single hydroxyurea injection. This poses a question about the nature of the stimulus, which after hydroxyurea administration triggers the CFUs from the resting G₀ state into the cell cycle. There is evidence that this stimulus is probably not represented by the damage caused to the various intensively proliferating cell populations of the bone marrow. This evidence is based on experiments which show that colchicine induced damage, of a degree similar to that after hydroxyurea, does not stimulate the CFUs proliferation rate to an extent comparable to hydroxyurea. The possibility that colchicine could block CFUs in the G₀ state or that it could interfere with the progress of CFUs through the G₁ and S phases of the cell cycle have been ruled out by experiments which demonstrated that colchicine (1 mg/kg b.w.), administered 10 min before hydroxyurea, does not reduce the number of CFUs triggered into the cell cycle as the consequence of hydroxyurea administration.     

Concentrating and Staining Bone Marrow; An On-the-Slide Technique

A 0.5-1 ml sample of bone marrow is aspirated into a syringe containing 3 drops of 15% K₂-EDTA and an additional 1-2 drops of the EDTA solution previously placed on a slide, is then drawn into the syringe. All of the contents are ejected onto this slide, which is carefully tilted 2 or 3 times to an angle of 5-10°, and the edge brought to the center of another slide. The slide with the aspirate is then slowly tilted to 80-90°. Most of the blood and part of the marrow will drain off, leaving spicules of marrow and some blood on the original slide. A small drop of this concentrated marrow is dragged off with the edge of a third slide and deposited about 2 cm from the edge of a fourth slide on which the smear is to be made. The smear is made by bringing a clean (smearing) slide to the slide with the deposited marrow with flat surfaces parallel and the edges at a 90° angle. With gentle pressure, the smearing slide is pushed toward the empty end of the slide upon which the smear is made. This separates the marrow from the circulating blood. Before staining the smear is air dried and heated in an oven at 120-125 C for 2 min; or alternately for satisfactory but less uniform results the smear is heated over a microburner for 10 sec; then the smear is covered with 1 part of undiluted Wright's stain for 30—45 sec which is then diluted with 2 parts of a solution of 0.1-0.2 gm of Na₂S₂O₃ in 1 liter of distilled water and stained for 10-13 min with this diluted stain. Smears made in this manner have 3 concentric zones; the central zone contains the myeloid tissue; the middle, erythropoetic tissue; the outer, a mixture of blood and marrow.     

Intraperitoneal Colchicine and Hypotonic KCl for Enhancement of Abundance and Quality of Meiotic Chromosome Spreads from Hamster Testes

Male meiotic chromosome spreads of the Syrian hamster (Mesocricetus auratus, 2n = 44) were prepared by introducing two modifications into the method of Evans et al. (Cytogenetics, 3: 289-94, 1964). The modifications were pretreatment of the intact animals with colchicine (4 mg/kg injected intraperitoneally) and the use of 0.563% KCl solution to cause swelling of the cells. In animals subjected to colchicine for 1 or 2 hr, there was a markedly increased yield of cells in 6rst meiotic metaphase. This increase was not present in animals subjected for 3 hr, but these animals showed a slight increase of cells in second meiotic metaphase. The use of hypotonic KCl resulted in much sharper chromosome definition than had previously been obtained with 1% sodium citrate solution     

A method of removing gels from narrow-diameter glass tubes as part of a mini-two-dimensional electrophoresis procedure

A glass chamber that facilitates the removal of polyacrylamide gels from narrow glass tubes after the completion of isoelectric focusing is described. The tube is firmly held at one end of the glass chamber and a syringe is connected to the other end. By slowly forcing water from the syringe into the glass chamber, sufficient pressure can be generated to cause the rod gel to move slowly out of the glass tube. The rod gel is then used for the second stage of a mini-two-dimensional electrophoresis technique. Rod gels of 1.7-mm diameter have been used most extensively and can be removed from the glass tubes with almost 100% success.     

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.     

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.     

A method for administration of prolonged intravenous infusion of prostacyclin (PGI2) to unanesthetized rats

Prostacyclin is short acting and chemically unstable. To study sustained effects in an intact animal, prolonged intravenous infusion may be required. The compound has adequate stability for 24 hr in pH 10.0 carbonate buffer at 0°. A “displacement syringe” is described wherein the prostacyclin solution is stored in a rubber bag inside the barrel of a 5 ml syringe. This device is placed in an ice bath. A syringe pump drives water into the barrel displacing an equal volume of solution out of the bag. Chronic venous cannulas, saddles, and flow-through swivels are used as for drug self-administration studies. A simple, inexpensive rack for use with conventional individual hanging cages is also described.     

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.     

A method for administration of prolonged intravenous infusion of prostacyclin (PGI2) to unanesthetized rats.

Prostacyclin is short acting and chemically unstable. To study sustained effects in an intact animal, prolonged intravenous infusion may be required. The compound has adequate stability for 24 hr in pH 10.0 carbonate buffer at 0 degrees. A "displacement syringe" is described wherein the prostacyclin solution is stored in a rubber bag inside the barrel of a 5 ml syringe. This device is placed in an ice bath. A syringe pump drives water into the barrel displacing an equal volume of solution out of the bag. Chronic venous cannules, saddles, and flow-through swivels are used as for drug self-administration studies. A simple, inexpensive rack for use with conventional individual hanging cages is also described.     

Preparation of bovine blood coagulation factors X1 and X2

A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg.     

The Xanthomonas campestris pv. vesicatoria citH gene is expressed early in the infection process of tomato and is positively regulated by the TctDE two-component regulatory system

Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease of tomato and pepper. Previously, we have reported the adaptation of a recombinase- or resolvase-based in vivo expression technology (RIVET) approach to identify Xcv genes that are specifically induced during its interaction with tomato. Analysis of some of these genes revealed that a citH (citrate transporter) homologous gene contributes to Xcv virulence on tomato. Here, we demonstrate that the citH product indeed facilitates citrate uptake by showing the following: citH is specifically needed for Xcv growth in citrate, but not in other carbon sources; the citH promoter is specifically induced by citrate; and the concentration of citrate from tomato leaf apoplast is considerably reduced following growth of the wild-type and a citH-complemented strain, but not the citH mutant. We also show that, in the Xcv-tomato interaction, the promoter activity of the citH gene is induced as early as 2.5h after Xcv is syringe infiltrated into tomato leaves, and continues to be active for at least 96h after inoculation. We identified an operon containing a two-component regulatory system homologous to tctD/tctE influencing citH expression in Xcv, as well as its heterologous expression in Escherichia coli. The expression of hrp genes does not seem to be affected in the citH mutant, and this mutant cannot be complemented for growth in planta when co-inoculated with the wild-type strain, indicating that citrate uptake in the apoplast is important for the virulence of Xcv.     

India Ink-Gelatin Vascular Injection of Skeletal Tissues

Dogs under sodium pentothal anesthesia were given 100 mg of heparin intravenously and, 15 min later, exsanguinated. A mixture consisting of Higgins India ink #4415, 1 part, and a 14% solution of calfskin gelatin, 1 part, was injected (at 37° C) into the femoral artery of a disarticulated hindlimb. A pump-type 5 ml hypodermic syringe was connected with a gravity-feed reservoir that contained the injection mass, with a filter in the outflow from the reservoir. Veins severed by the disarticulation of the limb permitted a free flow of the injection fluid out of the vascular tree. After injection of about 400 ml of fluid by pumping the syringe, the limb was chilled (—20° C) to solidify the gelatin. Specimens from cleaned bones were sawed out and fixed in Zenker-formalin solution for 24 hr, then decalcified with 6% sulfosalicylic acid. The technique was completed by embedding in celloidin and sectioning. Blood vessels in both cortical and trabecular regions were completely filled by the injection mass.     

The Effect of Colchicine on Regenerating Membranellar Cilia in Stentor coeruleus

SYNOPSIS. Stentors treated with toxic substances can be induced to shed their oral bands (19, Fig. 1), complex structures composed of many cilia organized into membranelles. Regenerating membranellar bands were observed in control stentors removed from toxic (urea-containing) medium at about 3.5 hours. At 8 hours regenerated control organisms were indistinguishable from normal unshed stentors. Experimental animals replaced into colchicine medium were inhibited from regeneration at low, nontoxic concentrations of this mitotic spindle inhibitor. Upon removal of the colchicine and replacement of the shed animals into normal medium or normal medium to which GTP had been added, complete and normal regeneration of the membranellar band ensued. Our observations are consistent with many suggesting that colchicine acts by reversibly binding with a protein during processes involving microtubule formation. Colchicine inhibition of membranellar band formation further indicates that oral membranelles are specialized evolutionary homologs to other centriole (= basal body, = kinetosome) derivatives such as mitotic spindle fibers, cilia and flagella, axopods, etc. (structures containing the ubiquitous microtubules of eukaryotic cells).     

Schistosoma mansoni: stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.     

An Improved Chemical Substitute for Fetal Calf Serum for the Micronucleus Test

The routine use of the micronucleus test in the mutagenicity evaluation of xe-nobiotics is limited by high cost and limited availability of fetal calf serum. On the other hand, there are disadvantages, such as hypotonic damage and clumping of cells, associated with the use of mineral medium substitutes for fetal calf serum. Alternatively, we recommend a chemical medium containing Hanks' buffered salt solution, 1% (w/v) bovine serum albumin, and 0.15% (w/v) ethylenediaminetetraacetic acid, final pH 7.4, to preserve morphology, density and homogeneity of bone marrow cells. Mast cell granules are efficiently removed from rat bone marrow preparations by washing twice with this medium. The morphological preservation of cells is further enhanced by fixation with 70 % (v/v) ethanol for 5 min. The proposed medium provides a cost-effective and convenient substitute for fetal calf serum with substantially improved quality of bone marrow preparations for the micronucleus test.