Water-Pretreatment Squash Technic: A New and Simple Practical Method for the Chromosome Study of Animals

To simplify the staining of animal chromosomes (especially in insect testes) the authors have borrowed (with necessary modifications) the squash technic of plant cytology. The method has four steps: (1) Water pretreatment. This step requires only about 5-10 minutes either in water at room temperature or in water kept at about 38°C. in a water bath. (2) Fixation. Ordinarily only 5 minutes in 10-15% aqueous solution of glacial acetic acid is necessary. (3) Staining. The fixed tissue is rinsed in two or three changes of distilled water and then placed in a solution of basic fuchsin: either 1% in 30% ethyl alcohol, or 0.2-0.4% in 5-10% lactic acid. In the former solution the staining period should be about 2 minutes: in the latter, 5-20 minutes. The time is not critical. (4) Squashing. The material is rinsed in several changes of distilled water, placed on a clean slide and squashed under a cover glass. Such preparations last 4-5 weeks, and a technic is described for removing the cover glass in order to mount in Euparal and to make them permanent. The authors list various species of vertebrates as well as invertebrates in which the technic has given good chromosome staining, as shown by illustrations.     

An Aceto-Carmine Squash Technic for Mature Embryo Sacs

A method is described by which, whole embryo sacs of Nicotiana, Petunia and no doubt of certain other genera can be obtained readily in aceto-carmine ovule squashes. Although application of the technic to megagametogenesis and fertilization stages is stressed in this paper, use of the method allows development to be traced from the archespore up to the second or third division of endosperm nuclei. The success of the technic depends on four phases:-1) fixation in a medium that causes cell and nuclear structures to become pliable, yet rigid enough that their spatial relationships are not greatly distorted in squashing; 2) heat, which apparently increases the cohesion of cytoplasmic and nuclear constituents; 3) maceration to separate the embryo sac from surrounding cells; and 4) the use of a stain that differentiates the various nuclear structures as well as those of the cytoplasm. Staining of the cytoplasm, essential in some embryological investigations, is one advantage of the aceto-carmine squash method over the Feulgen procedure. In contrast to the Feulgen ovule squash method the aceto-carmine technic will probably be most useful in genera having numerous small ovules. Advantages and defects of the aceto-carmine procedure as compared with the paraffin technic are discussed, likewise the possible usefulness of the former in studies of sterility and in certain other special connections.     

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.     

A Rapid HCl/Toluidine Blue Squash Technic for Plant Chromosomes

Fresh or pretreated root tips are simultaneously fixed and hydrolysed in 5 N HC1 for 15 min at room temperature. After washing they are macerated on a slide in a drop of 0.05% toluidine blue made up in McIlvaine citric acid-Na₂HPO, buffer at pH 4.0. Pressure on the cover slip completes the squash preparation. It is made permanent by removing the cover slip on dry ice, air drying and mounting in Euparal.     

Chromosome Spreading Induced by Vegetable Oils

Seeds soaked in the oil extracted from castor beans (Ricinus communis) for 2 hr were germinated in petri dishes on moist filter papers. Root tips were fixed in acetic alcohol (1:3) at 10-14°C, for 24 hr, washed successively with 70% alcohol (15 min) and water (10 min), hydrolysed in 1 N HCl at 60°C for 15 min and stained in leucobasic fuchsin for 30 min. The stained tip was squashed under a cover glass in a drop of acetocarmine and sealed with paraffin wax. The slides were made permanent by separating the cover glass in a mixture of acetic acid and n-butyl alcohol (1:1), passing through 2 changes of n-butyl alcohol and mounting in balsam. Such a method leads to contraction and spreading of chromosomes, without affecting either the clarity of the constriction regions or the anaphase separation of chromosomes.     

Cold and Chemical Pretreatments to aid Chromosome Counts in a Grass Leaf Squash Technique Incorporating Hot Pectinase Maceration

Leaf buds, comprising the basal 3-5 mm of the youngest leaves attached to short stems, were dissected out of fast-growing young tillers of certain grasses, including Festuca, Lolium and Phalaris spp. and various hybrids. They were kept overnight in distilled water at 0-2 C, treated in a mixture of equal parts by volume of saturated aqueous solutions of 5,7-dibromo-8-hydroxyquinoline containing a surfactant (Tween 80), and 1-bromo-naphthalene for 3-4 hr at 0-2 C, and fixed in Newcomer's fluid. The rinsed samples were hydrolysed in 1 N HCl for 8 min at 60 C and Feulgen stained for 1 hr. After rinsing, the buds were macerated in a filtered 3% solution of Pectinol 100-D (Rohm and Haas) in 0.1 M acetate buffer at pH 4.5 for 10 min at 60 C. Squashes were made in 45% acetic acid. The combined cold and chemical pretreatments resulted in strongly contracted, easily counted metaphase chromosomes, while intact cells with full chromosome complements were more readily retained during squashing after enzyme maceration at 60 C than at room temperature.     

Role of Water in Chromosome Spreading and Swelling Induced by Acetic Acid Treatment: A Ftir Spectroscopy Study

The so called chromosome preparation is a procedure consisting of three strictly connected stages that enables to obtain chromosomes of quality suitable for cytogenetic analysis. Interestingly, experimental evidence strongly suggested that chromosome spreading and swelling (key processes that allow their counting and detailed structural analysis) are induced in the last fixative-evaporation stage by the interaction, mediated by acetic acid, between water from the environmental humidity, and the cytoplasmic matrix and the chromatin. However, since a considerable variation in the quality of chromosome preparations is observed, strongly depending on the environmental conditions in which the procedure takes place, a better comprehension of the mechanisms underlying chromosome preparation is required. To this aim, here we analysed intact lymphocytes before and at each stage of the chromosome preparation protocol by Fourier transform infrared (FTIR) spectroscopy, a technique widely used for the study not only of isolated biomolecules, but also of complex biological systems, such as whole cells. Interestingly, we found that the chromosome preparation protocol induces significant structural changes of cell proteins and DNA, in particular due to the interaction with acetic acid. Moreover, noteworthy, through the monitoring of changes in the water combination band between 2300 and 1800 cm^–1, we provided evidence at molecular level of the crucial role of the bound water to the cytoplasmic matrix and to the chromatin in determining the chromosome spreading and swelling. Our FTIR results, therefore, underline the need to perform the last fixative-evaporation stage in standardized and optimized temperature and relative humidity conditions, thus providing chromosomes of high quality for the cytogenetic analysis that would lead in this way to more reliable results.Key words: Chromosome preparation, chromosome spreading and swelling, DNA conformational transition, FTIR (micro)spectroscopy, protein, DNA hydration     

Magnetite and Magnetotaxis in Algae

Magnetotactic algae of the genus Anisonema (Euglenophyceae) have been isolated from a coastal mangrove swamp in northeastern Brazil. The magnetotactic response is based on a permanent magnetic dipole moment per cell ~7 10^-10 emu. Each cell contains many magnetite (Fe₃O₄) particles organized in chains.