Technical Procedures for the Study of the Nucleolonema

In a recent paper¹ the authors have shown filamentous structures within the nucleolus of all cells. This structure was named the nucleolonema. In the present paper the technical procedures which prove their existence are described in detail.

There are two groups: a) Silver impregnation procedures and iron-pyrogallic stain for fixed material; b) phase contrast microscopy, dark-field illumination and oblique transillumination for fresh material.

The suitable objects are mentioned in every case in order to obtain the best results.     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Absorption Ratios of Biological Stains

Absorption ratios are supplied for most of the dyes used as biological stains.

The use of these ratios will enable the analyst to identify the stains conveniently, and will also frequently afford information of considerable value respecting their purity.     

Application of Fluorescence Microscopy and Photomicrography to Woody Tissues

A procedure is described for making preparations of woody tissues for visual observation or photography by incident-light fluorescence microscopy. The chief advantages of the technic are the following:

(1) Reliable recognition of anatomical characteristics in wood without ordinary time-consuming histological technics.

(2) Examination of relatively larger surface areas of wood blocks than by usual methods.

(3) Visual observation and, if desired, photography of tissues and cell structure in dry or in nearly natural or fresh condition.

(4) Marked color contrast without the use of stains in many tissues, including specific types of cells comprising them.

(5) Improved color contrast by use of Congo red with aspects not usually obtained by other methods.     

The “Dispersion Staining” Method for the Selective Coloration of Tissue

A method has been developed for the selective coloration of fixed tissue without the use of dyes. Microtome sections of formalin-fixed material are mounted under a cover glass in a mixture of two liquids such as diethylene glycol monobutyl ether with cin-namaldehyde and examined with the dark-field microscope. The refractive index of the liquid used for mounting must be of high dispersion and equal or close to the index of the specimen.

Tissue elements, dependent on their refractive index, whether slightly lower, the same as, or slightly above the mounting medium appear colored in shades of blue, red or yellow.

The optical principles involved in this optical dispersion method are similar to those involved in the production of colored light by the Christiansen filter.     

Studies on the Preparation and Recoloration of Fuchsin Sulfurous Acid

Some of the factors affecting the recoloration of Schiff's Reagent (fuchsin sulfurous acid or FSA) by formaldehyde have been studied spectrophotometrically to determine the optimal conditions for the reaction of this reagent with aldehydes.

Of the various reducing agents utilized in the preparation of the leuco dye from basic fuchsin, sodium sulfite and bisulfite proved to be the most satisfactory for obtaining in the reagent maximal sensitivity to recoloration with minimal quantitative variation of results.

The relative proportions of reducing agent and basic fuchsin present in die leuco dye determine its sensitivity to recoloration. Under the conditions of the present experiments, greatest reagent recoloration was obtained when the leuco dye contained 0.01 mole of sodium bisulfite and 0.001 mole of basic fuchsin per 100 ml., a ratio of 10/1.

The recoloration of a given amount of FSA is related to the amount of aldehyde and the temperature of the reaction.

The present experiments indicate the desirability of standardizing the composition of FSA and the conditions under which it is used, if the results of different investigators are to be readily reproduced or compared.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

A Modification of Alizarin Red S Technic for Demonstrating Bone Formation: Methods of Mounting, Photographing and Demonstrating the Specimens

This paper shows that by using solutions heated in the incubator during certain stages, the alizarin red S method of staining the ossified centers in embryos has been shortened, with a consequent saving in time.

New methods of mounting the specimens have been evolved and are described in detail.

The technic of photographing mounted and unmounted specimens is outlined and illustrated by diagrams.

Diagrammatic illustrations are provided of the various types of apparatus used, including a plan of the cabinet for demonstrating clearly the smaller embryos mounted between watch glasses. Photographic examples of the results achieved are also shown.     

Study of the Mode of Action of Some Nitrodiphenyl Ethers

Nitrosoderivatives of the nitrodiphenyl ether herbicides (nitrofen, bifenox) have been studied. UV irradiation in different organic solvents gives degradation products. In buffered aqueous media, in the presence of chloroplasts and spin traps such as DMPO, hydroxy and peroxy radicals have been characterized.

In organic media and in the presence of spin traps such as DMPO, PBN, 4-POBN, solvent radicals (CHCIl₂, CCI₃, CH₂O) have been formed.

Nitro-derivatives have been studied under UV irradiation and in the presence of tetramethylethylene (TME), alkenylhydroxylamines are formed which autoxidize in nitroxide radicals. The formation of the stable nitroxide radical occurs in the dark process after continuous irradiation. The intensity of the signal decreases strongly when a new irradiation is applied. Radical species, with analogous ESR spectral characteristics are formed on reaction with nitrodiphenyl ethers and fatty acids.

The reactivity of these herbicides in micellar media (SDS, Brij 35, and CTAB) has been investigated. The kinetics of formation of the ESR signal corresponding to the photoreduction of the nitrodiphenyl ether in the presence of TME behave differently in a micellar environment as compared to solution. The intensity of the formation of the nitroxide increases under irradiation and decreases in the dark; the rotational correlation time t_c has been determined for each type of micelle.

Synthetic nitrosodiphenyl ether made by the reduction of nitrodiphenyl ether using hydrogen gas and PtO₂ as a catalyst gives the corresponding amine, which is oxidized with rneta-chloroperbenzoic acid (m.CPBA). The nitrosodiphenyl ether in the presence of soja azolectin liposorne containing a fluorescent probe has been analysed. When this synthetic nitrosodiphenyl ether is added to a medium containing soja azolectin liposomes and a carboxyfluorescein, fluorescent probe placed inside the liposornes, a rapid increase in the fluorescence of the medium is observed. The nitrosodiphenyl ether induce a break in the liposorne membrane.     

Daily Changes in the Gonadotropin Levels and Response of Carp Oocytes to Hypophysial Homogenate

Daily changes in carp gonadotropin levels in adult female carp and daily changes in carp oocyte sensitivity to carp hypophysial homogenate, in vitro and in vivo, were investigated.

A total of three series of experiments were carried out. Gonadotropin levels were radioimmtmologically determined.

The results of series 1 and 2 experiments were subjected to statistical analysis with the use of cosinors circle and elipse of errors. It has been found that in the mature female carp in the pre-spawning period with the light periods being long (L:D = 16:8) the apogee for gonadotropin occurs 10 hr after the onset of the light period.

The sensitivity of the oocytes, in terms of the percentage of mature oocytes (after GVBD) following a 24-hr incubation of ovarian fragments with the hypophysial homogenate, reached the highest value at 1300, i.e. 9 hr after the onset of the light period.

It was also found that the injections of carp hypophysial homogenate made at 0900 were much more efficient in inducing ovulation than those at 2100.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.     

Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

Hematoxylin Substitutes: Fluorone Black and Methyl Fluorone Black (9-Phenyl- and 9-Methyl-2, 3, 7-Trihydroxy-6-Fluorone) as Metachrome Iron Alum Mordant Dyes

The phenyl and methyl trihydroxyfluorones, hitherto used histologically only in the rather difficult and unreliable Turchini tecbnics for discriminating deoxyribonucleic from ribonucleic acid, find a new use as iron mordant metachrome dyes which act as nuclear stains. Nuclear staining is unaffected by acid extraction of nudeic acids, as with hematoxylin lakes.

The two dyes, named by Liebermann and Lindenbanm 9-phenyl-2, 3, 7-trihydroxy-6-fluorone and 9-methyl-2, 3, 7-trihydroxy-6-Ruorone, have also acquired (illustrating with the phenyl homolog) longer chemical names of the form 2, 6, 7-trihydroxy-9-phenylisoxanthene-3-one (Eastman). Aldrich and Pfalz-Bauer adhere to the Liebermann-Lindenbaum nomenclature. The trivial name fluorone black is proposed for the phenyl homolog and methyl fluorone black for the methyl homolog.

The iron lake of fluorone black appears to be a useful substitute for iron hematoxylin, methyl fluorone black less useful. Neither dye has the diverse capability of hematoxylin.

Aided by a contract from the National Cancer Institute NO-1-CB-43912     

α-Chymotrypsin Immobilized on Chitin. Relationships Between the Enzyme Kinetic Constant and Support Structure

-Chymotrypsin was immobilized on chitin from squills, lobsters and prawns by means of glutaraldehyde. Hydrolase and peptide synthetase activities were determined in aqueous and homogeneous aqueous-organic media, respectively.

The results show -chymotrypsin immobilized on chitin from prawn to be the most active immobilized derivative based on its synthetase activity (90% yield of Bz-Tyr-Leu-NH₂ in carbonate buffer, p^H 9 containing 70% 1,4- butanediol).

The relationship between the kinetic constant of hydrolysis and chitin structure was also studied. -Chymotrypsin immobilized on prawn chitin was found to be the best derivative in kinetic terms.

The stability of the three derivatives was studied at 37C.     

Metallic Lakes of the oxazines (Gallamin Blue, Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2-3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3-5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

A Discussion of Some of the Factors Causing Variable Results with Flagella Stains

Most flagella stains would probably give more consistent results if they were of stable composition and more was known of the factors influencing the mechanism of their action. Many of the mordants that have been recommended change rapidly, both chemically and physically, after preparation.

The reaction of some mordants, such as that of Loeffler, seems to be an important factor in their use. It is complicated, however, by temperature effects, oxidizing and reducing agents, the age of the bacterial cultures and variations in different species.

Users of flagella stains will probably always have to select or alter methods to suit the cultures to be stained. Any method which makes use of a minimum of complicated solutions and operations is most likely to give consistently good results.

No clearing or decolorizing agent tried changed poor preparations to good ones, but sometimes good ones can be improved by this treatment.     

Eosin B

The two known isomeric dibromodinitrofiuoresceins have been prepared in a fairly pure state, and their absorption spectra determined.

Commercial samples of eosin B are not 4, 5-dibromo—2, 7-dinitrofluorescein, as stated in dye indices. They are mixtures which contain other bromonitro derivatives of fluorescein as well as di-bromodinitro derivatives.

The color acid method provides a substantially reliable means of determining actual dye content with commercial samples of the dye, but the reduction method may prove decidedly misleading.     

The Effect of Substrate Hydrophobicity on the Kinetic Behaviour of Immobilized Candida rugosa Lipase

Candida rugosa lipase (EC 3.1.1.3.) was immobilized in a hydrophilic polyurethane foam and used in the hydrolysis of olive oil, in H-hexane. The results obtained were compared with those from a previous study, in which the same lipase preparation was used in the esterification of ethanol with butyric acid.

The initial rate of hydrolysis increased exponentially with increasing olive oil concentration. In contrast, for the esterification reaction, Michaelis-Menten kinetics with inhibition by both substrates, had been observed.

The effect of medium viscosity, stirring conditions and size of immobilization particles could not explain the observed kinetics of the hydrolytic reaction. However, a direct relationship was observed between the log P values of the reaction medium and the initial rate of hydrolysis, i.e., activation of the immobilized Candida rugosa lipase appears to be promoted by a high hydrophobicity of the reaction medium.

In the case of the esterification reaction, no similar correlation was found.     

Ruthenium Red—p-Phenylenediamine Staining of Monolayers to Facilitate Handling and Selection of Specific Cells for Transmission Electron Microscopy

The handling of monolayers for transmission electron microscopy has presented many problems, the main one being difficulty in visualizing the monolayers after polymerization of their plastic embedment following conventional glutaraldehyde-osmium fixation.

The application of ruthenium red—p-phenylenediamine during processing produced intensely darkened cells which could be examined and photographed either in 95% ethanol or following Spurr embedment without further treatment or sectioning. This treatment also facilitated orientation of the monolayers when re-embedding, and permitted precise localization of monolayers within flat embedding molds when trimming and thin sectioning for transmission electron microscopy.

Increased color density is the combined result of more complete retention of soluble elements during initial fixation by ruthenium red and the formation of a colored reaction product between the bound ruthenium red and osmium which is further intensified by p-phenylenediamine.