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.     

A Consideration of French's Test of Basic Fuchsin for Endo's Medium

In describing a method of testing for the return of color in decolorized fuchsin for use in Endo Medium, French states that variations in hydrogen ion concentration fail to influence the appearance of color in this medium.

Duplications of this test were made using alcoholic and aqueous solutions of fuchsin and both sodium sulfite and sodium bisulfite as decolorizing agents.

In the decolorized alcoholic solutions of fuchsin the color failed to reappear when formalin was added, but a small amount of a weak solution of lactic acid caused the color to return.

Alcoholic solutions of fuchsin failed to decolorize in sodium bisulfite solutions until a few drops of NaOH were added. The color, then, reappeared immediately.

Solutions of peptones to which fuchsin had been added were substituted for the original fuchsin solution. Alcoholic and aqueous solutions of fuchsin were added to equal amounts of a 1% peptone solution. The peptone solutions varied in their hydrogen ion concentration and the results showed that those which were neutral decolorized readily while the more acid solutions were but partially decolorized.

Fuchsin decolorized according to results found in this test, was not satisfactory in the Endo medium, especially in the case of the aqueous solutions of fuchsin.

Experiments which were carried on by other workers and checked with this method all indicated that some acid is necessary to secure the restoration of color.     

The Specificity of the Feulgen Reaction for Thymonucleic Acid

The results of experiments on the specificity of the Feulgen reaction for thymonucleic acid do not substantiate the observations of Carr. The staining is not localized in the nucleus because of the destruction of cytoplasmic constituents following acid hydrolysis or because of the absorbing power of chromatin, since the cytoplasm and nucleolus can still be stained by numerous dyes. The effects of factors such as the acid hydrolysis and sulfurous acid washing baths upon the cytologic distribution of dye were studied on tissues stained with (1) fuchsin-sulfurous-acid (Feulgen) reagent, (2) fuchsin-sulfurous-acid reagent colorized by the addition of formaldehyde, (3) basic fuchsin in one-tenth normal HCl, and (4) basic fuchsin in distilled water. Under comparable conditions, important differences between these stains were found in the effects of preliminary hydrolysis; rapidity of staining and destaining; extractability of dye from tissues by water, alcohol, and sulfurous acid solution; rate of fading from exposure to light; localization of stain in tissues; and differences in hue. After treating tissues with desoxyribonuclease, an enzyme which acts only upon thymonucleic acid, cells do not stain with the Feulgen technic. Following removal of nucleic acid from chromatin by hydrolysis, attempts to demonstrate an absorption of thymonucleic acid upon the residual nuclear protein were unsuccessful.

The evidence for and against the specificity is discussed. In agreement with most other investigators, on the basis of the evidence in the literature as well as these experiments, it is concluded that when properly controlled the Feulgen reaction is relatively specific for thymonucleic acid.     

Double Staining for Comparative Measurements in Squash Preparations

Comparative measurements of nuclei or chromosomes following different treatments are seldom made on squash preparations, since variations which arise during preparation of the slides may easily mask genuine treatment differences. This drawback may be overcome by making use of dyes which, when substituted for basic fuchsin in Schiff's reagent, will give a Feulgen-type reaction with chromatin. By selecting dyes of contrasting colours, it is possible to intermingle cells from different treatments in the same squash preparation, and to perform comparative measurements on adjacent cells.

Suitable dyes which contrast well with basic fuchsin are toluidine blue, or azure A (which stain chromatin blue) and chrysoidin yellow (which stains chromatin yellow). These dyes are made up and used in the same manner as ordinary Feulgen reagent.

Samples of cells from the two treatments to be compared are fixed, washed and hydrolysed in 1 N HCl at 60 C. One sample is stained in regular Feulgen reagent, the other in the contrast dye, then both are macerated and thoroughly mixed on the same slide in a single drop of 45% acetic acid. A coverslip is added, and the preparation flattened to the required amount and made permanent after dry-ice removal of the cover. This technique may also be utilised for comparative grain counts in autoradiography, provided that the contrast dye does not cause chemical fogging of the film.     

Histological and Histochemical Uses of Periodic Acid

Periodic acid acts upon the 1,2 glycol linkage (-CHOH -CHOH-) of carbohydrates in tissue sections to produce aldehyde (RCHO+RCHO) which can be colored with Schiff s reagent. The method can be used on frozen or paraffin sections and is useful as a reaction for carbohydrates of tissues: glycogen (in paraffin section only), mucin, basement membrane, reticulin, the colloid of the pituitary stalk and thyroid, some of the acidophile cells of the human anterior hypophysis, the granular cells of the renal arteriole, etc.

In abnormal tissues, it colors many of the “hyaline” materials— amyloid infiltrations, arteriolosclerotic hyaline, colloid droplets, mitotic figures, etc.

The histochemical uses of the periodic-acid-Schiff's reagent (PAS) need careful control because of the possibility of attachment of iodate or periodate to tissue constitutents, producing a recoloration of the Schiff's reagent. Whenever possible the positive reacting material should be further identified by other methods since Lison showed other substances besides aldehydes can recolorize SchifFs reagent.     

Demonstration Of Golgi Zones By Three Technics For Carbohydrates

The carbohydrate of the Golgi apparatus of several organs of rats, rabbits, and frogs was selected as the principal test material for the behavior of three different technics: 1) periodic acid with colored fuchsin; 2) “direct” chromic acid piperazine silver; 3) periodic acid with leucofuchsin.

Parallel sections of organs in which positive reactions were observed, were treated before staining with a series of reagents to characterize them as glycoprotein.

The results obtained by the three technics under any constant set of conditions were essentially identical in all cases. It is concluded that discrepancies that may have been noted up to now are due to several factors, probably the most important being the tissue's physiological status and the influence of fixation. The study shows that HIO₄, -fuchsinl and chromic acid silver methods are, at least empirically, as valid as HIO₄, -leucofuchsin technics.

Considering the differences in the oxidative mechanism of chromic and periodic acids and other data, the possibility of two different chemical pathways leading to the same final result is discussed.

It has been found that colored fuchsin, as well as its leuco form, can be used in the histochemical demonstration of aldehydes after periodic acid treatment (Arzac, 1948). In a later report (Amc, 1950), a series of reactions were obtained with colored fuchsin which differed in several ways from the results of others using Hotchkiss' method. For example, Gersh (1949) reported the presence of probable glycoproteic granules in the Golgi apparatus of rabbit and guinea pig's intestine. Leblond (1950) also found positive Golgi reactions in different cells of male excretory ducts and in other organs of the rat. Such reactions had not been observed with the colored fuchsin technic in any of the two above-mentioned occasions.

Since the latter investigators used different fixatives, which might have caused the discrepancies, the experiment described below was undertaken to study: (a) the influence of fixation on the final re-actions elicited by HI04-fuchsin (colored and leuco-form) and chromic acid piperazine silver methods; (b) the results obtained in the demonstration of Golgi zones of several rat's, rabbit's and frog's organs by these methods.     

Basic Fuchsin as an Indicator in Endo's Medium

The writer has made an investigation of various samples of basic fuchsin for use in the Endo medium for differentiating the bacteria of the colon-typhoid group. Various different concentrations of the fuchsin samples have been used in making the media. The conclusions are as follows:

American made fuchsins differ markedly in their alcohol solubility properties. They contain materials which are very readily soluble in 95% alcohol, but which are precipitated by sodium sulphite.

This precipitation may be prevented by increasing the dilution of the fuchsin in alcohol.

In order to secure more dependable results in the use of decolorized basic fuchsin as an indicator in Endo Agar, it is advisable to test the fuchsin in different dilutions in alcohol in order to secure a completely decolorized solution. It is also advisable to carefully test those fuchsins which decolorize only in high dilutions with a known organism in Endo agar before relying on it as a satisfactory indicator for the presence of sewage organisms.     

Feulgen's Reaction Applied to Protozoa and Small Worms, Mounted In Toto in Venetian Turpentine

Permanent mounts of certain protozoa and small worms are obtained as follows: kill suspensions of the organisms with Feulgen's fixative (6% HgCl₂ in 2% aqu. acetic acid) for 3 to 24 hours. After pipetting off the fixative, add successively: 70% iodized alcohol; ditto, 30 minutes later; 50%, then 35% alcohol; 2 baths distilled water; normal HCl. Transfer to cold water and heat to 60°C for 4 to 5 minutes or longer. Cool under running water; and wash in distilled water.

Stain 1 to 3 hours in Feulgen's fuchsin sulfurous acid (1 g. of a suitable basic fuchsin, e. g. rosanilin hydrochloride, boiled in 200 cc. water, cooled, and allowed to stand 24 hours after adding 20 cc. normal HCl and 1 g. sodium bisulfite). Pass thru 3 baths of 200 cc. distilled water with 10 cc. normal HCl and 1 g. sodium bisulfite. Transfer to water and then to 35%, 70%, and 95% alcohols successively. Counterstain with fast green FCF, orange G or eosin Y in 95% alcohol. Pass thru two changes of absolute alcohol.

Transfer to 10% Venetian turpentine and place in a dessicator; mount after the turpentine has become concentrated.

If sections instead of total mounts are desired, the material should go from absolute alcohol, thru alcohol-xylol and xylol to paraffin (or preferably paraffin of M. P. 56°C with 3% bees-wax). The paraffin may be added to the material in the test tube, and cooled after the organisms have settled. Then break the tube, trim a block, and cut.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

Simplified Manufacture and Histochemical Use of the Schiff Reagent

Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8-2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.

To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.     

Comparative Spectroscopy of Basic Fuchsin, Schiff Reagent, and a Formalin-Schiff Derivative in Relation to Dye Structure and Staining

Ultraviolet (UV) absorption (200-330 nm) for 0.5 mg/ml aqueous solutions of basic fuchsia unadjusted and those adjusted to pH 0, 1.5, 8.5, and 11 were determined as well as spectra in the visible range (400-675 nm) for solutions with pH 1.9, 2.8, 3.9, 4.7, 5.5, 5.9, 6.5, 7.5, 9.3, 10.4, and 11. The UV absorbance of degassed Schiff reagent containing 1 or 0.5 mg dye/ml, and that of this reagent adjusted to pH 1.5, 2.3, 3.1, 4.5, 6.0, 7.1, and 8.4 were obtained for comparison. The progressive reaction of formalin with degassed Schiff reagent, followed spectrometrically for 2.5 hr, required 2 hr to reach completion. The degassed Schiff reagent contained only traces of-SO₃H as judged from its minimal absorbance between 280 and 295 nm. The UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical. The absorbance is that of basic fuchsin reduced by the addition of Cl^- or SO₃H^- to the central methane carbon and H to the amino groups, therefore the leuco structure of basic fuchsin so reduced shows the fomation-NH₃ groups. Infrared (IR) spectra of basic fuchsin, Schiff crystals, and a crystalline formalin-Schiff reaction product support these observations and indicate that the final colored product is a methylsulfonic acid derivative of basic fuchsin. Identical IR spectra were obtained for two types of crystals derived from Schiff reagents indicating that both are the same chemically, although only one became colored on exposure to air. When these crystals were redissolved and SO₂ added, a Schiff reagent of appropriate pH was produced. Since it is derived from a crystalline product, this type of reagent should be useful in histochemical studies     

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.     

A Spectrophotometry Analysis of Tissue Staining

By comparing spectral absorption curves of representative staining solutions and of substances stained with these solutions it is shown that information may be obtained regarding chemical changes associated with the staining process. The stains used in these determinations were acid fuchsin, anilin blue, azo-carmine G, basic fuchsin, eosin Y, orange G, picric acid and Sudan IV. The substrates stained were gelatin, tendon, blood plasma, thymus gland and fat.

Aqueous basic fuchsin and fuchsin-sulfurous reagent to which formalin was added (Setoff reaction) are different stains. The spectral absorption curves for staining solutions and substances stained with the solutions were comparable. Within the limitations of the spectrophotometry methods and stains employed, there was no evidence of significant chemical alteration in the chromophore radicals of the stains associated with the process of tissue staining.     

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.     

Chelating Agents as Histological and Histochemical Decalcifiers

The alterations caused by chelating agents (disodium ethylenediaminetetraacetate) used as decalcifying solutions at pH 7.0, in histological and histochemical technics have been studied comparatively. They have been controlled by the staining with hematoxylin and eosin, Gomori's aldehyde fuchsin, periodic acid-Schiff, metachromasia, and alkaline phosphatase. Their effect on the tissues was similar to that of buffered acid decalcifying solutions, such as that of Greep, Fischer and Morse (equal parts of 2% formic acid and 20% sodium citrate).

The use of 1% sodium diethylbarbiturate for 24 hr as a reactivating agent for alkaline phosphatase in the specimens treated with chelating agents is recommended.     

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.     

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.     

Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

The actual mechanism of the differentiation of lactose-fermenting and non-lactose-fermenting organisms on eosin-methylene-blue medium is not reported in the literature. The present study is an attempt to elucidate this problem.

The color of colon forms on E.M.B. agar was found to depend on two factors: (1) the reaction of eosin with methylene blue to form a dye compound of either acidic or neutral nature, and (2) the production, by lactose-fermenting colonies, of a sufficiently low pH so that this dye compound is taken up by individual cells of the colony. Non-lactose-fermenting organisms are not colored because the compound is not taken up in alkaline reaction.

An explanation is offered to account for the occasional blue colonies found on E.M.B. medium. It is suggested that these colonies form a relatively high pH and thus cause slight dissociation of the compound. This dissociation would allow independent staining of the colonies by methylene blue.     

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.     

Acylation of Amino Acids by Aminoacylase in Non-Conventional Media

N-Acylation of amino acids by aminoacylase (EC 3.5.1.14) isolated from pig kidney was investigated. In water containing organic solutions native aminoacylase proved to be unsuitable for acylation reactions. Covalent immobilization enhanced the stability of aminoacylase in organic solvents (dimethylformamide and dioxane).

The support gel beads showed extraordinary swelling behaviour in dimethylformamide solutions of different water content with respect to their liquid uptake and the temperature sensitivity of swelling.

N-Acylation activity of immobilized enzyme proved to be sufficient in case of L-methionine as substrate and acetate as acylating agent. In addition, the peptide (Ala-Ala) formation was unambiguous but the reaction was much slower than acylation under the given conditions.