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.     

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.     

Further Experiments with the Masson Trichrome Modification of Mallory's Connective Tissue Stain

Several dyes, notably ponceau 2R, azofuchsin 3B, nitrazine yellow, and Biebrich scarlet may replace imported “ponceau de xylidin” in the Masson ponceau acid fuchsin mixture. Of these Biebrich scarlet appears to be the best and may be used without acid fuchsin.

A mixture of equal parts of 5% solutions of phosphomolybdic and phosphotungstic acids is much superior to either acid alone and gives adequate mordanting in 1 minute at 22°C.

With the fast green modification, times in plasma and fiber stains can be reduced to 2 minutes each. With anilin blue a 4-minute plasma stain is required. One-minute final differentiation in 1% acetic acid is adequate.

Primary mordanting of formalin material may be accomplished by 5 minutes in saturated aqueous mercuric chloride or 2 minutes in saturated alcoholic picric acid. Three minutes washing in running water is required after these mordants.     

Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

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.     

Differential Stain for acid fast Bacteria and Spores

A modified method of staining acid-fast organisms is described. After staining with carbol-fuchsin as usual in the Ziehl-Neelson method, wash with water and while the slide is still wet cover with a saturated acetone solution of malachite green for three to five minutes. Wash and examine. The acid-fast organisms and spores are red in a green background. If the smear is thick and appears too dense, dry for three minutes and hold over the mouth of a bottle of ammonia until decolorized to suit. Upon exposure to the air the green returns. This can be prevented by keeping the smear alkaline, by the addition of sodium bicarbonate.

A second method is described for use with sputum in which acid-fast organisms are scarce. It permits the examination of thick smears and therefore increases the chances of finding tubercle organisms when few in number. Stain with carbol fuchsin as in the Ziehl-Neelson method. Decolorize with 30% phenol-disulfonic acid in water for a few seconds or until decolorized. Wash and examine at once. If color returns upon washing decolorize again. The tubercle organisms appear red in a colorless background.     

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.     

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.     

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.     

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.     

The Nature of the Reaction of the Colon Organism on Endo's Medium

Bacterium coli was grown in a medium of a composition similar to Endo's medium with the exception that the sulfite, fuchsin and agar were left out. When the fuchsin-sulfite mixture was added after 24, 36 and 48 hours, or after 3 and 10 days of incubation at 37°C., no reaction appeared. The substance that causes the reaction is only formed when the bacteria are grown in the presence of sulfite. When picric acid and ether are added to the red compound produced by the colon organism in Endo's medium and the mixture shaken, the color remains in the watery layer and thus the dye color that appears in the medium is not restored fuchsin, but a new substance. The work confirms Neuberg's and Nord's theory that the Endo medium acts as a trapping agent for the intermediate product, acetaldehyde, which causes the Endo reaction.     

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 Simplified Method for Staining Mast Cells with Astra Blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however, the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl₂ · 6H₂O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

An Easily Controlled Regressive Trichromic Staining Method

Three modifications of Mallory's connective tissue stain are described and some features of the action of picric acid are discussed.

In the first and most critical method the nuclei are stained in an iron hematoxylin and then differentiated in a picric acid solution containing orange G. This not only differentiates the nuclei, but stains all other elements yellow. The section is then washed in running water to remove the yellow color from all tissues except those which are to remain yellow in the final preparation (usually the erythrocytes). The section is next stained in an acid fuchsin mixture and then differentiated until the desired depth and contrast is obtained. Staining in anilin blue follows and this in turn is differentiated to suit. The section is then dehydrated and mounted.

In the second method the nuclei are stained in hemalum (e.g. Harris's) for a short time; the section is then rinsed and immersed in a mixture of picric acid and acid fuchsin and thereafter is differentiated; it is next passed into anilin blue w. s. and then differentiated and mounted as before. This is less critical than method I, but can be applied to large batches of slides at a time.

The third method is a one-solution method. After staining the nuclei in hemalum, the section is immersed in the “Picro-Mallory” solution, differentiated briefly, dehydrated and mounted. This modification, while being the least critical, is most suitable for routine use when the tissues have been fixed in a fluid containing chromate; the other commonly used fixatives, while giving useful results, are not so good.     

A New Microchemical Reaction for Cellulose

A microchemical test for cellulose applicable to fresh sections and commercial products is described. The test differs from the older technics in that materials tested are not permanently altered.

Two solutions are required: (1) 2% solution of iodine in 5% KI, diluted with 9 parts by volume of water containing 0.28% glycerin; (2) saturated aqueous LiCl.

Procedure: Apply 2 or 3 drops of solution 1 with a glass rod; allow the preparation to stand for 30 sec; blot with filter paper, drying as completely as possible. Apply one drop of solution 2, cover and examine. The color reaction will be obtained within 5 min. The reaction for pure cellulose is light blue. Reactions for 16 fibers are given in the table.

As a stain for demonstrating plant tissues the technic has been used in the Botany Department of Pomona College with much success; but this phase of the subject has not been extensively investigated.     

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.     

A simplified method for staining mast cells with astra blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Marchi's Staining Method: Studies of Some of the Underlying Mechanisms Involved

Spinal cord of cat and rabbit was stained, after experimental lesions, by variations of Marchi's method. The following conclusions were drawn:

1. The presence of an oxidizing agent (K₂Cr₂O₇, NaIO₃, or KCIO₃) in the osmic acid solution is of primary importance and a preliminary oxidation in Mueller's fluid is unnecessary or even detrimental.

2. Acetic acid added to Marchi's fluid, accentuates the action of the oxidizing agent in restraining the staining of normal myelin.

3. Too high concentration of oxidizing agent or of acid may inhibit staining of degenerating myelin.

4. Marchi's and Busch's methods have been modified as follows: Fix one day in 10% formalin and transfer without washing to the staining mixture, either A or B. Staining mixture A: Marchi's fluid plus 1 to 3% glacial acetic acid. B: An aqueous solution containing KCIO₃ 0.25%, osmic acid 0.33%, and acetic acid 1%. Stain about one week. These methods worked on spinal cord and medulla, but cannot be recommended for brain.

5. The detrimental effects of long post mortem autolysis or of prolonged fixation in formalin may be counteracted to some degree by increasing the concentration of the acid in Marchi's fluid up to 5% or of the KCIO₃ up to 0.4% in the modified Busch's fluid.