Studies on the Quantitative Nature of the Feulgen Stain

Bacterial suspensions were stained with Schiff's reagent according to the procedure suggested in essence by Dondero et al. (1954). Cell suspensions, Schiff's reagent, supernatant fluids and stained cells were analyzed by a micro-Kjeldahl procedure in an effort to quantitate the Feulgen reaction. The concentration of the bacterial suspension, type of fixative, time of hydrolysis and pH of cells and dye were varied and the effects analyzed quantitatively. While the cells were often stained deeply as determined by visual observation, the quantity of dye nitrogen in the cells was not large enough to be measured with the procedure employed. Significant quantitative results were obtained consistently only when the pH of the Schiff's reagent was raised. Feulgen reactions with solutions of formaldehyde and with solutions of DNA were also analyzed quantitatively after removing the colored compounds with charcoal. The analyses indicated that the DNA solution and the formaldehyde solution reacted differently with the dye.     

The Use of the Feulgen Technic with Certain Plant Materials

The Feulgen technic as modified by Heitz promises to become an extremely useful tool in the solution of certain cytological problems. A procedure is outlined for using this technic with root tip smears, and smears of plant microspores. The chief improvement suggested over previous methods is that the material be mounted in euparal, after immersion in 95% alcohol. The technic is of value in the study of chromosome fragmentation, chromatid coiling, centromeres, etc., in both somatic tissue and in microspores.     

The Production of Basic Fuchsin Suitable for the Feulgen Technic

Much difficulty has been experienced in obtaining basic fuchsins satisfactory for the Feulgen technic. A method of purification employing sulfur dioxide is described which has been found to improve many unsatisfactory fuchsins. It is also shown that if the best grade of commercial pararosanilin base is used and proper precautions observed in converting it to the chloride or the acetate, a product can be obtained which gives excellent results in the Feulgen technic. The methods by which the base can be converted to the chloride or acetate are described in detail. Precautions are given for avoiding, in the conversion of the base to the dye salts, contamination of the product with the impurities which interfere with the staining. The use of the acetate is recommended because of its greater solubility.     

Aldehyde Reactions in Tissues in Relation to the Feulgen Technic

The nature of the Schiff-positive radical responsible for the Feulgen reaction was studied by several standard tests for aldehyde. Oxidative, amine, alkali, and catalytic reactions for aldehydes were used. An aldehyde reaction, similar histologically to the Feulgen, could not be produced by the oxidative and amine technics. However, by means of alkali and catalytic methods, it was possible to block the Feulgen-positive tissue radicals ordinarily released by acid hydrolysis. A second hydrolysis of the tissues (after the blocking reactions) restored most of their original Feulgen-positive characteristics.     

Stain Technic for Nissl's Granules Following Alcohol-Formol Fixation

1. Nerve tissue is fixed 2-4 hrs in a 5% solution of strong formalin in commerical 95% alcohol.

2. If dehydration is perfect, either chloroform or xylol may be used as a clearing agent.

3. A slow method of paraffin infiltration is advisable.

4. Sections should be cut 10-12 microns in thickness.

5. Coplin staining jars should be annealed by placing them on a rack in a pan of cold water, bringing the water to the boiling point, and allowing the jars to stand in boiling water for twenty minutes.

6. One per cent aqueous solutions of either methylene blue or Grübler's Neutral Roth are used as specific stains for Nissl's granules.

7. These stains are heated to boiling in a beaker, the slides are placed in the Coplin jars which are partially submerged in boiling water, and the hot stain poured into the jars. The flame beneath the water bath is turned down and the slides left for 20 minutes.

8. The excess of primary stain is washed off in 25% and 50% alcohol and the slides passed rapidly thru the alcohol series to absolute alcohol, and finally to xylol.

9. When counterstaining is desired, nigrosin in 1% aqueous solution, methyl orange, saturated solution in 50% alcohol, or, a 0.5% solution of eosin in 50% alcohol are recommended. These stains are used cold, and the slides are merely dipped in them after the excess of primary stain has been washed out in 25% and 50% alcohol.

10. If a cold primary stain is desired, a saturated solution of thionin in distilled water, acidified with 1% carbolic acid, will prove specific for the Nissl substance. Sections should be stained 5–10 minutes in thionin, then passed rapidly thru to absolute alcohol, and xylol. The same counterstains may be used as in the hot method.

11. Sections prepared by the hot method show little tendency to fade after ten years use.

12. Excepting neutral red, all the stains used in this technic are carried by the National Aniline and Chemical Company and are satisfactory. Coleman and Bell neutral red may be substituted for Grübler's Neutral Roth with good results.     

A Modified Feulgen Technic for Small and Diffuse Chromatin Elements

A revised technic is proposed which differs essentially from the standard procedure only in the preparation of the staining solution and of the sulphurous acid bath, which are made by direct charging with SO₂ gas instead of HC1 and the sulphites ordinarily used. Tests on smear preparations of small amoebae, oocyte prophases of a parasitic wasp, and yeasts revealed Feulgen-positive results not usually obtained by the conventional method.     

Notes on Technic: Mahon's Myelin Stain for Celloidin-Embedded Nervous Tissue

Two methods commonly used to stain myelin sheaths are Kluver and Barrera's luxol fast blue (Kluver and Barrera 1953) and Weil's iron hematoxylin (Weil 1928). Both require differentiation of the stain; in addition, the Kluver-Barrera method specifies 16-24 hour staining. A third method for the selective staining of myelinated axons is that of Mahon (1938), which was introduced for use with paraffin-embedded autopsy tissue. The procedure possesses two distinct advantages since it requires: (1) no differentiation of the stain and (2) only 1 hour staining. Loyez's (1910) myelin stain for celloidin embedded tissue is similar to Mahon's but calls for long staining followed by differentiation. This report describes the application of Mahon's method to celloidin-embedded experimental tissue and emphasizes its utility for staining tissues to be used for reconstructing microelectrode penetrations (fig. 1) and for demonstrating the effect of experimental lesions (fig. 2).     

Feulgen nucleal reaction

Summary Electrophoretic means of separation revealed the presence of as many as five reaction products in Schiff-apurinic acid reaction at the maximum. They differed not only in their absorption maxima, but also in their ratios of apurinic acid phosphorus to fuchsin moiety. Some considerations on the reaction mechanism to account for the occurrence of these multiple reaction products have been made. The stoichiometry of Schiff-apurinic acid reaction was studied with respect to the main product responsible for the presentation of reaction color. A reaction product consisting of six or eight atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be formed, provided that the reagent of infinite concentration is used. From theoretical view point, a reaction product consisting of four atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be expected with the reagent of infinite concentration, provided that apurinic acid retains essentially the nucleotide sequence of its parent desoxyribonucleic acid except for some modification of the original purin nucleotide groups to react as aldehyde moieties, and provided that the reaction proceeds at a constant rate irrespective of the concentrations of the reagent.     

Robert Feulgen Lecture 1998

Attachment of leukocytes to the blood vessel wall initiates leukocyte extravasation. This enables leukocytes to migrate to and accumulate at sites of tissue injury or infection where they execute host-defense mechanisms. A series of vascular cell adhesion molecules on leukocytes and on endothelial cells mediate leukocyte attachment to the endothelium in a stepwise process. A large panel of about 40 known human chemokines is able to specifically activate certain leukocytes and attract them to migrate across the endothelial barrier and within tissue. The specific combination of molecular signals provided by the diversity of cytokines, adhesion molecules, and chemokines regulates the specificity and selectivity of the recruitment of certain subpopulations of leukocytes in vivo. This review will focus on selectins and chemokines which initiate the cell contact and regulate activation and chemoattraction of leukocytes. Accepted: 20 May 1999