Staining human lymphocytes and onion root cell nuclei with madder root

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

A Nissl Method Using Buffered Solutions of Thionin

Experiments were performed in an attempt to obtain a rapid method for staining the chromophilic substance of formalin-fixed nerve cells differentially without resorting to over-staining and subsequent acid-decolorizing. A satisfactory procedure using thionin in dilute buffered solutions was developed: Prepare a stock solution of the dye using 1 g. in 100 ml. of distilled water. Prepare veronal-acetate buffers (Michaelis, 1931) in the range of pH 5 to pH 3.S. To each 10 ml. of the buffer add 0.5 ml. of the stock dye solution. After rinsing in CO₂-free distilled water place mounted or unmounted sections in this solution. (Freshly fixed material, 10μ to 20μ thick, is completely stained in 10 to 20 minutes but over-staining does not occur when longer times are allowed.) Return sections to distilled water (2 changes) and wash until diffusion of excess dye is no longer visible. Wash in 70% ethyl alcohol (2 changes) until diffusion of excess dye is no longer visible. Dehydrate in 95% ethyl alcohol and normal butyl alcohol, clear and mount.

Optimum staining of chromophilic material occurs at pH 3.65. Glial processes are well stained at pH 4.6. Nissl bodies and glial cytoplasm are purplish blue, nuclear chromatin is blue, background is clear at pH 3.65 but pale blue at pH 4.9.     

Staining the Nucleus in Yeasts

Yeast cells kept young by repeated subculturing were centrifuged, washed twice in distilled water and smeared on slides coated with a little egg albumen. The cells were treated with 0.002 M 8-hydroxyquinoline for 1 hr, fixed first in OsO, vapour for 30 sec and then in chloroform for 30 sec. The slides were passed through descending grades of alcohol, washed in distilled water, then immersed in 0.17 M NaCl solution for 2 hr. at 57°C. They were again washed in distilled water and later hydrolysed in 1 N HCl at 60°C for 5-7 min. This was followed by washing in distilled water and in buffer. The slides were then kept for 3 hr in Giemsa stain comprising 96 ml of phosphate buffer of pH 7.0 and 4 ml of the stain. After dehydration, mounting was done in balsam. Nuclei were brightly stained and well differentiated; centrosomes were clear, and the process of nuclear division and movement to daughter cells could be studied. Pretreatment with 8-hydroxyquinoline increased the viscosity of the cytoplasm, while NaCl treatment and acid hydrolysis led to the complete removal of ribonucleic acid and basophilic material. A selective staining of chromatin was thus achieved. Structures resembling chromosomes could be seen when fixed and stained cells were squashed, soon after the removal of the slides from the stain, under a cover glass by applying uniform pressure with a rubber stopper. Fixation in osmic acid vapor and chloroform followed by acid hydrolysis and staining in leucobasic fuchsin also helps to obtain bright staining of the nucleus; however, the preparations are inferior to those obtained after 8-hydroxyquinoline, NaCl treatment and Giemsa staining.     

Lower azure B methylene blue ratios in Giemsa type blood and malaria stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure : methylene blue : eosin ratio of about 6 : 3 : 6.3 : 10; Lillie's 1943 formula had a 5 : 7 : 10 ratio. In the current series of tests 5 : 7 : 10 (I), 4 : 8 : 10 (II), 3 : 9 : 10 (III), 2 : 10 : 10 (IV), 1 : 11 : 10 (V), and 0 : 12 : 10 (VI) were used. Malaria and blood stains were better than the standard 5 : 7 : 10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10--30 min were used.     

A New Method Using a Modified Mayer's Hemalum at pH 6 for Demonstrating Mucous Neck Cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

A new method using a modified Mayer's hemalum at pH 6 for demonstrating mucous neck cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

Lower Azure B Methylene Blue Ratios in Giemsa Type Blood and Malaria Stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure:methylene blue: eosin ratio of about 6:3:6.3:10; Lillie's 1943 formula had a 5:7:10 ratio. In the current series of tests 5:7:10 (I), 4:8:10 (II), 3:9:10 (III), 2:10:10 (IV), 1:11:10 (V), and 0:12:10 (VI) were used. Malaria and blood stains were better than the standard 5:7:10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10-30 min were used.     

Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

Human chromosome banding with raw extract of fruits or leaves of papaya

One week old human chromosome preparations were treated with filtrate from one liquefied leaf (53 g) of papaya (Carica papaya) in 100 ml of distilled water, and stained with 1.5% Giemsa (pH 6.8). Good chromosome banding was obtained after 2 min of treatment. Solutions that have been frozen even for years are effective and the method is cheaper and easier than others.     

Light and scanning electron microscopic observations on the two contrasting types of sister chromatid differential staining after ultraviolet light irradiation

Chinese hamster cells were grown with 50 M 5-bromodeoxyuridine (BrdU) during the penultimate S phase to obtain chromosomes with the TB-TT chromatid constitution. Chromosome preparations made by the air-drying method were used to study the sister chromatid differential staining (SCD) resulting from ultraviolet (UV) irradiation followed by Giemsa staining by light and scanning electron microscopy (SEM). When chromosomes irradiated with UV light (253.7 nm, 5.2 J/m²/s) for more than 5 h were stained with 1% to 4% Giemsa in phosphate buffered saline (PBS) or in distilled water, the resulting SCD invariably belonged to the B-light type in which the TB-chromatid stained lightly. SEM observations of these chromosomes suggested that the B-light SCD was due to the selective photolysis of the TB-chromatid. On the other hand, when chromosomes were irradiated for only 10 min, and stained with 1% Giemsa in PBS, they showed a B-dark type SCD in which the TB-chromatid stained darkly. However, when chromosomes irradiated for 10 min were stained with 4% Giemsa in PBS or 1% Giemsa in distilled water, the resulting SCD again belonged to the B-light type. These findings indicate that when the irradiation dose is small, the resultant SCD is not a simple reflection of selective photolysis in the TB-chromatids and the type of SCD depends not only on the concentration of Giemsa but also on the salinity of the staining solution.     

Display of mitochondria in root tip cells

A method for displaying mitochondria and proplastids in root tip sections of Tradescantia paludosa and cereals was modified from Altmann and Volkonsky. Root tips were fixed in 3% glutaraldehyde in phosphate buffer, pH 7.1, or acetate buffer, pH 4.8, for 3 hr, rinsed and postchromed overnight in 3% potassium dichromate, all at room temperature (20 C), dehydrated through a tertiary butanol series and embedded in ester wax. Four-micrometer sections were stained in hot acid fuchsin in aniline water, rinsed, treated with 1% sodium phosphomolybdate for 30 sec, rinsed and stained progressively with azure B for 3-10 min before being made permanent. Mitochondria and proplastids were stained brilliant crimson against a light blue cytoplasm with deep blue chromosomes. Previously reported difficulties with Altmann staining techniques are attributed to the erratic action of the classical fixatives used.     

On the mechanism of differential giemsa staining of BrdU-substituted chromatids

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocyte spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with Giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.     

The Demonstration of Beta Cells in Pancreatic Islets and Their Tumors

The stain is applied routinely to tissues fixed in 10% buffered formalin (pH near 7.0) or in Bouin's fluid. Bring paraffin section to water as usual and mordant 72 hr in 5% CrCl₃ dissolved in 5% acetic acid. Wash in water and in 70% alcohol and stain 6 hr. Formula of staining solution: new fuchsin, 1% in 70% alcohol, 100 ml; HCl, conc., 2 ml and paraldehyde, 2 ml, mixed together and added to the dye solution; let stand 24 hr before use. After staining, wash in running tap water 5-10 min, rinse in distilled water and counterstain if desired. Dehydration in alcohol, clearing and covering completes the process. When the paraldehyde is obtained from a freshly opened bottle, standardized staining times can be used and thus eliminate the necessity of differentiating individual slides. The granules of beta cells stained deep blue to purple and were demonstrated in the pancreatic islet of man, dog, mouse, frog, guinea pig and rabbit.     

Improved Nuclear Staining with Mordant Blue 3 as A Hematoxylin Substitute

For progressive staining 1 g mordant blue 3, 0.5 g iron alum and 10 ml hydrochloric acid are combined to make 1 liter with distilled water. Paraffin sections are stained 5 minutes, blued in 03% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely resemble a good hematoxylin and eosin.     

Romanowsky-Type Staining: Effect of Adding Acid Dyes, Particularly Chromotrope 2R

Nuclear staining can be depressed and nucleolar staining accentuated by the addition of 1 part of 1% chromotrope 2R to 9 parts of Gurr's Improved Giemsa R66; this mixture then being diluted -with an equal quantity of distilled water and allowed to stand for 0.5-1 hr before use. Methanol-fixed smears of Walker rat carcinoma, used as test material, were stained 5 min. Experiments with undiluted and diluted Giemsa indicate that ionisation of the azure-eosin complex is necessary for the development of the metachromatic red nuclear color of Romanowsky-type staining. It is suggested that chromotrope 2R depresses this ionisation.     

Cytohistological and phytochemical study of madder root extracts obtained by ultrasonic and classical extractions

Introduction – Madder (Rubia tinctorum) has been used since ancient times as a source of pigments for dyeing and painting. Madder dyes are localised in roots and the native chemical population is composed of glycosiled and aglycone compounds. The aim of this study is to elaborate an efficient extraction process without any chemical denaturation of dyes. Objective – To compare an optimised ultrasonic process, using for madder dye extraction, with two conventional procedures and to determine the efficiency of ultrasound on these vegetable matrix. Methodology – Madder roots were extract in a methanol–water mixture in 37 : 63 (v/v) for ultrasound and 80 : 20 (v/v) for reflux and agitation. HPLC‐PAD analyses showed the anthraquinone proportion for each extraction process and their denaturing effects. Finally, cytohistological observations were made to show the consequence of each process on the cell organisation in madder roots. Results – The results showed that the amount of extracted dyes was higher with UAE than with agitation and reflux. HPLC‐PAD analysis revealed that the anthraquinone composition differed according to the extraction procedure. The UAE extracts presented an important richness in terms of anthraquinonic compounds that suggests a preserving effect. Cytohistological observations showed that the main alterations concerned the cell walls of phloem. After UAE the walls exhibited numerous pitted areas reflecting an ultrasound‐induced cavitation that enhances the extraction effectiveness of this method. Conclusion – The study has shown the improvement of madder roots extraction both quantitatively and qualitatively using the efficiency of ultrasound‐assisted extraction in comparison with magnetic agitation and reflux techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

A Simple Method for Histochemical Demonstration of Viral Inclusion Bodies in Cell Monolayers in Microtitration Plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.     

A simple method for histochemical demonstration of viral inclusion bodies in cell monolayers in microtitration plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.     

Topographic examination of sister chromatid differential staining by nomarski interference microscopy and scanning electron microscopy

BrdU-substituted Chinese hamster chromosomes were treated with a hot Na₂HPO₄ solution and stained with Giemsa to produce sister chromatid differential staining (SCD). The process of SCD was examined with the Nomarski differential interference microscope and the scanning electron microscope. After the Na₂HPO₄ treatment alone, unifilarly BrdU-substituted (TB) chromatids appeared somewhat more severely collapsed than the bifilarly substituted (BB) chromatids. Subsequent Giemsa staining, however, brought about pronounced piling up of the Giemsa dye on the TB-chromatids but not on the BB-ones, causing highly distinct differential Giemsa staining as well as a marked differentiation in surface topography between the sister chromatids. Removal of the Giemsa dye from the differentially Giemsa stained chromosomes resulted in a disappearance of such a pronounced topographic differentiation.