A hematoxylin staining procedure for maize pollen grain chromosomes

Studies of postmeiotic chromosome behavior have been impeded by the thick exine and abundant starch grains of maize pollen. Staining pollen grain chromosomes with acetocarmine is tedious and gives inconsistent, often unsatisfactory results. A hematoxylin stain, used in conjunction with the clearing agent chloral hydrate, has been successfully used by the authors to stain chromosomes, nuclei and sperm cells of the maize pollen grain. An ethanol-formaldehyde fixing fluid is used to fix and preserve the pollen samples. The procedure, which is rapid and simple, gives excellent preparations with both fresh and fixed material. Stained preparations do not get darker with time, as is typical of other hematoxylin stained materials.     

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

Effects of additives on alum hematoxylin staining solutions.

All additives tested (ethyl alcohol, glycerine, chloral hydrate, ethylene and propylene glycol, and citric, malonic and maleic acids) in varying degrees limited the conversion of hematein to insoluble compounds. Peak absorbances increased slightly in hematoxylin solutions containing citric, malonic and maleic acids, but decreased with other additives, and in controls. After four months storage the absorbance in all solutions increased about 50%, acidity increased and staining effectiveness increased.     

Block staining of mammalian tissues with hematoxylin and eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied. General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

A simple metachromatic and fluorescent staining method for microorganisms using carbocyanine dye

The cationic carbocyanine dye, 1-ethyl-2-[3-(1-ethylnaphtho[1, 2d]-thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1, 2d]thiazolium bromide, interacts with several classes of anionic polymers, exhibiting metachromasia. We were able to stain various kinds of microorganisms with this dye. Gram-negative bacteria were stained reddish purple, while Gram-positive bacteria were stained violet or bluish purple. Stains of molds were of various colors. Yeast vegetative cells were stained reddish purple, but zygotic asci were bluish. Chlamydia trachomatis inclusions, which are surrounded by cytoplasmic membranes, were also stained red. Microorganism and cell stains have different features and can be identified also by use of fluorescent microscopy. The new staining method we report here is rapid and simple enough for routine microscopical examinations of smears of clinical specimens including microorganisms.     

A simple fluorescence of staining technique for in situ soil microorganisms.

A simple, rapid straining technique using the magnesium salt of 1-anilino-8-naphthalene sulfonic acid is described. Treatment of soil with an aqueous, membrane-filtered solution (3.5 mg/ml) of the salt causes the soil microorganisms to fluoresce when examined with light from a mercury arc light source.     

Delineation of human chromosome contour by heat treatment and hematoxylin staining

An unusual form of staining was observed as a result of heat treatment: chromosome contour became easily recognizable. Chromosome contour is optimally delineated by heat treatment of metaphase preparations in 0.06 M phosphate buffer, pH 6.8–7.0 at 95° C for 10 min, and subsequent staining with Heidenhain's hematoxylin. This result is obtained by a process similar to that in which Giemsa has been reported to stain particular foci considered to represent selective staining of constitutive heterochromatin. Mordanting with 3% ferrous ammonium sulfate produced maximum staining. However, hematoxylin (Harris) also stained the periphery of chromosomes. A transitional temperature existed between 70° C and 80° C in which contour became evident. When metaphase preparations were heated at 90–95° C, contour occurred on some chromosomes within 3 min and was demonstrated in 100% of the chromosomes analysed after 10 min of heat treatment.Supported in part by a grant to C. Romero-Sierra from the Defence Research Board of Canada (DRB 9350-24) and to E. A. MacKinnon from the Medical Research Council of Canada (MA-4135).     

Improved nuclear staining with mordant blue 3 as a hematoxylin substitute.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% 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 results very resemble a good hematoxylin and eosin.     

Method of total staining of monolayer cell cultures using vanadium hematoxylin]

Original method of cytological staining for monolayer cell cultures and outgrowth zone of tissue explants with vanadium hematoxylin is described. This staining method is simple, universal, reproducible and give opportunity to stain rapidly practically all cellular elements of cultivated monolayer with high degree of cytological resolution of intracellular structures (nucleus, cytoplasmic organelles etc.).     

A new principle in polychrome staining: a system of automated staining, complementary to hematoxylin and eosin, and usable as a research tool

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 millimicrons acrylic sections.     

A simple and highly reproducible staining method for fungi and other polysaccharide-rich microorganisms in animal tissues

A newly devised, simple and highly reproducible method for fungal staining is reported. Grocott's method, in which methenamine-silver nitrate solution is employed, has been widely used for the staining of fungi in tissue sections, but it frequently produces heavy background staining because of sudden and progressive reaction in the methenamine-silver nitrate solution. We therefore replace the latter solution with an ammoniacal silver nitrate solution. This new method yields more consistent results in fungal staining without background staining, since the reaction time in the ammoniacal silver nitrate solution is prolonged. The present method is considered superior to Grocott's method with regard to its simplicity and reproducibility.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.