Since the earliest years see Krafts, this issue , Nature of the dyes in Romanowsky and still today, however, nearly all laboratory work- stains ers have used polychromed methylene blue as a source of azure B rather than the pure compound.
Early investigators, using spectroscopic analy- Polychrome methylene blue is a mixture of bluish Biotech Histochem Downloaded from informahealthcare. This view was confirmed using dyes derived blue by oxidative treatments such as exposure to from specific syntheses, followed by chromato- atmospheric oxygen under alkaline conditions or graphic purification; for a summary, see Wittekind acidic dichromate solution as reviewed by Lillie et al.
These workers demonstrated that Exact compositions, and in particular the the only dyes necessary for complete polychrome amount of azure B present, varies among manufac- Romanowsky staining were the basic cationic turers and lots. This has been well and repeatedly dye azure B and the acid anionic dye, eosin Y. In documented e. For personal use only.
Structures of dyes discussed including azure B and eosin Y. The ionic species shown are those likely to predominate under routine Romanowsky staining conditions. Only single resonance forms are shown.
Despite such techni- lation and eventually deamination of the tetram- cal variation, it is striking how many laboratories ethylated methylene blue parent compound.
Most are not carrying out Romanowsky staining in the resulting dyes, including azure B, are expected to precise sense, because their published works show be cationic under Romanowsky staining conditions cells with blue, not purple, nuclei.
Indeed, many and all compounds retain the fused tricyclic het- workers in reputable institutions are content to erocyclic ring structure. Dyes of standardized and traditional stains Halogenated-fluorescein acid dyes that have been used or investigated for use in Romanowsky Only azure B plus various halogenated fluoresceins Biotech Histochem Downloaded from informahealthcare. The sizes of the conjugated sys- the color purple.
Other components of polychrome tems and lipophilicities vary slightly as summarized methylene blue fail to either give the color purple in Table 1.
Analytical studies have shown that many or provide contrast between nucleus and cyto- commercial lots comprise mixtures of the nominal plasm. Methylene blue itself does not give purple dye with incompletely halogenated impurities that nuclei, but does provide sky blue basophilic cyto- are of lower lipophilicity Marshall and Lewis , plasms. Consequently, two types of standardized Marshall Romanowsky stains have been developed. The first are two-dye mixtures of azure B plus Technical features of Romanowsky eosin Y.
One such example, using azure B isothio- For personal use only. These differences probably B chloride plus the more widely available sodium arose from efforts to optimize staining when using eosinate Marshall et al.
The second type is dye lots that varied greatly in composition and, in a three-dye mixture, again containing azure B and eosin Y, but with methylene blue added Marshall et al.
The rationale was that when azure B Table 1. Some physicochemical features of dyes menti- is contaminated with other oxidation products, the oned in this review cytoplasmic staining becomes grayer and less con- trasted with purple nuclei; however, this is com- Dye properties1 pensated for by the blue staining dye.
The pure dye experiments coefficient. This dimethylsulfoxide. Both solvents, when diluted with polychromasia is used to distinguish populations buffer in the working solutions, permit generation of morphologically related cells, especially for iden- of the color purple. Dimethylsulfoxide, however, tification of the cells in blood and marrow smears Biotech Histochem Downloaded from informahealthcare.
Also, the color purple is unstable Another key feature is that considerable inten- in the presence of high concentrations of the lower sification of this purple staining, without over- alcohols, methanol and ethanol, as reported at least staining of blue and red colors, can be achieved as early as Lillie This is one reason in a technically straightforward way, e.
Such cell nuclei; they still used the traditional alcoholic intensification facilitates detection of small cells dehydrating agents e. These and cellular components near the limit of normal problems can be avoided by using isobutanol or iso- optical resolution. Examples include chromosome For personal use only. G-bands, neutrophil and platelet granules, and The color purple also is removed quickly by dilute bacterial spores.
Not only can such polychrome staining with In fact, the effects of pH are marked regardless intensification be obtained in a technically simple of the nature of the acid involved. The relation of way two dyes and a single staining solution , but pH to acidophilic—basophilic balance, and so to it can be achieved on films, smears, dabs etc. Erythrocytes, for paraffin or resin sections. A variety of fixatives example, become blue-green if the pH is too high. For a dramatic visual is pH sensitive.
After methanolic fixation, the color demonstration of the colored detail to be seen in often most is intense at pH , diminishes at pH Romanowsky stained resin embedded histology , and is completely inhibited at pH 3. Staining times Some physicochemical background Crucially, formation of the color purple follows an The physicochemical phenomena underlying the initial blue staining.
Preparations do not merely mechanisms of Romanowsky stains are considered become more intensely stained, they also become here in a general way. Not all phenomena discussed more purple as staining times are lengthened.
Acidic and basic dyeing mechanisms A simple way to conceptualize acid and basic dye- Fixation ing is to view it as an ion-exchange process Fig. Fixation also influences uptake of acid and basic Colored anions, e. Aldehyde the mobile inorganic anions to neutralize the fixed fixatives, e.
Colored cations, e. Acid and basic dyeing seen as ion exchange processes. Such a model accounts for both formation of dye—dye complexes such as occurs affinity, which as described is entirely an entropy with metachromasia, e. The formation of the depends only on the fixed charges on the biopoly- color purple, as described below, probably results mers, given that electrical neutrality of tissue and from the formation of a dye complex of a somewhat dye bath must be maintained.
Such an ion exchange picture also can explain the effects of pH on dye uptake. Consider cellular Acid dye-basic dye mixtures biopolymers carrying acidic and basic substitu- ents whose pKa values fall within the pH range of After mixing an acid anionic and basic cationic Romanowsky stains.
Their electrical charges vary dye in a single solution, one possible outcome is with pH, and as the charges vary, so will the ion the formation of an insoluble salt. All of the cat- exchange possibilities as illustrated in Fig. They have large do precipitate from aqueous solutions upon addi- aromatic systems that provide regions of both tion of eosin; the material precipitated contains hydrophobicity lipophilicity and polarizable the basic dye and acid dye in approximately electrons.
So acid and basic dyeing is not just ion molar ratio, which corresponds to neutral salts. First, there may be phobic effects between the hydrophobic species dye—biopolymer attractive forces such as those in aqueous solutions, such as the Romanowsky occurring between the polarizable aromatic rings staining solutions, which reduces precipitation. Second, The aqueous solutions mixed to produce such the hydrophobic effects that arise in aqueous milieu azure eosinates contain dye at high concentrations when both dye and biopolymer have hydrophobic and precipitation is rapid.
Dye concentrations regions can come into contact. Both of these pro- in staining solutions are lower and precipitation cesses are involved in dye binding that involves the is slower. This occurs first in the Fig. Effect of changes in pH on the ionic status of thinner portions of the preparation Wittekind , biopolymers, thus on uptake of acid and basic dyes.
Horobin et al. It is intriguing that similar rate effects also have been reported in cell-free DNA films of varying thickness Friedrich et al. The porosity of some acidophilic For personal use only.
Gregory and Maher The molar ratio of the basic and acid dyes in the precipitate is approxi- entities, typically protein or protein-rich, also has mately and the presence of excess acid or, been assessed using the refractive index as a mea- especially, basic dye tends to reduce precipitation sure.
Relative porosity fell in the sequence: colla- Lillie In keeping with a diffusion rate control model, staining rates also fall into this sequence Consequences of differential staining rates Horobin , cited in Horobin Two dye binding sites with equal affinity for a dye can nevertheless be colored differentially if they stain Rate effects technically produced at different rates.
In progressive staining, the site that binds dye fastest becomes selectively stained Morphology is influential in this case also. Thus after short periods of staining. If a preparation sections of bone marrow Wittekind , Wittekind is over-stained, then de-stained, sites slowest to de- et al. Moreover, as considered in more detail stain retain dye longest and so become selectively later, resin sections stain more slowly than paraffin colored. Note that similar outcomes could follow if sections.
By analogy, in the thicker portions of cell some cellular targets have a higher density of bind- smears, the nuclei stain blue, not purple Boon and ing sites than other targets with the same affinity for Drijver Formaldehyde fixation also influences dye. For reasons of simplicity, however, this is not retards the rate of formation of the color purple stated repeatedly.
Large dyes typically stain structure or remove the water completely such slower, perhaps a direct effect of particle size Seki as when lower alcohols are used for dehydration. Taking the rela- as a major contributor to the dye-dye binding tive molecular masses of the dyes as an indication involved in formation of the color purple, although of size, eosin Y is almost three times the size of in their picture the DNA formed part of the com- azure B, so the latter may be expected to stain con- plex Friedrich et al.
Because the conditions of siderably faster. The color purple produced within biological speci- Finally, note that additional azure B-eosin mens following azure B—eosin staining has an complexes may exist. For example, in strongly aci- Biotech Histochem Downloaded from informahealthcare. An alternative view, which regarded this This genome Woynarowski et al.
Examples of biological sites where Substrate selectivity for formation of the the color purple arises include chromatin and gran- color purple ules of neutrophils and platelets in blood smears and tissue sections, in the bands of G-banded chro- Whatever the origin of the color purple, whether it is mosomes, and in collagenous connective tissue and a complex or merely an azure eosinate salt, its sub- mucus granules of tissue sections.
Why, for example, is DNA-rich chromatin purple, mation of the color purple as resulting from com- but not the adjacent RNA-rich nucleoli? Although plex formation, the specific interactions between such questions cannot yet be answered definitively, the dyes, and perhaps also the biosubstrate, still are contributing possibilities can be proposed.
This concept was adopted specimen. Therefore, both affinities for and rates by Wittekind who noted the possible role of uptake into a substrate could control selectiv- of hydrogen bonding between the N—H groups of ity.
Rate effects certainly play a part. Chromatin, azure B and eosin for facilitating electron transfer. At the The latter feature would account for lack of purple stage of staining when chromatin becomes purple, staining when methylene blue is used with eosin basophilic cytoplasms still are blue, but if staining Y and its occurrence when azure A, with two N—H times are prolonged sufficiently, these cytoplasms groups, is used; however, the question of why less also become purple.
With extremely extended methylated dyes, e. Horobin and Walter The causes of such rate Wittekind also considered that azure B effects are varied and are considered as appropri- and eosin are associated in part due to hydrophobic ate for each of the staining applications, e. This markedly non-uniform staining inhibition Almost a century after the introduction of the poly- is due to non-uniform resin infiltration.
These properties cause resin to be objects such as the submicrometer lysosomes of largely absent from dense structures, e. Other staining of … nuclei and chromosomes with Giemsa regions, such as nuclear chromatin and the cyto- and similar mixtures are far greater than those nor- plasmic matrix, though not the small, dense ribo- mally attained with … methylene blue … perhaps somes, are infiltrated well by the resin Horobin Giemsa is not dyeing chromosomes in the normal , Gerrits et al.
It may be noted thiazines to eosin to form a precipitate, thereby free- that resin inhibition can arise from both a reduction ing the reactive groups on the DNA to bind more of free volume available for dye diffusion and dye- thiazine. In the context of staining blood smears straightforward.
A small, weakly hydrophilic dye and other complex tissues, one can, in fact, replace For personal use only. Access by the three times mers, e. Such a generalized template inhibited significantly in resin-containing structures effect mechanism is illustrated in Fig. As precise chemical nature of the azure B-eosin com- an example of the resulting staining outcomes, con- plex is not specified. This staining is blue In smears, represented by the template intensification effect, because the dense ribosomes impede entry of the consider the views of two well known figures in his- eosin Y.
In resin sections, however, because ribo- tochemistry and biotechnique. John R. Baker, in his somes are dense, they are, unlike the cytoplasmic classic monograph, considered that an azure—eosin matrix, poorly infiltrated.
Because routine staining complex was unlikely to transfer from solution onto times for resin sections are necessarily longer or DNA; however, he had no suggestions regarding staining temperatures higher than for smears, eosin what did happen Baker Moreover, Ralph D. Effects of resin embedding media Chemical and physical effects of fixation Resin plastic embedding media have an inhibitory Changing the fixative used could influence influence on dye uptake when sections are stained Romanowsky staining in two ways.
First, chemically, with the resin still present. For example, staining e. This has been observed heated Horobin and Boon It is not merely to lead to preparations with an overall bluer color- that many structures stain somewhat slower in resin ation Wittekind Intensification of the Romanowsky effect with continued staining seen as a template effect.
Note that the precise relation of azure B to eosin is not defined. These differ- scopic level, whereas aldehyde fixation produces a ences are outlined below. Empirically, alcoholic fixatives produce speci- Specimen pretreatment mens that tend to stain faster than formalin fixed preparations. Where two dyes of different sizes are Fixation of cell smears for hematology or cytol- used simultaneously, faster staining typically favors ogy routinely use methanol.
Chromosome spreads entry of the larger dye. Either of these mechanisms usually are prepared using methanol-acetic acid. If staining times are lengthened or eosin bind- Chromosome banding uses various pretreatments ing is enhanced by lowering the pH of the dye bath of the fixed chromosome spreads prior to staining Lillie , the purple staining is restored.
It has in addition, e. Hematological or cytological preparations are cell monolayers produced as brushings, dabs, smears, Staining mechanisms of Romanowsky spins etc.
For diagnosis of protozoal parasitic and related stains infections, thick, i. Cytogenetic chromosome spreads While all methods use the typical Romanowsky are monolayers of chromosomes. Glycol methacrylate swollen character of GAG-rich lysosomes in sections are stained with the resin in situ; these are partially aqueous media. Failure to stain pur- the eponymous resin sections.
Sections are typi- ple, as with the lysosomes of monocytes, also cally 0. The easy visibility of the purple staining of Staining conditions tiny granules, which occurs only if the batch of Romanowsky stain used contains sufficient Blood and marrow monolayers, e.
Stain- template effect. The staining of smears and sections, and in particular resin sections, Banding of chromosome spreads has on occasion been accelerated considerably by heating the solutions, sometimes with a microwave Several distinct phenomena need explanation. Why Biotech Histochem Downloaded from informahealthcare. Section staining some- do the chromosomes stain, and in particular, why times uses regressive procedures with differentia- do they stain purple?
And how is it that these tiny Wittekind et al. To retain the color purple, structures are stained so intensely as to be notice- however, contact with acetic acid differentiation able? To address these questions, four aspects of solutions must be minimized, and dehydration must mechanism are discussed below. The first step involves basic dyeing of DNA by drying or solvents such as butanol or isopropanol. Mechanistic summaries of each general method- 2.
The blue staining subsequently is converted to ology are given below. Note that if the chromosomes are covered with incompletely dispersed cytoplasmic material, Blood, bone marrow and cytological smears the entry of the larger eosin ions is retarded Explanations for development of the full and this step is inhibited Sumner Romanowsky polychromasia will be considered 3.
The question of selectivity is more conten- below. The specific issues are: why do any dyes tious. Various suggestions have been made to bind, and in particular, why do some substrates explain why dye binding favors certain lim- stain red, some blue and some purple? Stains can be either applied manually with the aid of cuvettes, or mechanically in automated staining systems. Romanowsky stains are neutral stains composed of a mixture of oxidized methylene blue azure dyes and eosin Y.
Romanowsky staining is the prototypical cytological staining method. Changes in the methodology have lead to the evolution of other linked staining methods. Buffer is essential to enable the dyes to precipitate out of the solution and bind to sample material. The azures are basic dyes that bind acid nuclei and result in a blue to purple color.
The eosines are acidic dyes that are attracted to the alkaline cytoplasm, producing red coloration. They generally provide excellent nuclear detail and also clear differentiation of the cytoplasm. The alcohol based 'fast' staining kits such as Diff-Quik are readily available and commonly used in practice.
Uses Bone marrow examination. Blood films. Stain cells to identify abnormalities in morphology and pathological changes. Advantages Easy to prepare. Readily available. Disadvantages Nuclear and nucleolar detail not as defined as Papanicolaou stain. Is adequate to distinguish neoplasia from inflammation. Can get variation in coloration with a change in staining times and pH.
Merck Millipore Manual. Website: www. Longman Singapore Publishers Pte Ltd. Osteoporosis: lateral radiograph. Endometrial aneurysm. Ovarian neoplasia. Epizootic enteropathy. Ovarian cysts. Anorectal papilloma. Scrubbing in. Damaged clipper blade. Closed gloving Open gloving Surgical asepsis and scrubbing.
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