Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

Mechanisms of Giemsa banding

Summary We investigated the capability of individual thiazins in Giemsa mixtures (methylene blue and azures A, B, and C) and of two related dyes (toluidine blue and thionin) to produce G-banding. We further tested the effects of variations of buffer composition and concentration, dye concentration, and staining time.G-banding was produced by all of the dyes at low concentrations, although differences were noted. Overall, methylene blue and azure B produced the best banding, azures A, C, and toluidine blue produced moderately good banding, and thionin produced poor banding. This order did not appear to be altered essentially by different treatments. The optimal conditions for G-banding for all dyes and treatments included the use of (1) 0.025–0.05M phosphate buffer, (2) dye concentrations of 0.002%–0.005%, and (3) staining times of 6–15 min.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 4. Binding of azure B to DNA

We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (CF) and the nucleic acid (CN) using absorption spectroscopy [CF and CN represent the total concentrations of the ye (F) and the mononucleotide units (N) of the DNA, respectively]. The binding isotherms of the dye to DNA in aqueous solutions were determined. In addition, we analysed the composition of insoluble DNA/azure B precipitates that are formed in presence of an excess of azure B. These precipitates are of particular interest, because Giemsa staining is usually performed using high dye concentrations. Azure B easily forms dimers in aqueous solutions. When determining the binding isotherms, the equilibrium between free monomers and dimers must be taken into account. Therefore, we determined the dimerisation constant (Kd) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T = 298 K (25 degrees C), Kd = 6.5 X 10(3) M-1 (experimental conditions: tris buffer, pH 7.2; concentration of Na ions, CNa = 0.002 M). As the CNa value increases, the dimerisation constant rises rapidly. When the azure B concentration is very low and there is an excess of DNA, ordinary Scatchard and Langmuir isotherms are observed. Monomer dye cations are bound to DNA, these cations being in equilibrium with free monomers in the solution. In order to obtain the Scatchard binding constant (Ks) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (epsilon Fb) of the monomer bound (b) dye molecules (F) at one analytical wave number (upsilon a). The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, CN----infinity. We obtained Ks = 1.8 X 10(5) M-1 and n = 0.18 (25 degrees C; tris buffer, pH 7.2; CNa = 0.002 M). At very low dye (CF) and competitor (CNa) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing CNa values the concentration of bound azure B cations decreases rapidly. The Na cations displace the bound dye cations and act as a competitor. The Ks value also greatly depends on the competitor concentration (CNa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Oblique banding pattern in collagen fibrils reconstituted in vitro after trypsin treatment.

Collagen fibres from rat tail tendon suspended in small pieces in a solution (pH 7.8) containing 0.5 M CaCl2 were treated with purified bovine trypsin at 20 degrees C for 20 h. After the enzyme treatment collagen from this solution was precipitated out and reconstituted in vitro into native-type fibrils. The banding pattern in these reconstituted fibrils was found to be oblique. This is comparable to that observed recently in fibrils reconstituted from cartilage collagen. On the other hand, normal transverse banding pattern was observed in the fibrils reconstituted in vitro from collagen solution of rat tail tendon which was not pre-treated with trypsin. No significant change was, however, observed in the segment long spacing fibrils precipitated from the enzyme-treated collagen solution. It is possible that the enzyme might affect the mode of organization of tropocollagen molecules during in vitro fibrillogenesis into native-type fibrils either by interacting with the "telopeptide" regions or with the non-collagenous components associated with the native protein and this could probably result into the formation of fibrils with oblique banding pattern.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : V. WHY DOES AZURE B PENETRATE MORE READILY THAN METHYLENE BLUE OR CRYSTAL VIOLET?

Glass electrode measurements of the pH value of the sap of cells of Nitella show that azure B in the form of free base penetrates the vacuoles and raises the pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, but the dye salt dissolved in the sap does not alter the pH value of the sap. It is concluded that the dye penetrates the vacuoles chiefly in the form of free base and not as salt. The dye from methylene blue solution containing azure B free base as impurity penetrates and accumulates in the vacuole. This dye must be azure B in the form of free base, since it raises the pH value of the sap to about the same extent as the free base of azure B dissolved in the sap in vitro. The dye absorbed by the chloroform from methylene blue solution behaves like the dye penetrating the vacuole. These results confirm those of spectrophotometric analysis previously published. Crystal violet exists only in one form between pH 5 and pH 9.2, and does not alter the pH value of the sap at the concentrations used. It does not penetrate readily unless cells are injured. A theory of "multiple partition coefficients" is described which explains the mechanism of the behavior of living cells to these dyes. When the protoplasm is squeezed into the sap, the pH value of the mixture is higher than that of the pure sap. The behavior of such a mixture to the dye is very much like that of the sap except that with azure B and methylene blue the rise in the pH value of such a mixture is not so pronounced as with sap when the dye penetrates into the vacuoles. Spectrophotometric measurements show that the dye which penetrates from methylene blue solution has a primary absorption maximum at 653 to 655 mµ (i.e., is a mixture of azure B and methylene blue, with preponderance of azure B) whether we take the sap alone or the sap plus protoplasm. These results confirm those previously obtained with spectrophotometric measurements.     

Studies of Chromosome Banding with Giemsa in Vicia faba and Allium cepa

Giemsa dye is a complex mixture containing methylene blue, its oxidation products-azure Ⅰ, Ⅱ, Ⅲ, and their eosinate. The results of our experiments have demonstrated that staining with methylene blue alone can give a faint trace of banding as well as azure Ⅰ, Ⅱ. No bands are obtained with eosin. Nevertheless, good chromosome bandings can be often produced by staining with methylene blue-eosinate or azure Ⅱ-eosinate. These data indicate that eosinate has an important effect for the formation of C-banding on plant chromosomes. In our experiments, the treatments of chromosomes with trypsin or papain have also resulted in good C-banding pattern when slides are stained with Giemsa. We found that the slides untreated with proteinase showed homogeneous intense chromosome staining and, on the contrary, the slides treated with proteinase led to palestaining chromosomes and presenting bandings. It has shown that proteinase, especially trypsin, not only can remove a large amount of chromosomal protein but also can remove DNA and results in C-bandings. Treated properly with trypsin and followed by the Feulgen staining, chromosomes can also produce the C-bandings, but chromosomes treated overtime with trypsin are stained more palely in Feulgen reaction or lead to colourlessness. The above results have further proved that trypsin technique removes large amounts of chromosome DNA and removes less from the C-band regions than from the non-band regions. In this paper we mainly discussed the effects of protein on mechanism of plant chromosome banding. We consider that the production of plant C-banding is probably due to the differential accessibility of nucleoprotein between euehromatin and heteroehromatin regions. It brings about selective removal of nucleoprotein from the chromosome arms. We have compared the effect of trypsin with papain and pepsin on producing bands. Good bands are produced by Giemsa staining chromosomes with trypsin, but no bands are obtained by staining chromosomes treated with pepsin. So the results have expressed that histones are possibly playing more important role in C-bandings.     

Counterstain-enhanced chromosome banding

Summary Chromosome staining, in which at least one member of a pair or triplet of DNA binding dyes is fluoescent whereas the others act as counterstain, is reviewed. Appropriately chosen combinations of fluorescent dyes and counterstains can be employed to enhance general chromosome banding patterns, or to induce specific regional banding patterns. Some pairs of dyes which exhibit complementary DNA binding specificity, A-T/G-C or G-C/A-T, provide enhanced definition of positive or reverse banding patterns. Dye combinations of the type A-T/A-T, that include two DNA stains with similar specificity but non-identical binding modes, produce a specific pattern of brightly fluorescnet heterochromatic regions (DA-DAPI bands). In man, the method highlights the C bands of chromosomes 1, 9, 15, 16, and the Y. Certain dye triplets of the type G-C/A-T/A-T, which include two spectroscopically separated fluorescent stains with reciprocal DNA base pair binding specificites and a non-fluorescent A-T binding counterstain, can be used to highlight selectively, in the appropriate wavelength ranges, either R bands or DA-DAPI bands.Applications of these techniques in human cytogenetics are described. The potential of the new methodology for detecting and analysing specific chromosome bands is demonstrated. The mechanisms responsible for contrast enhancement and pattern induction are reviewed and their implications for chromosome structure are discussed as they relate to the banding phenomenon and to the DNA composition of chromosomes.     

Plasma membrane domains participate in pH banding of Chara internodal cells

We investigated the identity and distribution of cortical domains, stained by the endocytic marker FM 1-43, in branchlet internodal cells of the characean green algae Chara corallina and Chara braunii. Co-labeling with NBD C(6)-sphingomyelin, a plasma membrane dye, which is not internalized, confirmed their location in the plasma membrane, and co-labelling with the fluorescent pH indicator Lysotracker red indicated an acidic environment. The plasma membrane domains co-localized with the distribution of an antibody against a proton-translocating ATPase, and electron microscopic data confirmed their identity with elaborate plasma membrane invaginations known as charasomes. The average size and the distribution pattern of charasomes correlated with the pH banding pattern of the cell. Charasomes were larger and more frequent at the acidic regions than at the alkaline bands, indicating that they are involved in outward-directed proton transport. Inhibition of photosynthesis by DCMU prevented charasome formation, and incubation in pH buffers resulted in smaller, homogenously distributed charasomes irrespective of whether the pH was clamped at 5.5 or 8.5. These data indicate that the differential size and distribution of charasomes is not due to differences in external pH but reflects active, photosynthesis-dependent pH banding. The fact that pH banding recovered within several minutes in unbuffered medium, however, confirms that pH banding is also possible in cells with evenly distributed charasomes or without charasomes. Cortical mitochondria were also larger and more abundant at the acid bands, and their intimate association with charasomes and chloroplasts suggests an involvement in carbon uptake and photorespiration.     

The Oxidation Products of Methylene Blue

The mechanism of the oxidation of methylene blue varies with the conditions. The formation of trimethyl thionin (azure B) and of asymmetrical dimethyl thionolin (azure A) is followed under alkaline conditions by that of dimethyl thionin (methylene violet) and under acid conditions by that of monomethyl thionin (named by authors azure C).

Simple and practical methods are given for the preparation of azure A and azure C. The latter product, which has not been obtained from methylene blue hitherto, has valuable staining properties as a nuclear and bacterial stain in tissue and may also be employed satisfactorily as a substitute for azure A in the MacNeal tetrachrome formula as a blood stain or substitute for the Giemsa stain.

Azure B has no particular merit in staining.

Azure C proves to be a very valuable stain. A procedure is given for its use with eosin Y and orange II as counterstains, by which it is possible to demonstrate bacteria in tissue and at the same time the cytological elements of the tissue.     

Endonuclease banding of isolated mammalian metaphase chromosomes

Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes. Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa. After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C-banding observed in fixed chromosomes exposed to the same treatments. Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes. In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions. Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA. From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin.     

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.     

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.     

Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears

Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).     

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.     

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy.

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.     

Early replication banding reveals a strongly conserved functional pattern in mammalian chromosomes

One of the best documented autosomal linkage associations in man is on chromosome 1p and in the mouse on chromosome 4. On mitotic chromosomes this genetic homology is shown more clearly by early replication banding (RBG; induced by incorporation of 5bromodeoxyuridine (BrdU) in the second half of the S phase) than by structural banding (induced on prefixed chromosomes by denaturation, RFA, or trypsin, GTG). To analyse this phenomenon in more detail, 11 chromosomal regions in man and the domestic cat with known genetic homology were compared. In four chromosome pairs RBG and GTG banding show the same degree of homology. In seven chromosome pairs the homology is more pronounced by RBG than by GTG banding. RFA banding does not reveal the same extent of homology as does RBG banding. These results clearly show a difference between the structural banding pattern, RFA and GTG, and the replication banding pattern, RBG. The following conclusions can be drawn: in chromosomal regions with homologous functions the DNA replicates in the same temporal order. Early replication banding (RBG) reveals a functional pattern in these regions which has been more strongly preserved during evolution than the underlying chromosomal DNA. Differences in chromosomal banding are most prominent in the GTG banding pattern, whereas similarities are most apparent in the RBG banding pattern.     

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.