Can azur-B eosin replace the May-Grünwald-Giemsa stain?]

A new method is described for staining blood and bone marrow smears. It is characterized by the presence of only two dyes, purified azure B and eosin in methanol, as stock solutions. Staining results are equivalent to those obtained by using the traditional dye mixtures according to May and Grunwald, Giemsa, Leishman or Wright. Different from these azure B-eosin staining can be standardized and is easier to be handled. Correlations between the azure-B-eosin and May-Grunwald-Giemsa (MGG) staining methods are briefly discussed.     

Stability study of Azure B, Eosin Y and commercial Romanowsky Giemsa stock solutions using high performance liquid chromatography

Summary The stability of Azure B and Eosin Y in stock solutions of the individual compounds as well as in mixtures of the two dyes was studied. The purpose of the study of these two essential constituents of the Romanowsky Giemsa stain, commonly used in cytology and histology, was to select a stable mixture as a definitive stock solution. Two specific high performance liquid chromatographic methods were used to monitor qualitative and quantitative changes in solutions. Several parameters influencing the stability of Azure B were examined e.g. the type of counter ion the presence of Eosin Y and the type of solvent used. The second part focused on the stability of Eosin Y in mixtures with different cationic dyes submitted to high temperatures. In conclusion, an Azure B SCN-Eosin Y acid mixture in dimethylsulphoxide (concentrations 0.75% and 0.12%, respectively) was selected as being the most appropriate composition of a stock solution for the Romanowsky Giemsa stain.     

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.     

On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azures B-Eosin Y stain as compared with other thiazine dye-Eosin Y combinations

Summary After incorporation into a polyacrylamide matrix, the biopolymers DNA, RNA, heparin, hyaluronic acid, collagen and the synthetic polymers poly(U) and poly(A, U) were stained with the pure thiazine dyes, Methylene Blue, the Azures and Thionin alone and combined with Eosin Y. Satisfactory spectrophotometric agreement was obtained between the staining reactions of the biopolymers in the artificial matrix and those in their natural surroundings. This was especially true with respect to the specificity of the Azure B-Eosin Y dye-pair, which is based on the generation, on suitable substrates, of a purple colour, the Romanowsky-Giemsa effect (RGE), with an absorbance maximum near 550 nm. In the model experiments, DNA, heparin, hyaluronic acid and collagen were found to be RGE-positive and poly(U), poly(A, U) and RNA RGE-negative.A theory of RGE is proposed which complies with the new and earlier observations: after saturation of available anionic binding sites and aggregate formation by Azure B, electron donor acceptor complexes are formed between Eosin Y and Azure B via hydrogen-bridge formation of the aminosubstituent proton of Azure B and between Eosin Y and the biopolymer surface. Charge-transfer complex formation may also account for the qualitative identity of Azure B-Eosin Y and Azure A-Eosin Y spectra of substrates, which are coloured purple. Quantitatively, Azure A-Eosin Y is less efficient in giving RGE. The generation of RGE is time-dependent. Equilibrium staining is attained after about 120 h. The implications of the results for the biological application of Romanowsky-Giemsa staining are discussed briefly.     

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May–Grünwald–Giemsa stained cutaneous tissue smear

Objective:  Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK).
Methods:  We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May–Grünwald–Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report.
Results:  Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted ( P  =   0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK.
Conclusion:  Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases.     

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital,Argentina

The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.     

Assessing HER-2 status in fresh frozen and archival cytological samples obtained by fine needle aspiration cytology

Determination of HER-2 status is an essential prerequisite in considering a patient's eligibility for anti-HER-2 therapy; few studies have focused on its evaluation on cytological material. We present a new method of assessing HER-2 status on cytological samples, by fluorescence in situ hybridization (FISH) with an automated detection system, using fresh frozen (FF) and May–Grünwald–Giemsa (MGG) stained smears, and we evaluate the reliability of HER-2 determination on fine needle aspiration cytology (FNAC). The pre-treatment protocol for FF smears is easier and faster than for MGG stained slides. However, with the described procedure, FISH is also feasible on archival MGG stained slides. We conclude that with this method, cytological samples obtained by FNAC, either FF or MGG, are a reliable option for assessing HER-2 status.     

The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.     

Thyroid fine needle aspiration: the morphological features on ThinPrep® slide preparations. Eighty cases with histological control

This study had several purposes: to define cytomorphological features of thyroid cells that might be modified by alcohol fixation; to optimize May-Grünwald–Giemsa (MGG) staining on ThinPrep^® (TP; Cytyc Inc., Bexborough, MA, USA) slides and to compare the diagnostic accuracy of slides prepared by a liquid-based method with those obtained by conventional technique. This study included 120 cases of ultrasound-guided fine needle aspiration (FNA) of the thyroid and 55 FNAs performed on surgically resected thyroid specimens. Histological control was available in 80 cases. In the first group of 120 FNAs, a split-sample technique was used for the TP. Three screenings were performed: first, an individual screening of the conventional smears (CS) and of the TP, a second screening to compare cells observed on the TP with the histological control and a third screening to assess the previously defined diagnostic criteria. Twenty-seven TP cases (22%) were considered unsatisfactory for diagnosis compared with 10 in CS (8%). The high rate of unsatisfactory cases with TP is likely to be due to the use of the split-sample technique. The sensitivity was 94% for CS and 81% for TP. The specificity was 67% and 60% for CS and TP, respectively. Two occult papillary carcinomas were missed by both methods. As for the MGG staining, the modified technique used for TP resulted in the same quality as the standard procedure. Conversely, TP did however induce uncommon morphological features. In this study, sensitivity and specificity levels are higher for CS than for TP; the difference may be explained by the fact that the methanol fixative used for TP induces some cytological alterations, especially in oncocytic tumours and lymphocytic thyroïditis.     

Studies on the relationship of human natural killer and lymphokine-activated killer cells with lysosomal staining and analysis of surface marker phenotypes

We have previously demonstrated that natural killer (NK) cells are lysosome-rich and stain more intensely with lysosomotropic agents such as neutral red and quinacrine (Qu) than do non-NK cells. In this study we combined the quantitation of Qu staining with surface marker staining to define subpopulations of NK cells. While all NK activity was contained within the Qu+ population, most but not all NK cells expressed the surface marker CD16. A subpopulation of NK cells was found to be Qu+CD16- composed of medium- to large-sized cells with a granular appearance on Giemsa staining. Culture with interleukin-2 (IL-2) induced enhanced cytotoxicity in peripheral blood lymphocytes (PBL) against NK-sensitive and NK-resistant tumor cells. Like NK cells, these lymphokine-activated killer (LAK) cells were predominantly Qu+CD16+. However, some LAK cells were Qu+CD16-. The Qu+CD16+ cells were typical large granular lymphocytes (LGL). The Qu+CD16- cells were also large lymphocytes, more than 50% of which were proliferating. However, the granulation in some Qu+CD16- cells, as detected by Giemsa staining, was more prominent and numerous than others in the same population. No LAK activity was ever detected in Qu- cells, which were uniformly small lymphocytes. Quantitation of Qu staining in effector cells was therefore demonstrated to have a good correlation with NK and LAK functions, and with surface markers can help to characterize both types of cells. Moreover, these results indicate that both NK and LAK populations include a small subset of CD16- cells in each.     

Polychromatic Stains for Thin Sections of Beta Embedded in Epoxy Resin

Thin sections of leaves and anthers of Beta vulgaris L., fixed in glutaraldehyde-OsO₄ and embedded in epoxy resin, were stained with different stains at pH ranges from 5 to 9 at 50 C to select those that provided polychromatic staining of suitable intensity. The thionin derivatives, Azure B, Toluidine Blue O, and polychrome Methylene Blue provided adequate staining, as did the commercially prepared stain Paragon PS 1301. Azure B stain was superior for sugar beet 0.5μ monitor sections: cytoplasm appeared grey; nuclei, blue-gray; nucleoli, blue; chloroplasts, blue-green; primary walls, blue; and secondary walls, light blue. Choice of one of the stains mentioned probably would depend upon the plant material under study.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40° C is recommended for stallion semen.     

Quantitative microspectrophotometry of RNA in plant tissue

Synopsis Chemical estimation of nucleic acid, essentially RNA, in fixed tissue from Jerusalem artichoke tubers, coupled with an examination of the types of RNA in the fixed tissue by gel electrophoresis, demonstrates that ribosomal and soluble RNA are preserved in this tissue after various fixation procedures including methanol, ethanol-acetic acid and aqueous formaldehyde. Tissue fixed in ethanol-acetic acid or formaldehyde is resistant to loss of nucleic acid by aqueous extraction but tissue fixed in all three standard fixatives loses nucleic acid in citrate buffer under conditions used for Azure B staining. The presence of Azure B in the buffer does not wholly prevent this loss. Tissue fixed in formaldehyde or mixed fixatives containing formaldehyde is resistant to loss of nucleic acid during treatment with EDTA to obtain cell suspensions.Preliminary experiments with Azure B, Gallocyanin chrome-alum and Methylene Blue showed that the Gallocyanin technique is the most practicable for demonstrating RNA cytochemically. Its specificity was confirmed by ribonuclease extraction of the tissue. Optimum staining conditions, requiring treatment of the tissue in Gallocyanin chrome-alum solution overnight at 40°C, are established for the artichoke tissue and for paraffin sections of pea shoot apices.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa.

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40 degrees C is recommended for stallion semen.     

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F₁ hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.     

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.     

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.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. I. Staining Properties of Commercial Samples and Their Component Dyes

SYNOPSIS. Studies on the composition of commercial Giemsa stain and its effect upon staining quality are reported. These studies were supplemented by observations on the preparation of the components of Giemsa stain and their staining properties in aqueous solution, in Nocht's solution, and in laboratory prepared Giemsa stains containing one azure component. Five groups of commercial batches were differentiated on the basis of their staining reactions on thick and thin films of bovine blood containing Babesia bigemina and B. argentina. Spectrophotometric and chromatographic analysis showed that four groups differed in the proportions of the thiazine components present, while the fifth-group did not appear to be Giemsa stain. Comparison of their staining effects with those obtained with each component in laboratory prepared stains indicated that the major effects of commercial batches on both blood cells and parasites were due to the thiazine component or components in highest proportions, with satisfactory staining of protozoa associated with those batches containing high proportions of methylene blue and azure B and low proportions of the remaining thiazine components.
The function of each component of Giemsa stain is defined and the need for the proper balancing of thiazine eosinates with free azure is shown. Close correlation was obtained between analysis by spectrophotometry and chromatography and direct staining tests when samples initially with low MX values were re-examined spectrophotometrically after removal of their methylene violet content. The existence of a leuco form of eosin is reported and its possible significance to the Romanowsky effect is discussed.     

The role of chromosomal proteins in the induction of a differential staining of sister chromatids by light

Summary Fixed chromosomes of human lymphocytes, cultured in the presence of bromodeoxyuridine (BrdU) during two cell cycles, were exposed to near-ultraviolet irradiation, stained with Giemsa, and after destaining, were subjected to either Coomassie Blue or Feulgen-Schiff staining. A differential reaction of sister chromatids was first revealed by Coomassie Blue staining. Differential staining with Giemsa required a longer irradiation time. This appeared to be reduced after the addition of dithiodipyridine to the cells during their last few hours of culture. The differential pattern obtained after Coomassie Blue staining was the inverse of that obtained after Giemsa staining. From these findings we concluded that the induction of sister chromatid differentiation by light in BrdU-substituted DNA containing chromosomes occurs primarily via chromosomal proteins, presumably by differential breakage of their disulphide bonds. The results of the Feulgen-Schiff staining indicated that differential depurination of BrdU-containing DNA could occur, although only after very prolonged irradiation. A faint though distinctly differential Feulgen-Schiff pattern of sister chromated staining, resulting from differential removal of DNA, was observed after photosensitization by specific DNA-binding dyes. Thus, DNA seems to be affected only under more extreme conditions.     

The role of chromosomal proteins in the C-banding of Allium cepa chromosomes.

When chromosomes of Allium cepa are subjected to a C-banding procedure (incubation in saturated barium hydroxide followed by phosphate buffer at 60 degrees C for 1 h) and then treated with Giemsa stain, bands appear at the telomeres of all chromosomes. Microspectrophotometric measurements of Feulgen-DNA content, demonstrated that the C-banding procedure extracted DNA from the nuclei. Staining of banded chromosomes with several DNA-specific stains showed that this loss was differential, with the band DNA exhibiting more resistance to extraction than that of the rest of the chromosome. The C-banding procedure did not extract chromosomal proteins, however, and no difference in mass per unit length could be detected by Nomarski optics between band and interband regions. Several experiments demonstrated that chromosomal proteins play a significant role in C-banding. First, treatment of chromosomes with pronase before C-banding resulted in the elimination of differential staining with Giemsa. Furthermore, in preparations where the DNA was completely hydrolysed with hot TCA, the remaining chromosomal proteins were found to exhibit a differential affinity for Giemsa stain. Amido black staining demonstrated that total chromosomal protein was uniformly distributed after the hot TCA digestion, but the proteins localized in the telomeres had a greater affinity for the Giemsa stain than the bulk of the chromosomal proteins. When the TCA-digested chromosomes were subjected to the C-banding procedure before staining, the differential affinity of the telomeres for the Giemsa stain was lost. Thus, C-banding appears to be the result of a complex interaction between protein and DNA in which the greater resistance to extraction of the band DNA is necessary to stabilize and preserve chromatin protein which exhibits a differential affinity for Giemsa stain.