Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

The staining of the synaptonemal complex for light microscopic study in the mouse

Demonstration of the synaptonemal complex for light microscopy has until now been based on staining with silver. After fixation at pH 9-10 it is also possible to visualize synaptonemal complexes with several nonspecific protein stains such as Coomassie brilliant blue, Giemsa, fast green, light green and Stains All. Although staining with silver gives the best contrast between synaptonemal complexes and the background, the other dyes have a number of advantages, such as more even staining, easy extractability, and lower cost than silver.     

The Staining of the Synaptonemal Complex for Light Microscopic Study in the Mouse

Demonstration of the synaptonemal complex for light microscopy has until now been based on staining with silver. After fixation at pH 9-10 it is also possible to visualize synaptonemal complexes with several nonspecific protein stains such as Coomassie brilliant blue, Giemsa, fast green, light green and Stains All. Although staining with silver gives the best contrast between synaptonemal complexes and the background, the other dyes have a number of advantages, such as more even staining, easy extractability, and lower cost than silver.     

Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa

Gel electrophoresis of nucleolar isolates from Zajdela ascites hepatoma cells followed by various staining procedures revealed a common set of bands that stained selectively with silver and Giemsa. The gel bands, corresponding to molecular weights of 104, 78, 37, and 29 kilodaltons (kd), appeared to contain phosphoproteins that were at least partly associated with oligo-deoxyribonucleotides. Enzyme digestion studies showed that the Giemsastainability was due to the phosphorylated state of the proteins. The positive selective silver-staining reaction in gels could be most likely attributed to the high content of carboxyl groups present in these phosphoproteins. The significance of these findings in relation to cytological results produced by selective silver staining of nucleolus organizing regions (NORs) and by Giemsa N-banding is discussed.     

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes

Summary A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

Visualization of centriole-bodies using silver stain

Duplicate staining of human metaphase chromosomes, first with Giemsa followed by silver, revealed the presence of two small silver-stained bodies not seen in the Giemsa stained metaphases. Similar bodies were subsequently found in the metaphases of several animal groups. The size, structure, spatial relationships to the nucleus, behavior throughout the cell cycle, and apparent universal presence of these bodies suggest that they are either centrioles or associated centriolar structures. These centriole-bodies can be seen throughout the cell cycle, even in hypotonically spread C-metaphase chromosome plates. The silver stain procedure allows enough resolution to distinguish parent and daughter centriole-bodies at interphase, thus permitting visualization of the replication, maturation and separation stages of these bodies with the light microscope.     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.     

Karyotypes,C-banding and nucleolar numbers inGuizotia (Compositae)

Karyotypes, constitutive heterochromatin and nucleolar numbers of five recognized taxa and two systematically new populations ofGuizotia have been studied using Giemsa or aceto-orcein staining, C-banding and silver nitrate staining. All accessions have 2n = 30 chromosomes, but satellite chromosome number and nucleolar number varied from four to eight. Centromere positions varied from predominantly median to submedian and subterminal in different materials. The satellites and an interstitial region in the short arm of one chromosome pair were C-banded in all materials. Telomeric and centromeric C-bands were also observed. The material could be classified into three groups, indicating possible phylogenetic relationships.     

A Method for Combined C-Banding and Silver Staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

A method for combined C-banding and silver staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

Structural changes of the nucleolar organizing regions in rhabdomyosarcoma RA-23 tumor cells induced by okadaic acid

It has been recently shown that okadaic acid (OA), an inhibitor of dephosphorilation, is capable of inducing changes in the nucleolar organizer regions (AgNORs) of some mammalian cells. Our work was focused on studying the structural changes in AgNORs of tumour cells of rat rhabdomyosarcoma RA-23 by their exposure to 100 nM OA. A standard silver staining procedure of interphase AgNORs in tumour cells was used. We measured no less than 100 tumour cells in each clone. In the examined tumour cell populations, the index of interphase AgNORs varied from 1.54 to 4.35. A clear structure and form of AgNORs was not observed in 30% okadaic acid-treated tumour cells, as opposed to 10% of the control ones. AgNORs in these cells looked like a mixture of thin threads encompassing some dark dots lying, mostly, separately. Additional procedures of tumour cell staining with Giemsa and fluorescent dye Hoechst 33,258, respectively, revealed that such structures were not chromosomes. Meanwhile, the frequency of cells at the stage of prophase exceeded 3%, as opposed to the control, where the frequency of cells at this stage was less than 0.5%. Thus, we can conclude that we have detected specific changes in AgNORs and chromatin structure of okadaic acid-treated tumour cells.     

Efficacy of immunohistochemical staining in detecting Helicobacter pylori in Saudi patients with minimal and atypical infection

Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H&E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H&E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H&E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H&E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H&E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H&E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.Key words: Helicobacter pylori, immunohistochemistry, modified Giemsa stain, real-time polymerase chain reaction, chronic gastritis     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

Effects of acetic acid-alcohol,trypsin, histone 1 and histone fragments on Giemsa staining patterns in chromosomes

Summary In an effort to minimize subjective bias, a classification scheme was devised to assess Giemsa staining patterns obtained with experiments involving acetic acid-alcohol and exogenously applied histone 1 and polypeptides. A single rinse of metaphase preparations with acetic acid-alcohol quantitatively reduced Giemsa dye binding. Acid-alcohol irrversibly changed the conformation of HI and its ability to interfere with trypsin G-banding. Our results suggest that, in addition to protein extraction, acid-alcohol may alter the conformation of acid-insoluble components of metaphase chromosomes. The carboxy-terminal polypeptide (residues 73–212) from NBS cleavage of H1 was an effective inhibitor of Giemsa staining and trypsin G-banding. However, this polypeptide which is preferential for supercoiled DNA was much less efficient in inhibiting Giemsa staining of trypsinized metaphase chromosomes. The molecular consequences of these experiments are discussed.     

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.     

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.     

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

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

Phosphoprotein pp135 is an essential component of the nucleolus organizer region (NOR)

The association of phosphoproteins pp135 and pp105 with distinct substructures of the nucleolus was studied by cytochemical and immunological methods at the light microscopic and electron microscopic level. Both phosphoproteins exhibited a very high affinity for silver and Giemsa staining compared to other nucleolar proteins. Immunolocalization of pp135 and pp105 during mitosis by light microscopy revealed a tight association of pp135 with the silver staining nucleolus organizer region (NOR), whereas pp105 (cross-reacting with C23) appeared to be only partially associated with the NOR, exclusively at telophase. At the immunoelectron microscopic level the distribution of pp135 and pp105 was investigated in interphase nucleoli. Phosphoprotein pp135 was located in the fibrillar shell and pp105 in the fibrillar shell and the granular zone. The fibrillar centers were essentially free of both phosphoproteins..