Plastic Sectioning for Light Microscopy of Pyrenomycetes

In preparation for light microscopy, ascocarps of Sordaria fimicola Ces. & DeNot. were embedded in Spurr's medium and sectioned at 1-1.5 μm on an ultramicrotome. Sections were floated on Giemsa staining solution at 60 C for 10-30 min, washed in distilled water, affixed to slides by drying, and mounted in immersion oil. Best preservation of the delicate sterile tissues of the centrum was obtained by fixation in 1% KMnO₄ for 2.5-3 hr, followed by the Giemsa stain. This method is suggested for future studies on the morphology of perithecial ascomycetes.     

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.     

An improved banding technique exemplified in the karyotype analysis of two strains of rat

Using an improved Giemsa banding technique karyotypes were prepared from cells of two strains of laboratory rat (AS and Hooded Lister). Slides, aged for 7 days at room temperature were incubated in 2 x SSO at 60 °C for 3 hours and then exposed to 1% trypsin for 90 seconds at 10 ° C. Following Giemsa staining, consistent banding patterns were found in both early and late metaphase cells without loss of chromosome morphology. No major differences were found in the Giemsa banding patterns of the rat strains studied. Some variability in the banding pattern was observed for the small subterminal autosome (B5).     

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.     

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.     

A Buffered Giemsa-Bodian Stain for Neurological Material

A buffered Giemsa counterstain for the Bodian method is described. It is very useful for bringing out Nissl substance and nerve fibers in the same section. Bouin perfused or formalin fixed material from mammals, amphibia, reptiles and fish was used. After fixation, all tissues were decalcified for at least a week in 50% formic acid (1 part) and 20% sodium citrate (1 part). This was washed out thoroughly. The method described by Bodian (1936) was followed except for the following minor changes: Winthrop Protargol (Strong Protein Silver) Batch N346BJ was used exclusively; all glassware was cleaned with acid prior to setting up the stain; before developing, the sections were washed in warm tap water for 10 minutes; the gold chloride was chilled before use. The sections were then put into buffered Giemsa for 24 hours. Stock Giemsa: 0.75 grams powdered Giemsa (Coleman and Bell, Certification No. CGe-3) was dissolved in 50 ml. of glycerin overnight in the oven, then 50 ml. of methyl absolute alcohol were added. To 3 ml. of Giemsa stock solution, 87 ml. of distilled water buffered to pH 5.3 with 10 ml. of Sorensen's buffers were added and the solution filtered. Coleman buffer tablets gave best results at pH 5.0. Sections were then rinsed in 95% alcohol, two changes of absolute alcohol, two changes of xylene, and were then mounted in Clarite.     

Light and scanning electron microscopic observations on the two contrasting types of sister chromatid differential staining after ultraviolet light irradiation

Chinese hamster cells were grown with 50 M 5-bromodeoxyuridine (BrdU) during the penultimate S phase to obtain chromosomes with the TB-TT chromatid constitution. Chromosome preparations made by the air-drying method were used to study the sister chromatid differential staining (SCD) resulting from ultraviolet (UV) irradiation followed by Giemsa staining by light and scanning electron microscopy (SEM). When chromosomes irradiated with UV light (253.7 nm, 5.2 J/m²/s) for more than 5 h were stained with 1% to 4% Giemsa in phosphate buffered saline (PBS) or in distilled water, the resulting SCD invariably belonged to the B-light type in which the TB-chromatid stained lightly. SEM observations of these chromosomes suggested that the B-light SCD was due to the selective photolysis of the TB-chromatid. On the other hand, when chromosomes were irradiated for only 10 min, and stained with 1% Giemsa in PBS, they showed a B-dark type SCD in which the TB-chromatid stained darkly. However, when chromosomes irradiated for 10 min were stained with 4% Giemsa in PBS or 1% Giemsa in distilled water, the resulting SCD again belonged to the B-light type. These findings indicate that when the irradiation dose is small, the resultant SCD is not a simple reflection of selective photolysis in the TB-chromatids and the type of SCD depends not only on the concentration of Giemsa but also on the salinity of the staining solution.     

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.     

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.     

Reverse differential staining of sister chromatids after substitution with BUdR and incubation in sodium phosphate solution

Chinese hamster strain cells were cultured in the presence of BUdR and air-dried on slides. The chromosome preparations were incubated in 1 M NaH₂PO₄ at 88 °C for 4–6 min and stained with Giemsa. The reverse type of sister chromatid differential staining occurred, in which unifilarly BUdR-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Feulgen reaction performed on the same chromosomes after removing Giemsa stain showed the same type of differential staining.     

Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

Staining human lymphocytes and onion root cell nuclei with madder root.

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5-10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.     

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.     

Premature release of Plasmodium falciparum from swollen erythrocytes induced by some antimalarials

Qinghaosu and chloroquine, but not pyrimethamine, treatment of Plasmodium falciparum cultures resulted in the formation of swollen red blood cells (RBCs) and the expulsion of degenerate trophozoites and schizonts, but not ring-stage parasites, from the infected RBCs. The parasite release resulted in the formation of RBCs with holes, that had otherwise retained their structural integrity. Membranes of swollen RBCs and their ghosts associated with parasites were efficiently visualized by Giemsa staining of thin smears for 18-24 hr but not by standard Giemsa staining for 20 min.     

Dientamoeba fragilis is more prevalent than Giardia duodenalis in children and adults attending a day care centre in Central Italy

Giardia duodenalis is a well recognised enteropathogen, while Dientamoeba fragilis is rarely detected and consequently it is not recognised as an important human pathogen. In 2002-2003, a survey has been carried out on enteroparasites in faecal samples of outpatients attending a day care centre in the town of Perugia (Central Italy). To improve the detection level, at least three samples from each patient were collected at different days and within two hours from defecation. The coproparasitological examination has been carried out by direct microscopic examination, faecal concentration, and Giemsa and modified Ziehl-Nielsen stainings of faecal smears. The genotypes of Giardia duodenalis isolates were determined by PCR of the beta-giardin gene. Of 1,989 enrolled people (966 children, 1,023 adults), 165 persons (8.3%; 153 adults, 15.0%; 12 children, 1.2%), were positive for parasites, but only 1 12 adults (73.2% of those infected) and eight children (66.7% of those infected) harboured D. fragilis and G. duodenalis. Both the Assemblages A and B were detected in 18 G. duodenalis isolates examined at the beta-giardin gene. The higher prevalence of D. fragilis infections than that of G. duodenalis is probably related to the method used, a procedure, which is rarely followed in laboratories for the diagnosis of enteric parasites. These epidemiological data suggest that when faecal samples are examined after a period of time and without Giemsa staining, most D. fragilis infections goes undetected.     

Epidemiological study of malaria in north Sulawesi, Indonesia by fluorescence and Giemsa staining.

An epidemiological study of malaria infection was conducted in the Likupang District, Minahasa Regency, North Sulawesi Province, Indonesia, during August 2-15, 1991. In this study, 510 people of six villages, representing ages between 1 month to 84 years cooperated voluntarily. Blood smears stained with Giemsa and acridine orange (AO), revealed 33 and 83 malaria parasite positives respectively. This significant difference was due particularly to the fact that AO staining examined under either a daylight- or halogen-illuminated microscope equipped with interference filters was sensitive to detect low-density parasitemia in many subjects previously diagnosed negative by Giemsa staining in the field. The low malaria prevalence obtained by Giemsa staining may have been attributable to the lack of standard-quality diagnostic tools in the field or inadequate observation of the slides. In both staining methods, Plasmodium falciparum was found to be the predominant species, while the remainings were P. vivax or a mixture of both. Subjects infected with P. vivax revealed higher density of parasitemia and gametocytemia than those with P. falciparum.     

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.     

Staining the Nucleus in Yeasts

Yeast cells kept young by repeated subculturing were centrifuged, washed twice in distilled water and smeared on slides coated with a little egg albumen. The cells were treated with 0.002 M 8-hydroxyquinoline for 1 hr, fixed first in OsO, vapour for 30 sec and then in chloroform for 30 sec. The slides were passed through descending grades of alcohol, washed in distilled water, then immersed in 0.17 M NaCl solution for 2 hr. at 57°C. They were again washed in distilled water and later hydrolysed in 1 N HCl at 60°C for 5-7 min. This was followed by washing in distilled water and in buffer. The slides were then kept for 3 hr in Giemsa stain comprising 96 ml of phosphate buffer of pH 7.0 and 4 ml of the stain. After dehydration, mounting was done in balsam. Nuclei were brightly stained and well differentiated; centrosomes were clear, and the process of nuclear division and movement to daughter cells could be studied. Pretreatment with 8-hydroxyquinoline increased the viscosity of the cytoplasm, while NaCl treatment and acid hydrolysis led to the complete removal of ribonucleic acid and basophilic material. A selective staining of chromatin was thus achieved. Structures resembling chromosomes could be seen when fixed and stained cells were squashed, soon after the removal of the slides from the stain, under a cover glass by applying uniform pressure with a rubber stopper. Fixation in osmic acid vapor and chloroform followed by acid hydrolysis and staining in leucobasic fuchsin also helps to obtain bright staining of the nucleus; however, the preparations are inferior to those obtained after 8-hydroxyquinoline, NaCl treatment and Giemsa staining.     

Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.     

Frequencies of micronuclei detected on Mytilus galloprovincialis by different staining techniques after treatment with zinc chloride

The frequencies of micronuclei induced by ZnCl2 and detected on the gill tissue of the marine mussel Mytilus galloprovincialis with different staining techniques (acridine orange, gallocyanin chromallum, Feulgen, Giemsa) were compared. At least in the used system, the Feulgen and gallocyanin chromallum methods gave a frequency of micronuclei significantly lower than that obtained with the acridine orange and Giemsa techniques. No significant difference between the frequencies obtained with acridine orange and Giemsa was shown. So, though the acridine orange is surely the method which provides the more reliable data, in environmental screening works the Giemsa technique may be more suitable for its simplicity.