Mitotic Frequency Determinations and Micro-Photometric Feulgen Dye Measurements in Root Tips

Treating a Feulgen stained (10 min. acetic-alcohol fixation, 8 min. hydrolysis) onion root tip with 5% aqueous pectinase for 6 hours causes it to disintegrate on shaking in 1 ml. water into a suspension of stained cells, either individual or in small groups. Vicia faba and pea root tips require 15 minutes hydrolysis and 12 hours pectinase treatment. Absence of cell destruction allows absolute cell-number determination by Brown and Rickless' counting chamber technic. By centrif uging the cells down, resuspending them in 2 drops of a Karo syrup-phosphate buffer mixture (2 parts Karo to 1 part 0.5M, pH7 buffer) and mounting a small drop of the now concentrated suspension, a semipermanent slide containing a concentration of well-spread, randomized cells suitable for rapid mitotic frequency determination is obtained. Scoring about 1,000 cells as to nuclear stage gives a representative, workable statistical sample and takes only 20-30 minutes if interphases are recorded on a mechanical tally. Permanent slides can be prepared by carrying the intact, stained, pectinase-treated root through a water wash, 70%, 95%, and absolute alcohol, shaking and performing the centrifuge procedure with Diaphane in place of the Karo mixture. Although the pectinase treatment appears to introduce some error, the Diaphane mounts are adequate for conventional microphotometric Feulgen dye (DNA equivalent) determinations. The three dimensional character of the cells is retained in all preparations.     

Isolation of protoplasts from the placental cells of Lycopersicum pimpinellifolium Mill

Living protoplasts were isolated from the interplacental regions of Lycopersicum pimpinellifolium berries by the removal of the walls from cells in tissue slices treated for 1–2 h with 10 % pectinase in 0.5 M sucrose solution. Protoplasts thus isolated, then washed and transferred to microculture chambers for observation were invariably spherical. Each protoplast contains a nucleus, a number of chloroplasts of variable shape and a vacuole with smaller vacuoles contained therein. Phase contrast optics reveal cytoplasmic granules, the size of mitochondria, which serve to indicate such dynamic processes as cyclosis. Treatment with ox bile salt and sodium lauryl sulphate cause rapid disruption of the protoplasts producing subprotoplasts and isolated tonoplasts and serves to confirm the absence of rigid cell walls.     

A fixation method for visualization of yeast ultrastructure in the electron microscope

A primary fixative containing glutaraldehyde (3%), acrolein (1.5%), and paraformaldehyde (1.5%) buffered in 0.05 M sodium cacodylate at pH 7.2 was applied to the cells ofCryptococcus vishniacii for 2 hours on ice. The cells were then treated with a 6% aqueous solution of potassium permanganate for 1 hour at room temperature. This method preserves most of the yeast cell fine structural components including cell walls and membrane, nuclear membrane, mitochondria, endoplasmic reticula, microbodies, vacuoles, nucleoli, and ribosomes. However, it leads to disruption of capsular materials and loss of some of the lipid and glycogen granules.This study was supported by a National Aeronautics and Space Administration (NASA) research grant NAGW-26.     

Cytoplasmic Components of the Broad Bean Root Tip Cells

The cytoplasmic components of broad bean (Vicia faba L.) root tip cells were studied light- and electronmicroscopically. Using a light microscope, differences were revealed between cells from the cortex and from the central cylinder. For electron microscope studies the cells near the boundary between the mentioned parts of the root tip were selected at a distance of about 2 mm from the initials. The orientation of objects during embedding made possible fairly accurate localization. No peculiar, strikingly osmiophillic bodies were seen, which could be identified without doubt as osmiophillic platelets. It follows that some of the current cytoplasmic components, perhaps more or less altered were described as osmiophillic platelets. After fixation with KMnO₄, in which case the electronmicrographs are most instructive, the leucoplasts show several inclusions, mostly only partially limited against the matrix; it is not clear whether the true membrane is concerned here. The origin of intravacuolar membraneous formations is discussed.     

Luxol Fast Blue Mbs and Phloxine; a Stain for Mitochondria

The applicability of Luxol fast blue MBS as a 0.1% solution in 0.05% acetic acid to the staining of mitochondria, first recognized in rat kidney by Shanklin and Nassar (Stain Techn., 34: 257-60. 1959), was confirmed in various organs (formalin-Zenker and Regaud's fixations; paraffin embedding) of the mouse and bullfrog. In liver cells and in the epithelium of renal tubules, mitochondria were stained green, selectively and clearly. The dark cells of the renal tubules and the middle piece of sperms in both animals were conspicuously demonstrated by their dense assemblages of green granules. The periodic acid-Schiff procedure proposed by Shanklin and Nassar as a counterstain was replaced by staining in 0.5% aqueous phloxine, 2-3 min; differentiation in 5% phosphotungstic acid, 2 min; and washing in water, 5 min. This simplified and accelerated the techique, and gave a better color contrast. Advantages of Luxol fast blue MBS and phloxine staining over traditional methods for mitochondria in paraffin sections are: durability of the stain, high specificity, simplicity of procedure, and constant result.     

Different amounts of DNA in each mitochondrion in rice root

The DNA content of individual mitochondria in rice root cells was analyzed by fluorescence microscopy. Differences in DNA content were detected between individual mitochondria. Some mitochondria contained no detectable nucleoid (DNA-protein complexes). The percent of mitochondria with DAPI(4',6-Diamidino-2-phenylindole) -stained nucleoids varied over the length of the root (root base, 33%; middle portion of root, 41%; root tip, 91%). The mean amounts of DNA per mitochondrial nucleoid were equivalent to 46.4 kbp in the root base, 52.0 kbp in the middle portion of root and 124.2 kbp in the root tip. The amount of DNA in individual mitochondria and the ratio of mitochondria with visible nucleoids were higher in the root tip than in other parts of the root. The estimated amount of DNA in almost all of the observed mitochondria was smaller than the amount of DNA equivalent to the rice mitochondrial genome size (490 kbp), even in root tip.     

Protoplast isolation,culture and regeneration from Egyptian cultures of Pea and Beans

Abstract

A protocol of protoplast isolation from Egyptian varieties of pea and bean is reported. Protoplast cultures were established from apical shoots of pea (Pisum sativum) and suspension cultures of bean (Phaseolus vulgaris). To isolate protoplasts of pea, apical shoot tissues were digested for 10 h using enzyme solution containing 1% pectinase, 0.5% cellulase, 0.5% hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. For protoplast isolation from suspension culture of bean, collected cells were incubated for 6 h in digestion solution containing 0.5% pectinase, 0.25% of each of cellulase and hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. Purified protoplasts were cultured in liquid culture medium. Microcalli were obtained after 30 days of culture. Calli colonies with a diameter of about 5 mm were developed after one month of culturing on solid B5 medium containing 2% sucrose, 2 g/l casein hydrolysate, 0.7% agar and supplemented with either 1 mg/l of each 2,4-D and kin in case of pea or 2 mg/l 2,4-D+0.5 mg/l kin in case of bean. Protoplast derived callus of pea was successfully differentiated into shoot and root, and highest frequency of shoot organogenesis was recorded on medium containing 0.5 mg/l NAA+2 mg/l BA. Protoplast derived callus of bean, on the other hand, gave rise to a high frequency of root formation when cultured on medium containing 1 mg/l NAA, but attempts to regenerate shoots from this callus was unsuccessfull.     

Histochemical evidence for lipoidal material in secretory granules of rat salivary glands

Synposis The granules of parotid acinar cells and submandibular granular tubule cells of rats contain one or more periodic acid-Schiff positive substances that are extracted during fixation with lipid solvents or acidic solutions or if frozen sections are stained in aqueous solutions. The granules in these cells can be stained by Schmorl's reaction, Luxol Fast Blue and a permanganate-Aldehyde Fuchsin sequence. The results obtained with these stains after a variety of fixation procedures strongly suggest that the secretory granules of these two cell types contain several components and that in parotid acinar and submandibular granular tubule cells, at least one of these components is a lipoidal substance.     

Biochemical and Cytological Changes Accompanying Growth and Differentiation in the Roots of Zea mays

The apical meristem of the root affords an excellent material with which to study changes in cellular components accompanying growth and differentiation. The ontogeny of cytoplasmic particles can be followed, since the younger cells are constantly dividing and reforming new cytoplasm. Electron microscope pictures of these newly formed cells reveal a dense background of microsomal granules and small, thin walled vesicles of the endoplasmic reticulum. Two types of mitochondria are noted and, as the cells enlarge, mitochondria regarded as immature can no longer be seen, but only mitochondria with well developed cristae. The development of these cristae was found to be associated with an increase in respiration of the tissue as well as with increased rates of oxidation and phosphorylation of isolated mitochondria. As the cells grow and mature, the mitochondria make up an increasing percentage of the total cytoplasmic protein, and this increase probably accounts to a great extent for the increase in tissue respiration. Concomitantly, there is a decrease in microsomal granules. All these changes have been verified by electron microscope pictures of cells in situ, chemical analysis of isolated particulates, and metabolic studies of tissue and isolated fractions.     

Ultrastructural studies of oocytes and embryos derived from females flies carrying the grandchildless mutation in Drosophila subobscura

The maternal effect mutant grandchildless in Drosophila subobscura has been analyzed with the electron microscope. The original mutation was linked to a visible genetic marker and established in a balanced stock. Oocytes and early embryos were examined by both transmission and scanning electron microscopy. The earliest defect is seen in mutant eggs and occurs at the end of oogenesis. In the cortex, at both the anterior and the posterior tips, regions appear which are free of ribosomes, mitochondria, and other cytoplasmic organelles. Most of the polar granules are included in these regions at the posterior tip. Following oviposition, this cytoplasmic segregation is no longer observed and most polar granules have disappeared. The few remaining granules are presumed to derive from the peripheral polar plasm which does not become segregated. During embryogenesis there is a retarded movement of nuclei to the anterior and posterior cortices. At the posterior tip nuclei are delayed in reaching the lateral sides and never move directly into the posterior polar plasm. Pole cells never form. After the last syncytial division the lateral nuclei move under the posterior polar plasm to complete the blastoderm. The posterior polar plasm itself protrudes during blastoderm formation as long cytoplasmic extensions which separate from the blastoderm as cytoplasmic blebs. Neither polar granules nor mitochondria are found in these blebs. The grandchildless phenotype is due to the failure of nuclei to migrate directly into the posterior polar plasm. The defect in the polar plasm presumably is related to the process in mature eggs whereby portions of the cortex become segregated at both anterior and posterior tips. This process may change the properties of the posterior polar plasm so that nuclei do not penetrate into it.     

Feulgen Cytophotometry of Pine Nuclei. II. Effect of Pectinase Used in Cell Sfparation

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that hut denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.     

Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.     

紫茎泽兰叶片水浸液对蚕豆的化感效应

外来入侵植物可以通过淋溶、自然挥发、根系分泌和植株凋落物分解等途径向周围环境释放化感物质,抑制伴生植物的生长、发育。该研究以不同浓度紫茎泽兰(Eupatorium adenophorum)叶片水浸液处理蚕豆(Vicia faba)种子,研究紫茎泽兰叶片水浸液对蚕豆根尖细胞微核、染色体畸变、细胞凋亡、蚕豆幼苗叶片叶绿素和N含量、光合生理特性、生物量的影响。结果表明:(1)紫茎泽兰叶片水浸液处理显著抑制蚕豆根尖的伸长和细胞的有丝分裂,并诱导蚕豆根尖细胞染色体畸变和细胞微核的产生,有丝分裂指数随着叶片水浸液浓度增加而减小,根尖细胞微核率随叶片水浸液浓度增加而增大,高浓度叶片水浸液处理对蚕豆根尖细胞的凋亡及坏死有明显影响。(2)紫茎泽兰叶片水浸液处理引起蚕豆幼苗叶片的叶绿素和N含量显著降低,并导致蚕豆幼苗最大净光合速率和生物量的显著下降。总之,紫茎泽兰叶片水浸液可能引起蚕豆根尖的氧化损伤和抑制根尖的伸长,且叶片水浸液的抑制作用呈现一定的剂量效应。紫茎泽兰叶片水浸液对蚕豆根尖的损伤和抑制作用可能影响了植株对氮素的吸收,进而对蚕豆幼苗光合生理表现以及生物量积累产生显著负面效应。     

A morphological study of the mycorrhiza ofEntoloma clypeatum f.hybridum onRosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.     

Cytotoxic and apoptotic activities of extract of <Emphasis Type="Italic">Amaranthus</Emphasis><Emphasis Type="Italic">spinosus</Emphasis> L. in <Emphasis Type="Italic">Allium cepa</Emphasis> and human erythrocytes

The present study examined the apoptosis inducing effects of Amaranthus spinosus L. aqueous extract in Allium cepa root meristematic cells and human erythrocytes. Cytogenetic assay revealed many apoptosis inducing cytogenetic aberrations viz., cytoplasmic breakage, cytoplasmic disintegration, cytoplasmic shrinkage, receding of cytoplasm, cytoplasmic vacuolation, enucleated cell, ghost cell, nuclear vacuolation, nuclear fragmentation and nuclear disintegration. A remarkable modification of red blood cell surface morphology was observed in the result of RBC assay. The treated RBCs show membrane blebbing and shrinkage, features typical for apoptosis in nucleated cells. Significant induction of cell death was observed in treated Allium root tip cells after Evans blue staining, disclosing the membrane damage potential of the plant extract. TTC assay results in reduced mitochondrial/metabolic activity in Allium root tip cells after treatment, designating the adverse effect of plant extract on mitochondrial respiratory chain. These results confirm the apoptosis inducing potential of A. spinosus extract. Confirming the present results by further in vitro studies, it can be effectively targeted against cell proliferation during cancer treatment by inducing apoptosis. Thus from the present investigation it can be concluded that the aqueous extract of A. spinosus exhibited apoptosis induction and cytotoxic activities.     

Differentiation of Nucleic Acids by Staining at Controlled pH and by A Schiff-Methylene Blue Sequence

Fixation with Bouin's fluid preserves cytoplasmic and nucleolar ribonucleic acid (UNA) particularly well. RNA may be demonstrated preferentially in Bouin fixed tissue by staining with 0.02% thiazine dye in aqueous McIIvaine phosphate-citrate buffer between pH 3 and 4. Methylation blockage of basophilia other than that of nucleic acids permits staining of RNA with thiazine dyes near neutrality. The deoxyribonucleic acid (DNA) of chromatin undergoes a Feulgen type hydrolysis in the tissue block during 24 hr fixation with Bouin's fluid. This hydrolysis by picric acid permits Schiff staining of the DNA wthout further acid hydrolysis. Consequently after Bouin fixation it is possible to demonstrate DNA and RNA specifically by a Schiff-methylene blue sequence. Thus a Schiff stain without further acid hydrolysis followed by 0.02% methylene blue in phosphate-citrate buffer at pH 3.0 to 3.5 colors DNA magenta in contrast to the blue of RNA.     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

The use of chromic potassium sulphate in bone electron microscopy

The ultrastructure of endochondral bone was studied using an aqueous solution of chromic potassium sulphate as the decalcifying agent. 0.5 mm thick sections of rat tibiae were fixed in buffered glutaraldehyde, immersed in an aqueous solution of 1% chromic potassium sulphate pH 3.4, dehydrated and embedded in Poly Bed 812 without exposure to osmium tetroxide. In unstained sections we observed clusters of crystal like structures throughout the osteoid and calcifying cartilage matrix as well as solitary needle shaped structures in association with collagen fibrils. Stained sections revealed nuclei, endoplasmic reticulum, membrane limited dense granules, mitochondrial particles and other cell components typical of bone cells. It appeared that the chromic potassium sulphate method preserves the relationship between hard and soft tissues well, gives fine cytological detail and produces images of intracellular and extracellular deposits identical to untreated crystallites. It is concluded that the chromic potassium sulphate method is indicated for ultrastructural studies of bone.     

太湖蓝藻滤液的遗传毒性研究

蓝藻爆发是环境污染引发的重要事件之一,随之产生的蓝藻毒素又直接危及区域水安全.该论文采用蚕豆和大蒜根尖微核试验研究了太湖蓝藻暴发期间蓝藻滤液的遗传毒性.结果表明,同阴性对照相比,所有试验处理对蚕豆根尖细胞微核发生率的影响显著增加;对大蒜根尖细胞微核发生率而言除蓝藻滤液8倍稀释液的影响不显著外,其它水平效应显著高于阴性对照,而且表现出一定的剂量效应.暴发期蓝藻滤液原液对蚕豆根尖细胞微核发生率影响显著高于阳性对照(0.8mg·mL^-1环磷酰胺)的效应,从而说明蓝藻暴发时期蓝藻滤液具有较强的遗传毒性.通过微核试验效果分析,蚕豆作为植物监测系统的敏感性和稳定性都优于大蒜材料.     

Fine structure of the parotid gland in the crest-tailed marsupial-rat (Dasyuroides byrnei).

The parotid gland of Dasyuroides byrnei was examined by light microscopy, and transmission and scanning electron microscopy. The acini were composed predominantly of seromucous cells with a few mucous cells. The seromucous cells were light or dark cells containing acidophilic spherical granules of moderate to high electron density and had well-developed cytoplasmic organelles-ordinary mitochondria and large mitochondria with tubular cristae, RER with vesicular or tubular elements, and Golgi apparatus with lamellae, vesicles and vacuoles. The mucous cells had basophilic amorphous granules of low electron density, like those of ordinary mucous cells. The intercalated ducts were composed of simple cuboidal light cells having a few electron-dense granules. The striated ducts consisted of tall columnar light cells containing numerous vesicles and mitochondria with tubular cristae, the same as found in acinar seromucous cells.