Calcofluor Staining Research Articles

Isolated Maize Zygotes Mimicin VivoEmbryonic Development and Express Microinjected Genes When Culturedin Vitro

We established conditions for the regeneration of natural maize zygotes isolated from pollinated plants with the goal of investigating the molecular control of early embryogenesis in higher plants. Viable zygotes were excised from embryo sacs by minimal enzymatic digestion and microdissection. Viable zygotes transferred to coculture with androgenic microspores from barley developed into embryo-like structures in 61% of the cases. No development was observed when zygotes were cultured in the presence of maize anthers undergoing androgenetic embryogenesis. Zygote-derived embryo-like structures regenerated into fertile plants through secondary embryogenesis when transferred to solid medium. The first zygotic division was asymmetrical and bipolar structures similar to pretransitional embryos observedin plantawere later produced as observed using light and electron microscopy. Conditions for efficient microinjection of DNA into zygotes were established. Calcofluor and PATAG staining of zygotes showed that cell wall regeneration occurred as early as 20 min after enzymatic isolation and that after 2 hr, each zygote was bordered with cell wall material. Through quantitative microphotometry, DNA synthesis during the first cell cycle of the zygote was shown to occur between isolation and 12 hr of culture. Microinjection of two types of reporter genes (GUSgene and anthocyanin regulatory genes) demonstrates transient expression in plant zygotes. On average, 3.5% of microinjected zygotes showed transgenic expression. Reporter gene expression was observed in zygotes at different time points of their first cell cycle.

Biofilm structural heterogeneity visualized by three microscopic methods

The structural heterogeneity of a microbial biofilm was demonstrated by scanning electron microscopy, confocal scanning laser microscopy (CSLM), and cryoembedding followed by sectioning and microscopic examination. Biofilm was composed of a binary population of Pseudomonas aeruginosa and Klebsiella pneumoniae grown in a continuous flow annular reactor. The three microscopic methods provided a consistent picture of the biofilm as a non-uniform structure characterized by variable thickness and variable local cell and polymer densities. Significant changes in these parameters occurred in the biofilm over distances of 10 μm or less. Though the biofilm was several hundred microns thick in places, areas of bare substratum were also observed on the same sample coupon. Cell-free pores and channels in the biofilm interior were evident. Specific staining of cellular nucleic acids with ethidium bromide and extracellular polymeric substances (EPS) with calcofluor showed that cell and EPS distributions did not always overlap. The ethidium bromide-stained region was contained within the larger region of calcofluor staining; thus, some cell-free areas actually were filled with EPS. CSLM and cryoembedding approaches are superior to SEM in their ability to image the biofilm interior and in their potential to provide quantitative information.

The effect of natural and simulated insect herbivory, and leaf age, on the process of infection of Rumex crispus L. and R. obtusifolius L. by Uromyces rumicis (Schum.) Wint.

summaryThe development of uredinia of Uromyces rumicis (Schum.) Wint. was studied on Rumex crispus L. and R. obtusifolius L. plants inoculated in the laboratory. Fewer uredinia developed on leaves injured by simulated insect herbivory, those fed upon by Gastrophysa viridula Degeer (Coleoptera: Chrysomelidae), and young, incompletely expanded leaves, than on uninjured, fully expanded control leaves. The effect of simulated herbivory and leaf age on the process of U. rumicis infection was investigated using Calcofluor staining and epifluorescence microscopy. Simulated insect herbivory reduced significantly the proportion of sporelings producing appressoria and the proportion of sporelings with appressoria that entered the substomatal cavity. In R. obtusifolius, leaf injury also reduced the proportion of penetration hyphae that formed substomatal vesicles. The proportion of sporelings that produced intercellular hyphae in injured leaves was reduced by 58 % in R. crispus and 89 % in R. obtusifolius. G. viridula feeding induced similar resistance in R. obtusifolius leaves.Simulated insect herbivory reduced both uredinial density and the area occupied by intercellular hyphae by between 65% and 89%. Injury had a greater effect than leaf age on sporelings in the pre‐haustorial stages of development. However, the total area occupied by intercellular hyphae in young leaves was reduced by between 42% and 78% compared to mature leaves, owing to a lower uredinial density and, in R. obtusifolius, also to a significant reduction in the size of individual uredinia.

Flunarizine affects F-actin pattern in Mucor rouxii germlings

The effect of the Ca2+ antagonist flunarizine on Mucor rouxii germlings was analyzed. In sporangiospores cultivated in aerobiosis, the drug blocked germ tube emergence, but enlarged spheres continued to grow isodiametrically. The formation of broad protuberances and irregular buds was observed. Calcofluor staining indicated aberrations on the surface of the flunarizine-treated cells. This was not a simple switch to the yeast form, since labelling with rhodarmine-tagged phalloidin revealed various ring-shaped structures of F-actin, indicating furrowing of the plasma membrane. Flunarizine apparently did not interfere with nuclear divisions or with the integrity of microtubules. In young germlings, flunarizine treatment induced cessation of tip growth and promoted septation associated with the rearrangement of F-actin. Apical growth was reestablished after either the drug was removed or an excess of Ca2+ ions was added to the medium. The results suggest that tip-related arrangement of F-actin requires flunarizine-sensitive Ca2+ entry.Key words: Mucor rouxii, F-actin, Ca2+ antagonist, flunarizine, germination.

Stage-specific requirement for myosin II during Dictyostelium development

Dictyostelium cells that lack a functional myosin II heavy chain are motile and are capable of aggregation, but fail to undergo further multicellular development. We have used a Dictyostelium mutant expressing a cold-sensitive myosin heavy chain to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature-shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between those stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. Calcofluor staining and reporter gene fusions reveal that these structures contain defective spores and a miniature stalk.

Pathogenicity of Paecilomyces farinosus toward Cephalcia abietis Eonymphs (Insecta, Hymenoptera): Enhancement of Bioactivity by in Vivo Passaging

Culture media influenced the germination of conidia, appressorial development and mycelial growth of Metarhizium anisopliae. Although the addition of KC1 to Sabouraud dextrose agar (SDAM) significantly reduced germination of the conidia of three isolates in vitro and lowered germination and appressorial development on the cuticles of Myzus persicae and Meligethes aeneus, conidia grown on SDAM and minimal medium (MM) were more aggressive than conidia derived from SDA or yeast extract agar. No relationship was found between either germination or appressorial development and LT50 values, suggesting that virulence depends on the speed of infection. Calcofluor staining and lectin binding showed that culture conditions also influenced the adhesion of Metarhizium anisopliae to insect cuticles through alteration of surface carbohydrates such as 1–4-β-glucans. Dark, pigmented conidia from MM bound less lectins and calcofluor whereas the paler conidia from the other media fluoresced more intensely. Conidia from SDAM fluoresced 20-fold brighter and adhered more readily to insect cuticle compared with conidia derived from MM. Although inocula from both media showed elevated pathogenic activity against aphid and pollen beetle, MM-grown conidia were more aggressive since high mortality was induced by comparatively fewer conidia. When fluorescent intensity of calcofluor-stained conidia was compared to the number of conidia adhering to the insect cuticles, the relationship was significant, indicating that this dye has practical implications for the assessment of fungal strains in screening programmes for commercial development.

Evaluation of Calcofluor staining in the diagnosis of fungal corneal ulcer

For laboratory diagnosis of mycotic keratitis, demonstration of fungal pathogens on direct microscopy and their isolation by culture is essential. The addition of Calcofluor white (CFW) stain to the diagnostic armamentarium has significantly increased the sensitivity of smear examination on direct microscopy. During a period of 1 year, 143 consecutive patients with corneal ulcers were investigated by direct microscopy with potassium hydroxide (KOH) wet mount, Calcofluor white stain and routine cultures of corneal scrapings. Fungi were detected as aetiological agents in 21 (15%) patients. Different species of the genera Aspergillus (35%), Fusarium (23%), Acremonium (12%), Paecilomyces (12%), Cladosporium (6%), Alternaria (6%) and Pseudallescheria (6%) were the common isolates. Calcofluor white stain on direct microscopy detected fungi in 20 (95.2%) patients in comparison with 15 (71.4%) patients by both KOH wet mount examination and culture. Calcofluor white stain was significantly more sensitive than KOH wet mount in demonstrating fungal pathogens.

Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin.

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37 degrees C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23 degrees C and 37 degrees C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.

Isolation and characterization of mutants supersensitive to the spindle poison, isopropyl N-3-chlorophenyl carbamate (CIPC) in the fission yeast Schizosaccharomyces pombe.

Mutants supersensitive to the spindle poison, Isopropyl N-3-chlorophenyl carbamate (CIPC) of the fission yeast Schizosaccharomyces pombe were isolated and characterized genetically. Fourteen different recessive loci were assigned for the mutation (donated as cps1 to cps14) and two, cps1 and cps3, were mapped precisely on the chromosomes. Nine mutant strains were also supersensitive to phenothiazine derivatives, inhibitors of calcium-binding protein calmodulin. Four of nine strains were incapable of growing in the presence of 10 microM calcium ionophore A23187, at which the drug had no effect on cell growth in other strains. Fluorescence microscopy using the DAPI and Calcofluor staining methods showed two strains out of four to be defective in normal cell division; most stationary-phase cells of the cps6 mutant were seen to be bi- or tetra-nucleate, being partitioned with one or three septa, respectively. In the other mutant (cps8), enlarged cells were unequally partitioned with multisepta, and each compartment contained several daughter nuclei. The septa appeared aberrant in position within the cell, and situated diagonally but not vertically along the long cell axis.

Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa.

In filamentous fungi, chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development. To investigate the role of chitin synthase in cell wall biogenesis in Neurospora crassa, we cloned a chitin synthase structural gene and examined the consequences of its inactivation. Using degenerate oligonucleotide mixtures designed on the basis of conserved sequences of the Saccharomyces cerevisiae CHS1 and CHS2 polypeptides, a DNA fragment encoding a similar predicted amino acid sequence was amplified from N. crassa genomic DNA. This product was used to probe N. crassa libraries for a gene homologous to one of the yeast genes. Full-length genomic and partial cDNA clones were identified, isolated, and sequenced. The amino acid sequence deduced from a cloned 3.4-kb gene [designated chitin synthase 1 (chs-1)] was very similar to that of the S. cerevisiae CHS1 and CHS2 and the Candida albicans CHS1 polypeptides. Inactivation of the N. crassa chs-1 gene by repeat-induced point mutation produced slow-growing progeny that formed hyphae with morphologic abnormalities. The chs-1RIP phenotype was correlated with a significant reduction in chitin synthase activity. Calcofluor staining of the chs-1RIP strain cross-walls, residual chitin synthase activity, and the increased sensitivity of the chs-1RIP strain to Nikkomycin Z suggest that N. crassa produces additional chitin synthase that can participate in cell wall formation.

Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation

Michiels, K., Verreth, C. & Vanderleyden, J. 1990. Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation. Journal of Applied Bacteriology69, 705–711.Surface polysaccharide production by Azospirillum is demonstrated by fluorescence of colonies grown on media containing the fluorescent dye Calcofluor, which binds to β‐linked polysaccharides. Mutants showing decreased and increased levels of fluorescence are obtained from Azospirillum lipoferum strain Sp59b by chemical mutagenesis, and from A. brasilense strain 7030 by Tn5 mutagenesis.The A. brasilense 7030 fluorescence mutants produce wild‐type levels of exo‐polysaccharide in their culture supernatant fluids, but are affected in flocculation in liquid culture. On the basis of these observations, we postulate that an A. brasilense surface polysaccharide, different from the exopolysaccharide, is involved in both Calcofluor staining and flocculation.It is shown by DNA hybridization that the genetic loci affected in the A. brasilense 7030 fluorescence mutants are different from the A. brasilense exoB and exoC loci, which are involved in exopolysaccharide production.

Detection of chitin deacetylase activity after polyacrylamide gel electrophoresis

Mucor racemosus and Rhizopus nigricans were used as sources of chitin deacetylases. Crude protein extracts were subjected to polyacrylamide gel electrophoresis at pH 8.9 (Davis system) or 4.3 (Reisfeld system) under native conditions. After electrophoresis, an overlay gel containing 0.1% ( w v ) glycol chitin as substrate was incubated in contact with the separation gel. Chitin deacetylase activity was revealed by uv illumination with a transilluminator after staining for 5 min in 0.01% ( w v ) Calcofluor white M2R. Chitosan (deacetylated chitin) generated by chitin deacetylases appeared more fluorescent than the intact chitin embedded in the overlay gel. Chitosan in a separate overlay gel was also subjected to a nitrous acid treatment which specifically depolymerizes chitosan while leaving chitin intact. Hydrolysis of chitosan by nitrous acid followed by Calcofluor staining yielded dark (nonfluorescent) bands (chitin deacetylase activities) in the fluorescent chitin-containing gel. Both assays revealed the presence of several chitin deacetylases from Zygomycetes. The same assays were performed after denaturing electrophoresis in 12% ( w v ) polyacrylamide gels containing 0.1% ( w v ) glycol chitin. Enzymes were renatured in buffered 1% ( v v ) purified Triton X-100. Chitin deacetylases with estimated molecular weights between 26,000 and 64,000 were detected after Calcofluor staining. The assays were also performed in two-dimensional gel electrophoretic systems. Chitin deacetylases can be rapidly revealed by using the assay involving the nitrous acid treatment. However, both assays (with and without nitrous acid treatment) should be run to conclusively demonstrate chitin deacetylase activity after polyacrylamide gel electrophoresis.

FACTORS INFLUENCING THE HARDNESS (MILLING ENERGY) AND MALTING QUALITY OF BARLEY

The hardness of samples of 41 barley cultivars grown in Australia was determined by measuring milling energy. The milling energy was negatively correlated (r= −0.49) with the starch and positively correlated (r=0.50) with the β‐glucan content. The correlation with protein content was not significant. This suggests that a low starch content and a high β‐glucan content may contribute to hardness but other factors may probably also be involved. The extent of modification of these barley samples measured by Calcofluor staining after steeping and 48 h of germination was correlated with the grain hardness (r= −0.56). Factors contributing to grain hardness may limit the rate of endosperm modification during malting, indicating the value of selecting softer cultivars for malting. 1990 The Institute of Brewing & Distilling

Diffuse growth, a new pattern of cell wall deposition for the Rhodophyta

Abstract By means of calcofluor staining, cell elongation measurements, and microspectrofluorometry, diffuse cell wall deposition is demonstrated for the first time in the red algae in Audouinella dasyae (Acrochaetiaceae, Acrochaetiales), Spermothamnion repens and Tiffaniella snyderae (Ceramiaceae, Ceramiales). Measurements of cell length indicate that cell elongation is occurring, but neither visual inspection nor microspectrofluorometry of cells stained with calcofluor show band deposition. The Rhodophyta thus possess all three mechanisms of cell wall deposition known to operate in plants: apical cell wall deposition in all apical cells examined to date, and either band deposition or diffuse deposition in intercalary cells. These results suggest that patterns of wall deposition may be a useful systematic character in Ceramiaceae, with the tribe Spermothamnieae possessing diffuse growth, and other tribes investigated to date exhibiting band growth.

Malt Modification by Calcofluor Staining

Malt Modification by Calcofluor Staining

The effects of taxol and colchicine on microtubule and microfibril arrays in elongating plant cells in culture

Treatment with 10 μM taxol or 3 mM colchicine results in the development of similar aberrant morphotypes in Vicia hajastana (vetch) suspension cultures. After 2 days exposure to either drug, cells exhibit a disruption in polarity (i.e., swelling or spherical morphology). In taxol-treated cells, microtubules are abundant and brightly stained. In the presence of colchicine, microtubules are absent. Calcofluor staining illustrates that in the presence of taxol the microfibrils appear as thick ordered bundles, while in the presence of colchicine, microfibril bundles are smaller and exhibit less detectable order. The presence of thick bundles of microfibrils is confirmed in taxol-treated cells by the analysis of birefringence patterns. Walls of colchicine-treated cells exhibit a patchy birefringence pattern in polarized light, with adjacent patches exhibiting different order. Although taxol-treated cells contain abundant microtubules, their morphology and wall deposition resemble those of cells that lack microtubules. These observations indicate that the dynamics of microtubules may be important to their proper functioning in cell wall deposition. The differences in wall morphology between taxol- and colchicine-treated cells may reflect the different mechanisms of action of these two drugs.

Rhizobial purine and pyrimidine auxotrophs: nutrient supplementation, genetic analysis, and the symbiotic requirement for the novo purine biosynthesis

Previously described Rhizobium leguminosarum bv. phaseoli mutants elicit nodules on bean without infection thread formation. These mutants were shown to be purine or, in one case, pyrimidine auxotrophs. Each of the seven purine auxotrophs grew normally when supplied the penultimate precursor of inosine, 5-aminoimidazole-4-carboxamide riboside. Four seemed blocked early in the purine pathway, because they were also thiamine auxotrophs. Reversion analysis and genetic complementation using cloned wild-type DNA showed that in each mutant a single mutation was responsible for both the symbiotic defect and purine or pyrimidine auxotrophy. The mutations were mapped to five dispersed chromosomal locations. The previously reported weak Calcofluor staining of these mutants on minimal agar appeared to be caused by partial growth on contaminating nutrients in the agar, rather than deficient exopolysaccharide production. Nodulation by the mutants was not enhanced by supplying purine or pyrimidine compounds exogenously. Furthermore, with or without added purine, the purine auxotrophs grew in the root environment as well as the wild type. However, nodulation by the purine auxotrophs was enhanced greatly in the presence of 5-aminoimidazole-4-carboxamide riboside. The results suggest that undiminished metabolic flow through de novo purine biosynthesis, or a particular intermediate in the pathway, is essential in early symbiotic interactions.

Malt Modification by Calcofluor Staining

Malt Modification by Calcofluor Staining

Estimation of Malt Modification Using Calcofluor Staining

"Estimation of Malt Modification Using Calcofluor Staining." Journal of the American Society of Brewing Chemists, 45(3), p. 101