Calcofluor Staining Research Articles

Calcofluor staining of cellulose during microcyst differentiation in wild-type and mutant strains of Polysphondylium pallidum.

Calcofluor White ST was used to monitor the morphological events in the biogenesis of cellulose in the microcyst wall of the wild-type strain (WS-320) and two developmental mutants (mic-1 and mic-2) of Polysphondylium pallidum. During encystment, the cell surface acquires a Calcofluor-specific material which appears to be cellulose because of its sensitivity to purified cellulase. Cellulose-containing vesicles appear distributed throughout the cytoplasm of encysting cells of the three strains. Later, the cellulose-rich vesicles appear near the cell surface. Subsequently, the cell surface stains with Calcofluor, and the vesicles are no longer detectable. Intracellular vesicles resembling the cellulose-rich vesicles in size, in the timing of appearance, and in cellular location are also seen in thin sections. These vesicles are surrounded by a single unit membrane, and their amorphous matrix, which contains a dense irregular core, further implicates them as the basis for the bilayered microcyst wall.

The ultrastructure of germinating barley seeds. I. Changes in the scutellum and the aleurone layer in Nordal barley

The ultrastructure of the scutellum and the aleurone layer has been examined in seeds of Nordal barley following malting for 0, 30, 72, 120 and 162 hours at 15°C. Thick sections from the seeds were stained with Calcofluor White M2R New and examined in the light microscope to determine the extent of cell wall degradation in the endosperm. The changes in the scutellar parenchyma cells include degradation of the protein bodies and the appearance of vacuoles and prominent amyloplasts. The analysis of a serially sectioned epithelial cell from the scutellum 72 hours after the start of malting revealed a large number of Golgi apparatuses, preferentially located near the cell wall. It is suggested that the presence and location of the Golgi apparatuses are related to enzyme secretion. Following malting for 72 hours or more, the epithelial cells contain lomasomes and membrane aggregates, termed membrane bodies. The analysis indicated that lomasomes originate from the membrane bodies and are involved in the growth of the cell walls of the epithelium. Organelles indicating cellular activity, i.e., rough endoplasmic reticulum, mitochondria and Golgi apparatuses, first appear in the epithelial cells of the scutellum, and later in the cells of the aleurone layer. Furthermore, the degradation of the endosperm starts in the vicinity of the scutellum as judged by the calcofluor staining. Therefore, it is concluded that the scutellar epithelium provides the enzymes during the initial degradation of the endosperm. In seeds malted for 162 hours, aleurone cells with a cytoplasmic organisation indicative of active metabolism are located in the embryo part of the seed, whereas aleurone cells adjacent to unmodified endosperm in the distal end of the same seed show little or no structural sign of activity.

Protoplast Formation and Cell Wall Regeneration in Chlorosarcinopsis halophila Studied by Scanning and Transmission Electron Microscopy, and Calcofluor Staining

Protoplasts (90% +) were induced in 4 h in the salt-tolerant single-cell green alga, Chlorosarcinopsis halophila , by incubation in seawater + 0.6 M mannitol + 3% Cellulysin. Cell wall regeneration (90% +) in seawater without protective osmoticum, started in 4 h, and was completed by 80% of the protoplasts in 72 h. Regenerating protoplasts with incomplete new walls started to divide in 8–12 h. The entire process was studied concurrently be scanning and transmission electron microscopy and by Calcofluor fluorescence. The outer common wall surrounding the 2–8 cell cluster, and the inner cell walls, were completely digested by the Cellulysin. Protoplasts were formed by dividing cells as commonly as by clustered cells. Protoplasts showed dense accumulation of lipid bodies. There were two pyrenoids per chloroplast. A conspicuous micro-tubule organizing center was associated with the nucleolus. There were transient Calcofluor-positive wall plaques formed prior to cell walls.

Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.

The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors.

Zygote formation inCosmarium botrytis studied by scanning and transmission electron microscopy, phase-contrast, and Calcofluor staining

Rapid zygote formation byCosmarium botrytis was induced in a liquid medium by incubation in 5% CO2. Conjugation and zygote formation were studied by SEM, TEM, phase-contrast, and Calcofluor fluorescence microscopy. It was observed that the cells divided immediately prior to conjugation and formed Calcofluor fluorescent conjugation papillae as soon as the primary wall was shed. The conjugating cells and the resultant zygote were envelopped by a non-fluorescent mucilagenous envelope which was eventually pierced by the zygote spines, but never shed. The very young smooth-walled zygote had a thick Calcofluor fluorescent wall. At that stage the zygote could be plasmolysed in 0.4 M mannitol, but no protoplast could be induced to emerge even with the addition of up to 5% Cellulysin; probably indicating that the zygote wall composition and structure is different from that of the secondary wall of the vegetative cells, particularly in the absence of mucilage pores.

Cell differentiation during fruiting body formation in Polysphondylium Pallidum

The spatial pattern of cellular differentiation was studied during fruiting body formation in Polysphondylium pallidum using 3 different staining methods: Calcofluor fluorescence (cellulose accumulation), neutral red (prestalk cells) and immunofluorescence (prespore cells). Neutral-red staining revealed the existence of a clear prestalk region which becomes evident during aggregation and continues throughout culmination. Immunofluorescent staining demonstrated that cells in the prestalk region gradually lose their presporeness (fluorescence) as they are transformed into differentiated stalk cells. Calcofluor staining revealed that stalk cell differentiation begins during the mid-aggregation phase and that the mode of formation of the main stalk and the side branches differs slightly in morphology. Calcofluor staining also demonstrated the development, during aggregation, of a thick cellulosic girdle with lateral tubular extensions which surround the aggregation streams. The above results are discussed in terms of our present knowledge about differentiation and morphogenesis in cellular slime moulds.

Vital and Calcofluor staining ofCosmarium and its protoplasts

Intact cells, protoplasts, primary and secondary walls ofCosmarium species were stained with 13 vital stains and with Calcofluor. The intracellular contents of viable cells and protoplasts were not stained with any of the test dyes although the primary and secondary walls often were stained. Protoplasts disintegrated after short periods in acid stains but did survive for up to 24 hours in several dilute basic dyes. Once the protoplast membrane was damaged, the nuclei and cytoplasm were brightly stained with most dyes, whereas the vacuoles remained unstained. Calcofluor was particularly useful in determining the porous structure and pattern of the primary walls.

Microfibril deposition on cultured protoplasts ofVicia hajastana

Cell wall regeneration by protoplasts fromVicia hajastana suspension cultures was investigated with Calcofluor White ST staining and platinum-palladium surface replicas. Microfibril deposition was initiated after 10–20 minutes of culture and within 20 hours protoplasts were covered with a heavy mat of microfibrils. The early stages of microfibril formation could not be detected with Calcofluor staining.

Production of Guard Cell Protoplasts from Onion and Tobacco

Guard cell protoplasts (GCP) from young cotyledons of onion and tobacco were isolated in culture microchambers where optimal isolating and culture conditions could be determined in situ. The digestion course was quantified by following under polarized light the loss of.retardation of the birefringent cellulose of the guard cells. The assay showed that driselase has a 5-fold higher cellulytic activity than cellulysin. Driselase is, however, harmful to the GCP. Calcofluor staining was less adequate for establishing digestion courses because it increases sharply after exposing guard cells to cellulysin.Osmotic conditions were crucial for GCP survival. Onion guard cells fragment in the presence of strong plasmolyticum (>0.45 m) indicating cytoplasmic connections between neighboring guard cells and/or cytoplasmic attachments to the wall. Tobacco guard cells plasmolyzed with 0.7 m mannitol revealed several areas of strong attachment to the wall which resulted in severe damage to the cells. Healthy tobacco GCP are obtained by an initial digestion with 4% (w/v) cellulysin in 0.23 m mannitol for 2 to 3 hours followed by an increase in the osmoticum to 0.7 m to stabilize the forming protoplasts.Onion GCP were obtained by digesting paradermal slices with 4% (w/v) cellulysin in 0.23 m mannitol. Protoplasts can be osmotically released by replacing the enzyme solution with 0.23 m mannitol at early stages of digestion. They are also available after prolonged digestion (6-12 hours). Paradermal slices also yield mesophyll and epidermal cell protoplasts but they can be selectively washed away if a pure preparation of GCP is desired. Onion GCP have been kept alive in a simple culture solution for up to 10 days.

Detection of areas of wall differentiation in fungi using fluorescent staining.

Fungal colonies were stained with a fluorescent brightner, Calcofluor White M2R New. When viewed using ultraviolet light certain hyphal regions showed intense fluorescence, whereas others showed a lower intensity fluorescence. The apical 10 μm of leadingBotrytis cinerea hyphae showed intense fluorescence. Developing septa and side branches also showed this intense localised fluorescence. Calcofluor staining of hyphae and their subsequent growth illustrated the phenomenon of tip extension.

Tuberculosis Status and Coinfection of Pulmonary Fungal Infections in Patients Referred to Reference Laboratory of Health Centers Ghaemshahr City during 2007-2017.

Pulmonary tuberculosis is one of the most important health concerns. Pulmonary fungal infections have clinical and radiological characteristics similar to tuberculosis which may be easily misdiagnosed as tuberculosis. This study aimed to evaluate tuberculosis (TB) status and coinfection of TB with pulmonary fungal infections in patients referred to reference laboratory of health centers Ghaemshahr city during 2007-2017. This cross-sectional study was conducted during eleven years, between 2007-2017, on 3577 patients with suspected TB referred to health centers of Ghaemshahr City. For isolation, sputum smear preparation and Ziehl-Neelson staining in companying with microscopy direct observation and KOH 10% + white Calcofluor staining was used. The culture of fungi was performed on Sabouraud Dextrose agar, Czapek and chrome agar media. Then, data were analyzed using SPSS software (version 16) through Student's t-test, Fisher test, and Odds Ratio. P values <0.05 were considered statistically significant. Of 3577 patients, 10731 smears were prepared, 3.6% (n=130) of patients were identified as smear-positive pulmonary tuberculosis, 86.4% (n=3090) were smear negative and 10% (n=357) drug-resistant TB. The mean age of patients was 48±1.8 years. With increasing age, the prevalence of TB has increased which was statistically significant (P value <0.05). Prevalence of tuberculosis in the age group over than 57 years in males and females had an ascending trend. Amongst the positive tuberculosis patients, 16/130 cases (12.3%) had the coinfection of TB with fungi microorganisms. Our findings showed the coinfection of fungi agents in patients with tuberculosis that should be considered.