Auramine O

Flowers for histochemical examination were selected according to the pollen tube kinetics results, at anthesis, two, and three days after pollination. Two flowers - 10 styles - per day were fixed in 2.5% glutaraldehyde in 0.03M saline phosphate buffer pH7.3 for 4 h [78 (link)]. Then the pistils were washed in 0.03M saline phosphate buffer and sequentially dehydrated in an ethanol series (30%, 50%, 70%, and 96%), leaving them one hour in each ethanol concentration. The gynoecia were left for five days in the embedding solution at 4ºC, and then embedded in JB4 plastic resin (Polysciences Inc., 0226A). Both longitudinal and transversal sections 2μm thick were cut on a LEICA EM UC6 ultramicrotome with a glass knife and then placed onto distilled water on a glass slide previously coated with 1% gelatine. Polysaccharides were stained with periodic acid shift reagent-PAS [79 (link)] counterstained with 0.02% Toluidine Blue for general structure, and proteins with 0.25% Naphtol Blue Black in 1% acetic acid [80 (link)]. Also 0.07% calcofluor white for cellulose [48 (link)] and other polysaccharides [52 (link)], 0.01% auramine in 0.05M phosphate buffer for cutin and lipids [81 (link)], and 0.01% acridine orange in 0.03% phosphate buffer, pH7.4 [82 ] were used to observe the stylar morphology.
Slides were observed under bright field LEICA DM2500 microscope carrying 100W light source, and photographs were obtained with a Leica DFC320 camera linked to the software Leica Application Suite. Fluorescence observations were done with the same microscope provided with an epifluorescence source and connected to a CANON Power Shot S50 camera linked to the CANON Remote Capture software. Filters used were 355/455 nm for calcofluor white, and 450/510 nm for auramine and acridine orange stained sections.

For confocal microscopy of mature pollen grains, pollen was placed into a 5-μl drop of auramine O solution (0.001%; diluted in water from the 0.1% stock prepared in 50 mM Tris-HCl), allowed to hydrate, covered with a #1.5 coverslip, and sealed with nail polish. Pollen then was visualized with a 100x oil-immersion objective (NA = 1.4) on Olympus Fluoview1000 or Nikon A1+ confocal microscopes using FITC excitation/emission settings and 3x confocal zoom. To count apertures, images from the front and back view of the pollen grains were taken. If some apertures were present on sides of a pollen grain not directly visible by focusing on the front and on the back, then z-stacks were taken (step size = 300 nm) and 3D images were reconstructed using NIS Elements software v.4.20 (Nikon) and used for aperture counting. To determine the size of pollen grains in plants of different ploidy and genotypes, we used the surface areas of “front-view” pollen images (the surface closest to the microscope objective) as a proxy for cell size and measured these areas using NIS Elements and ImageJ.
For imaging tetrads and dyads, anthers were dissected out of stage-9 flower buds [56 (link)] and placed into the Vectashield anti-fade solution (Vector Labs, Burlingame, CA) supplemented with membrane stain CellMask Deep Red (Molecular Probes, Eugene, OR) (5 μg/ml) and calcofluor white (0.02%). Tetrads were released by covering anthers with a coverslip and applying gentle pressure on a coverslip. For imaging apertures at the tetrad and dyad stage, Vectashield was supplemented with DAPI (1 μg/ml). Tetrads and dyads were imaged on confocal Nikon A1+ with 100x oil-immersion objective (NA = 1.4) and 5x confocal zoom. YFP was excited with 514-nm laser and emission was collected at 522–555 nm, DAPI and calcofluor white were excited with 405-nm laser and collected at 424–475 nm, and CellMask Deep Red dye was excited with 640-nm laser and collected at 663–738 nm. Z-stacks of tetrads were obtained with a step size of 300 or 500 nm and 3D-reconstructed using NIS Elements v.4.20 (Nikon) or IMARIS (Bitplane) software.
To measure levels of YFP fluorescence, the INP1pr:INP1-YFP and DMC1pr:INP1-YFP tetrads were prepared simultaneously and imaged on the same day under identical acquisition conditions on confocal Nikon A1+. The mean YFP signal intensity of diffuse cytoplasmic INP1-YFP fluorescence was determined with the help of NIS Elements v.4.20 (Nikon) for one microspore per tetrad (n≥15 tetrads), using a single optical section with diffused fluorescence for each microspore. The optical sections containing assembled puncta of INP1-YFP at the periphery of microspores were excluded from the analysis.
To determine karyotypes of elongated cells in anther filaments [57 (link)], flowers were fixed in a 3:1 mixture of ethanol: acetic acid for 5 min and neutralized in 50 mM potassium phosphate buffer, pH 7.0. Anther filaments were dissected and placed in a drop of DAPI (10 μg/ml) in 80% glycerol. Confocal z-stacks of elongated cells were obtained and chromosomes counted using maximum intensity projections. Microspore mother cells undergoing meiosis were prepared and visualized as described [58 (link)].

A cross-sectional study was conducted. Faecal sampling was carried out from 2016 to 2018 and obtained from 350 patients with an HIV/AIDS positive status associated with diarrhoea attending inpatient (hospitalisation) and outpatient care units at El Hadi Flici (ex El- Kettar) hospital, Alger city, Algeria. After obtaining informed consent, patients completed a comprehensive questionnaire on age, sex, contact with animals (pets and farm animals) and drinking water sources. Clinical characteristics, including diarrhoea, weight loss, vomiting, abdominal pain and nausea, types of HAART drug regimens and laboratory characteristics, including blood CD4+ T-cell counts, were recorded by the physicians in charge. Cryptosporidium microscopy-based screening was performed in El Hadi Flici Ex El- Kettar hospital, Alger city, Algeria. All specimens were smeared onto glass slides, stained using the modified Ziehl Nielsen (mZN) and auramine techniques [15 (link)] and examined using light (1000×) and fluorescence (100× and 400×) microscopy, respectively (Figure 1). A sample was considered Cryptosporidium-positive if typical 4–6 μm diameter oocysts were visible. Positive samples were transferred to the Centre National de Référence–Laboratoire Expert crypyosporidioses (CNR-LE) (Rouen University Hospital, France) for molecular analysis.

Each sputum sample should contain 5–10 ml of sputum. Direct smear, prepared with sputum, was stained using auramine and then examined by light-emitting diode (LED) microscopy (Carl Zeiss AG, PrimoStar, Germany). The solid culture with LJ medium was performed following the guidelines from the Chinese anti-TB Association (13 ) and the MGIT960 culture was conducted according to the manufacturer's protocol. Briefly, after liquefied and decontaminated by the hydroxide-sodium citrate-N-actyl-L-cysteine method, 0.5 ml of sputum sediment was inoculated onto an LJ slant and incubated at 37°C for up to 8 weeks in an incubator, and/or inoculated 0.5 ml into a 7-ml MGIT tube and incubated in the BACTEC MGIT 960 system (BD Diagnostic Systems, Sparks, MD, USA). Positive cultures of MGIT960 were confirmed by examining Ziehl-Neelsen (ZN)-stained smears for AFB.