Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Volumetric histological characterization of optic nerve degeneration using tissue clearing: literature review and practical study.

Tissue clearing technologies can greatly improve the depth and accuracy with which the three-dimensional structure of tissues, especially those of th*-e nervous system, can be visualized. A review of the present literature suggests that the growing diversity and sophistication of various approaches have contributed to the expansion of this method to a greater variety of tissue types, experimental conditions, and imaging modalities. In the proof-of-concept study presented in this paper, a simplified and modified version of the tissue clearing method CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) was used in conjunction with fluorescent staining and immunohistochemistry to illustrate the three-dimensional structure and molecular characteristics of inflammatory and degenerative activity in the mouse optic nerve. Based on the studies summarized in this mini-review, and our impression from using the mCUBIC method, it appears that tissue clearing could be a viable approach revealing three-dimensional histological features of myelin-rich tissues under normal conditions and after injury.

New Cationic fac-[Re(CO)3(deeb)B2]+ Complex, Where B2 Is a Benzimidazole Derivative, as a Potential New Luminescent Dye for Proteins Separated by SDS-PAGE.

Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be used to separate proteins based mainly on their size such as in denaturing gels. Different staining methods have been reported to observe proteins in the gel matrix, where the most used dyes are generally anionic. Anionic dyes allow for interactions with protonated amino acids, retaining the dye in the proteins. Fluorescent staining is an alternative technique considered to be sensitive, safe, and versatile. Some anionic complexes based on d6 transition metals have been used for this purpose, where cationic dyes have been less explored in this context. In this work, we synthesized and characterized a new monocationic rhenium complex fac-[Re(CO)3(deeb)B2]+ (where deeb is 4,4'-bis(ethoxycarbonyl)-2,2'-bpy and B2 is 2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol). We carried out a structural characterization of this complex by MS+, FTIR, 1H NMR, D2O exchange, and HHCOSY. Moreover, we carried out UV-Vis, luminescence, and cyclic voltammetry experiments to understand the effect of ligands on the complex's electronic structure. We also performed relativistic theoretical calculations using the B3LYP/TZ2P level of theory and R-TDDFT within a dielectric continuum model (COSMO) to better understand electronic transitions and optical properties. We finally assessed the potential of fac-[Re(CO)3(deeb)B2]+ (as well as the precursor fac-Re(CO)3(deeb)Br and the free ligand B2) to stain proteins separated by SDS-PAGE. We found that only fac-[Re(CO)3(deeb)B2]+ proved viable to be directly used as a luminescent dye for proteins, presumably due to its interaction with negatively charged residues in proteins and by weak interactions provided by B2. In addition, fac-[Re(CO)3(deeb)B2]+ seems to interact preferentially with proteins and not with the gel matrix despite the presence of sodium dodecyl sulfate (SDS). In future applications, these alternative cationic complexes might be used alone or in combination with more traditional anionic compounds to generate counterion dye stains to improve the process.

"Sporotan" a new fluorescent stain for identifying cryptic spores of Rhodobacter johrii.

We propose a new fluorescent stain "sporotan" and staining protocol which aid in the identification of cryptic endospores which are otherwise mistaken as poly-ß-hydroxyalkanoate granules.

Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels.

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.

Assessment of Cell Viability with Single-, Dual-, and Multi-Staining Methods Using Image Cytometry.

The ability to accurately measure cell viability is important for any cell-based assay. Traditionally, viability measurements have been performed using the trypan blue exclusion method on a hemacytometer, which allows researchers to visually distinguish viable from nonviable cells. While the trypan blue method can work for cell lines or primary cells that have been rigorously purified, in more complex samples such as PBMCs, bone marrow, whole blood, or any sample with low viability, this method can lead to errors. In recent years, advances in optics and fluorescent dyes have led to the development of automated benchtop image-based cell counters for rapid cell concentration and viability measurement. In this work, we demonstrate the use of image-based cytometry for cell viability detection using single-, dual-, or multi-stain techniques. Single-staining methods using nucleic acid stains such as EB, PI, 7-AAD, DAPI, SYTOX Green, and SYTOX Red, and enzymatic stains such as CFDA and Calcein AM, were performed. Dual-staining methods using AO/PI, CFDA/PI, Calcein AM/PI, Hoechst/PI, Hoechst/DRAQ7, and DRAQ5/DAPI that enumerate viable and nonviable cells were also performed. Finally, Hoechst/Calcein AM/PI was used for a multi-staining method. Fluorescent viability staining allows exclusion of cellular debris and nonnucleated cells from analysis, which can eliminate the need to perform purification steps during sample preparation and improve efficiency. Image cytometers increase speed and throughput, capture images for visual confirmation of results, and can greatly simplify cell count and viability measurements.

Antibacterial Efficacy of Pastes Against E Faecalis in Primary Root Dentin: A Confocal Microscope Study.

OBJECTIVES: Management of abscessed primary teeth often present endodontic failure owing to questioned efficiency of dressings or obturating pastes to eliminate Enterococcus faecalis, a resistant bacterium, residing in depth of dentinal tubules. The present study evaluates the antimicrobial efficacy of two antibacterial and two obturating pastes in dentinal tubules of primary teeth infected with Enterococcus faecalis using viability stain and confocal laser scanning microscope (CLSM). STUDY DESIGN: Total 28 samples were prepared.Four groups with 6 samples each were made according to antibacterial pastes i.e. 1% or 2%Chlorhexidine (CHX) + calcium hydroxide (CH), CH + iodoform (Metapex) and Zinc Oxide Eugenol (ZOE). Dentinal tubules from the root canal side were infected with E. faecalis by centrifugation of the bacterial suspension. Two specimens from each group were subjected to 1, 7 and 15 days antibacterial pastes exposure. Viability staining followed by CLSM were used to quantitatively analyze the dead cell count directly inside dentin. RESULT: Univariate analysis showed that all medicaments were significantly effective (p<.05). Kruskal wallis ANOVA test did not show significant difference among four medicaments at day 1 while it was significantly different at day 7 & 15. Paired sample student's t-test revealed significant difference in efficacy between 1& 15 days for 1%CHX+CH; between 1&15,and 7&15 days; between all days for ZOE. Ranking of antimicrobial efficacy of tested medicament was (most effective to the least): 1%CHX+CH(15)> ZOE(15)> Metapex(15)> 2%CHX+CH(15)> 2%CHX+CH(7)> 2%CHX+CH(1)> 1%CHX+CH(7)> 2%CHX+CH(15)> Metapex(1)> ZOE(1)> ZOE(7). CONCLUSIONS: All medicaments were effective against E. faecalis in dentine of primary teeth and their efficacy increased with longer contact with 1%CHX+CH being most effective at day 15. Inclusion of 1% CHX in dressings or obturating pastes might minimize the endodontic relapse and maximize the tooth retention in functional state in pediatric dentistry.

Highly sensitive fluorescent stain for detecting lipopolysaccharides in sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A sensitive and simple technique was developed for the visualization of gel-separated lipopolysaccharides by using a hydrazide derivative, UGF202. As low as 0.5-1 ng total LPS could be detected by UGF202 stain, which is 2- and 16-fold more sensitive than that of the commonly used Pro-Q Emerald 300 and Keenan et al. developed silver stain, respectively. The results indicated that UGF202 stain could be a good choice for LPS determination in polyacrylamide gels.

Environment and body contamination: a comparison of two different removal methods in three types of personal protective clothing.

BACKGROUND: This study aimed to examine the body contamination rates and environmental contamination levels during the removal of 3 types of personal protective clothing (PPC) by the individual accustomed removal method (IARM) and gown removal methods recommended by the Centers for Disease Control and Prevention (CDC). METHODS: Fifty participants performed IARM and CDC-recommended gown removal methods to remove 3 types of PPC (ie, cotton gown, water resistant gown, and plastic apron) in random order at 2 separate sessions after applying Glo Germ simulated germ lotion on the gown's surface. A video demonstrating the CDC-recommended gown removal method was shown between the 2 sessions. After PPC removal, fluorescent stains were counted by an ultraviolet scan under dim light. RESULTS: Following IARM, contaminants were splashed in the surroundings, particularly on the front part of the subject. The plastic apron and cotton gown obtained the highest and lowest contaminative hazards, respectively, to the hands, shoes, and environment. Females, nurses, and senior staff had serious hand or shoe contamination. The CDC removal method more significantly reduced body and environmental contamination of small fluorescent stains (<1 cm(2)), but not of large patches (>1 cm(2)), than IARM. CONCLUSION: The effect of gown removal, PPC type, discarding PPC location, training of infection control measures, hand hygiene, and special work shoes should be considered daily.

Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins.