Acridine orange staining denoted to dna binding dye of the stained free promastigote looked green before entering to the fibroblasts after 12 h culture figure 4a. Acridine orange ao was first extracted from coal tar in the late nineteenth century and was used as a fluorescent dye. How to make the 3d model of acridine orange structural diagram. Acridine orange 10 mgml in water thermo fisher scientific. Evaluation of acridine orange, lysotracker red, and.
Cinkotai and j e maciver, journaljournal of clinical pathology, year1987. Acridine orange stain principle, procedure and result. Diagnosis of malaria by acridine orange fluorescent. Acridine orange staining solution for lysosome and dna. Lipophilic tracer dil and fluorescence labeling of. A simple technique for quantifying apoptosis in 96well plates. Tbe acridine orange used was a product of kepec, chemische fabrik gmbh, c. Staining procedure for a fluorescence microscopy for 24well plate. Fluorescence microscopy a fluorescence microscope is required for examining fluorochromestained smears.
Reticulocytes can be counted manually by fluorescence microscopy on appropriately stained films. Csf smear stained with grams stain and acridine orange stain. Its metachromatic shift to red fluorescence is concentrationdependent and, therefore, acridine orange fluoresces red in avos, such as autolysosomes. Acridine orange hemizinc chloride salt for nucleic acid. Nucleus staining fluorescent dyes with aromatic amino or guanidine groups, such as propidium iodide pi, ethidium bromide eb, diaminophenylindole dapi, acridine orange ao, and hoechst dyes, interact with nucleotides to emit fluorescence. Rapid virtual hematoxylin and eosin histology of breast.
Comparison of acridine orange staining using fluorescence. Protocol for acridine orange staining after dna denaturation acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and single. Ratiometric analysis of acridine orange staining in the. The fluorescent dye, 10 nonyl acridine orange nao, has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. In comparison acridine orange fluorescence staining was found to be more sensitive over giemsa staining especially in low. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological. Novel acridine orange staining protocol and microscopy. Principle, procedure, results and applications august, 2015 nisha rijal staining techniques in microbiology 1 acridine orange is a dye that intercalates or binds with the. This method measured with accuracy the increase in autophagy induced by. Fluorescence microscopy of acridine orange stained nuclei. Reliability of acridine orange fluorescence microscopy in. Acridine orange is a cellpermeant nucleic acid binding dye that emits green fluorescence when bound to dsdna and red fluorescence when bound to ssdna or rna. Acridine orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status.
Eb and pi molecules intercalate inside the dna double helix. Zachleder and others published fluorescence microscopy of acridine orange stained nuclei in chlorococcal algae find, read and cite. Acridine orange ao is a fluorescent dye that was discovered by a group of botanists. Pdf comparison of acridine orange fluorescent microscopy.
Felipe1,3 and guido lenz1,4, abstract acridine orange is a cellpermeable green fluorophore that can be. Fluorescence microscopy fm with acri dine orange ao as the fluorochrome was developed as a specific cytochemical stain ing method for use in exfoliative. The in vitro localization of acridine orange ao in living cells was monitored by means of fluorescence microscopy, quantitative cell viability studies, and photofluorimetric measurements following dyecell. While the procedure of mayor and hill is simple compared with the techniques of biochemical analysis, it involves numerous treatments and the use of fluorescence microscopy. The method for processing and evaluating breast tissue using the rapid staining and imaging technology. Fluorescence methods for performing a reticulocyte count. Acridine orange staining in clinical microbiology a mini.
Staining of bacteriophage nucleic acids with acridine orange. Acridine orange ao stain and fluorescence microscopy have been widely used for the evaluation of gynecologic exfoliative cytology. This cellpermeant cellular stain can be utilized in conjunction with a number of. Mercury vapor or halogen bulb light source about 150 hours of use newer mercury bulbs. Acridine orange is a cellpermeable green fluorophore that can be protonated and trapped in acidic vesicular organelles avos. Acridine orange staining procedure acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination.
The nuclei of the tissue that comprises the structure are rodshaped, 1020 m. Cell viability assay under 3 min with nc3000 chemometec. Ability of substances to absorb short wavelengths of light ultraviolet light and emit them at a longer wavelength. Fluorescence response of acridine orange to changes in ph.
A regular acridine orange staining at neutral ph stained everything brilliant orange, cell nuclei, bacteria, cell debris, etc. Fluorescence light microscopy with differential uptake of fluorescent dna binding dyes such as ebao staining is a method of choice for its simplicity, rapidity, and accuracy. Pdf fluorescence microscopy of acridine orange stained nuclei in. Some microorganisms fluoresce naturally under uv light pseudomonas. The technique of staining with acridine orange for fluorescence microscopy of fresh animal and plant cells, chiefly for the detection of ribonucleic acid in the cytoplasm, was brought to a high degree of. This is my grade 12 chemistry project sch 4u on building a 3d model of acridine orange. Nonviable intracellular bacteria wouldhave stained red as. Detection of leukocytes stained with acridine orange using unique. Acridine orange, a cellpermeable metachromatic fluorescent cationic dye that intercalates dna and rna, is used in fluorescence and epiflouresence microscopy. Mercury vapor or halogen bulb light source about 150 hours of use newer mercury bulbs about 2,000 hours of use led bulbs about 15,000 hours of use excitation and emission barrier filters are necessary for. Its metachromatic shift to red fluorescence is concentration.
Direct acridine orange fluorescence examination of blood. Comparison of acridine orange fluorescent microscopy and gram stain light microscopy for the rapid detection of bacteria in cerebrospinal fluid. Nuclear staining of dermatophytes with the fluorescent dye. Acridine orange, which is a metachromatic intercalator sensitive to dna conformation, has been widely used to detect apoptosis in wildtype, mutant, and drugtreated zebrafish brand et al. Zachleder and others published fluorescence microscopy of acridine orange stained nuclei in chlorococcal algae find, read and cite all the research you need on researchgate. After 2 minutes of staining, the slides are washed gently with water and dried and then examined in a fluorescent microscope. Ao interacts with dna and rna by intercalation or electrostatic. Acridine orange, a vital stain, will intercalate with nucleic acid, changing the dyes optical characteristics so that it will fluoresce bright orange under ultraviolet light. For fluorescence microscopy and microphotography the large universal. Pdf fluorescence microscopy of acridine orange stained. Intracellular distribution of the fluorescent dye nonyl. Three fluorescent dyes, acridine orange ao, lysotracker red dnd99 ltr, and quinacrine qa, were evaluated as acidic vesicle tracers for confocal fluorescence imaging. Pdf simple technique for fluorescence staining of blood. Ratiometric analysis of acridine orange staining in the study of acidic organelles and autophagy marcos p.
Simple technique for fluorescence staining of blood cells with acridine orange. A comparison was made between the acridine orange stain, gram stain and. Acridine orange staining method to reveal the characteristic. The renewed interest in the use of fluorochromes for malaria diagnosis prompted us to evaluate the acridine orange fluorescence technique on blood slides, and to compare it. On the other hand, acridine orange had been used for. Acridine orange fluorescence stain for the diagnosis of. This makes acridine orange staining a quick, accessible and reliable method to assess the volume of avos, which.
The acridine orangecrystal violet staining technique revealed viable greenfluorescing intracellular organisms table 1. First, the cells were grown in rpmi 10%fbs in confocal dishes, treated with compound with or without with wortmannin 0. The metachromatic fluorophore acridine orange ao has demonstrated green fluorescent staining of dormant bacillus spores and orange to red staining of transcriptionally active vegetative. The nc3000 viability and cell count assays use the cell stain. Novel acridine orange staining protocol and microscopy with uv surface excitation allows for rapid histological assessment of canine cutaneous mast cell tumors croix griffin1, richard levenson2. Ratiometric analysis of acridine orange staining in the study of. I used acridine orange staining to detect autophagy. Bacteria stain orange against a green to yellow background of human cells and debris. It is then put in trough with acridine orange staining working solution i. Microscopy with traditional methods for microbiological.
299 656 265 1113 1520 547 681 1312 1411 195 908 694 1129 1353 52 5 509 996 90 395 845 926 447 233 122 929 150 606 134 758 74 572 475 447 930 177 518 1432 1246 1385 622