In order to perform standardization in analytic methods of cryopreserved human semen, to investigate the differences of resistance to cryoinjury, to define the stage of critical cryoinjury during cryopreservation and to evaluate the quality change after thawing by time interval, the reviability of 30 normal and 30 abnormal semen were evaluated by the supravital stainings of spermatozoa using acridine orange and eosin yellow and the motility assay simultaneously according to the stages of freezing-thawing and the time interval after thawing. The results were summarized as follows: 1. Vitality was estimated higher than motility at all specimens and the gap between two became greater as motility decreased. 2. Reviability of abnormal semen was estimated lower than that of normal semen(p<0.05). 3. The critical cryoinjury to spermatozoa was noticed at the stage of freezing from 4 degrees C to -10 degrees C (p<0.05). 4. The significant decrease in quality of normal cryopreserved semen was noticed between 30 to 60 min. after thawing (p<0.05). These results suggest that the cryoinjury to human semen is different in nature, therefore it is advisable that the quality of cryopreserved human semen should be evaluated by vitality and motility assay simultaneously. And the resistance of abnormal semen to cryopreservation is so low that it would be difficult to be used clinically with satisfaction. Moreover the laboratory studies should be concentrated on freezing method to achieve better reviability and it is desirable in practice that post-thaw semen should be used within 30 min, after thawing.
Acridine Orange ; Cryopreservation ; Eosine Yellowish-(YS) ; Freezing ; Humans* ; Semen ; Spermatozoa*

We investigated the role of autophagy in SNUC5/5-FUR, 5-fluorouracil (5-FU) resistant SNUC5 colon cancer cells. SNUC5/5-FUR cells exhibited low level of autophagy, as determined by light microscopy, confocal microscopy, and flow cytometry following acridine orange staining, and the decreased level of GFP-LC3 puncta. In addition, expression of critical autophagic proteins such as Atg5, Beclin-1 and LC3-II and autophagic flux was diminished in SNUC5/5-FUR cells. Whereas production of reactive oxygen species (ROS) was significantly elevated in SNUC5/5-FUR cells, treatment with the ROS inhibitor N-acetyl cysteine further reduced the level of autophagy. Taken together, these results indicate that decreased autophagy is linked to 5-FU resistance in SNUC5 colon cancer cells.
Acridine Orange ; Autophagy* ; Colon* ; Colonic Neoplasms* ; Cysteine ; Flow Cytometry ; Fluorouracil* ; Microscopy ; Microscopy, Confocal ; Reactive Oxygen Species

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the mycobacteria. However, this method has several issues of false negative results, and hence a comparative experiment of the Ziehl-Neelson's AFB staining and the fluorescence staining method was done to remedy this problem. As the fluorescence staining method brightly highlights the AFB in a dark field, and also as it is observed with the lower power objective, it is a method that can better the observation and shorten the time of observation as well. The fluorescence staining method that was used in this experiment did a comparative analysis of the Auramine O-Rhodamine B and the Acridine Orange. The results showed that although the Auramine O-Rhodamine B allows easier observation of the AFB with a high fluorescence expression rate for the multibacillary leprosy sample, the darkness on the periphery makes it hard to observe anything else, while also making it hard to observe the cell changes and paucibacillary leprosy of the AFB. However, the Acridine Orange staining method highlights the cells in dark green and changes the color of the AFB from bright red to orange making it easier to observe bacilli. The results of the study show that the Acridine Orange method is superior to the Auramine O-Rhodamine B method in detecting acid fast bacilli in specimen.
Acridine Orange ; Benzophenoneidum ; Citrus sinensis ; Darkness ; Fluorescence ; Leprosy, Multibacillary ; Leprosy, Paucibacillary ; Mycobacterium ; Mycobacterium leprae

Several observation suggest that the antimelanocyte autoantibodies could play a role in melanocyte destruction. Some experiments indicate that melanocyte antibodies from patients with vitiligo can kill melanocyte in vitro. In these experiments, we demonstrated that vitiligo patient's sera containing antimelanocyte antibodies can lyse cultured human melanocytes by complement activation. Melanocyte cytotoxicity was measured using the ethidium bromide/ acridine orange viability assay. Significant melanocyte cytotoxicity was seen in sera from patients with both active and inactive vitiligo(p<0.01). Melanocyte cytotoxicity measured with complement-mediated cytotoxicity decreased after systemic steroid treatment(p<0.05) ; however melanocyte cytotoxicity showed no significant change with systemic PUVA therapy.
Acridine Orange ; Antibodies ; Autoantibodies* ; Complement Activation ; Ethidium ; Humans ; Melanocytes ; PUVA Therapy ; Vitiligo*

Purpose: Colonization of denture soft lining materials by Candida albicans can result in clinical problem, and deterioration of the materials. This study aimed to compare the retention and penetration of C. albicans into four denture soft lining materials commonly used. Materials and methods: Four denture soft lining materials(Coe-comfort., Coe-soft., GC soft liner., and Tissue conditioner.) discs were prepared to glass slide and dental stone. Adherence of yeast to surfaces was monitored after one hour incubation of standardized washed cell suspension with test disc surfaces. Adherent cells stained with acridine orange were counted fluorescence microscopy. Penetration of yeast into materials bonded with acrylic resin after 1, 2, 3, 4, 5, 6 and 7 days incubation was observed through sections stained using acridine orange and estimated to quantitative analysis using radioisotope. Results: There was statistical significance in cell numbers between smooth and rough surfaces(p<0.05). Higher numbers of cells were observed on rough surfaces. There was statistical significance in adherent cell numbers into smooth and rough surfaces individually(p<0.05). According to the increase of incubation periods, the cells penetrated into denture soft lining materials were shown to increase. The differences among all kinds of soft liner were statistically significant(p<0.05),and the largest number of cells penetrated into soft liners was observed in the Coe-soft. Conclusion: Initial adherence and penetration of yeast into denture soft lining materials has been influenced by surface roughness and chemical composition of them. The selection of appropriate materials and their fabrication may promote clinical performance.
Acridine Orange ; Candida albicans* ; Candida* ; Cell Count ; Colon ; Dentures* ; Glass ; Microscopy, Fluorescence ; Stomatitis, Denture ; Yeasts

BACKGROUND: In South Korea, indigenous malaria has been reappeared since 1993 and more than 350 cases diagnosed in 1996. For the diagnosis of malaria the classic methods such as thin and thick blood smears with Giemsa or Wright stain has been routinely used. Since recently fluorochrome staining has been shown to be more sensitive, easy to do, and less time-consuming, we applied the new method, Acridine orange stain, for diagnosis of clinically suspected cases. METHODS: Thin and thick blood smears were prepared from civilian patients of Kyunggi Province (n=20) and Republic Of Korea army patients pre- (n=67) and post-treatment (n=13) of malaria. The slides were fixed by methanol and stained by either Giemsa or Acridine orange solution (10-50 g/mL). For comparison, an expert on malaria diagnosis examined them by light and fluorescent microscope, respectively. RESULT: Acridine orange stain was found to be a rapid technique, and as sensitive (83%) as thick smears (83%) for diagnosis of malaria. The detection limit of acridine orange stain was 23.5 parasites/ul of blood. The staining time was much shorter (30 sec) than that of Giemsa stain (30-60min). CONCLUSION: Acridine orange stain is evaluated as a simple, rapid, and sensitive method for malaria diagnosis compared with Giemsa stain.
Acridine Orange* ; Azure Stains ; Diagnosis* ; Gyeonggi-do ; Humans ; Korea ; Limit of Detection ; Malaria* ; Methanol ; Republic of Korea

We assessed the comparatice sensitivity and specificity of acridine orange and Gram stains of corneal scrapings from 29 cases of presumed infectious keratitis. The corneal scrapings were placed on two clear glass slides and allowed to air dry. The slides were fixed in 95% methanol for 2 minutes and each slide was stained by Gram and acridine orange stains respectively. Culture was done with another corneal scraping. Cultures were positive in 19 of 29 cases(66%). Among them 14 cases (74%) were positive in Gram stain and 18 cases (95%) were positive in acridine orange stain. So the sensitivity of acridine orange was greater than that of Gram stain. In the specificity, Gram stain (80%) was better than acridine orange stain(50%). This study demonstrates acridine orange stain as well as gram stain is useful as a simple, rapid screening test for the diagnois of suspected infectious keratitis.
Acridine Orange* ; Coloring Agents* ; Diagnosis* ; Glass ; Keratitis* ; Mass Screening ; Methanol ; Sensitivity and Specificity

OBJECTIVES: The objective of this study was to identify the possible role of stem cell and myeloperoxidase (MPO) in the metabolic activation of styrene, hydroquinone and trichloroethylene, by investigating the effects of stem cell from umbilical cord blood and MPO on the frequency of sister chromatid exchange (SCE) and micronuclei (MN) induction in cultured human peripheral lymphocytes exposed to these chemicals. METHODS: Isolated lymphocytes from whole blood were cultured for 72 hours. The cells were treated with 1.50 mM styrene, 50 microM hydroquinone and 1.50 mM trichloroethylene dissolved with acetone (30 microl in total volume) at 24 hours after the beginning of culture. Control group was treated with acetone only. Immediately after adding these chemicals, 1.3X1 06 cells/ml and 2.6X1 06 cells/ml stem cell or 1.0 and 2.0 unit MPO with H2O2 (for substrate) were added to the cultures. Slides were stained with Giemsa's solution, and acridine orange for sister chromatid exchange, and for micronucleus analysis, respectively. RESULTS: The results were as follows: 1) Myeloperoxidase and stem cell did not significantly affect the frequencies of SCE or MN in the control group. 2) The frequency of SCE or MN with exposure to styrene did not different from control in the absence of stem cell or MPO. Sister chromatid exchange induced by styrene was significantly increased by adding stem cell or MPO in dose-dependent relationship. The frequency of MN induced by styrene significantly increased in the presence of 2.0 unit MPO. 3) The frequency of SCE was significantly increased with exposure to hydroquinone than acetone treated control in the absence of stem cell or MPO. Sister chromatid exchange induction by hydroquinone significantly increased dose-dependently in the presence of stem cell or MPO. There was a tendency of increase of the MN frequency induced by hydroquinone in the presence of stem cell or MPO, but not significant. 4) It was found that trichloroethylene itself did not increase SCE or MN frequency. Frequency of SCE induced by trichloroethylene was significantly increased with adding stem cell (low and high) and 2.0 unit MPO. Even though stem cell or MPO increased the frequency of MN of lymphocyte exposed to trichloroethylene, the difference was not significant. CONCLUSIONS: Authors found that the frequencies of both sister chromatid exchange and micronucleus induced by styrene, hydroquinone, and trichloroethylene were increased significantly with the treatment of stem cell or myeloperoxidase. It was suggested that myeloperoxidase may therefore play an important role in the metabolic activation of styrene, hydroquinone, and trichloroethylene and myeloperoxidase probably be involved in the myelotoxicity of these chemicals.
Acetone ; Acridine Orange ; Biotransformation ; Fetal Blood ; Humans ; Lymphocytes* ; Peroxidase* ; Siblings* ; Sister Chromatid Exchange* ; Stem Cells* ; Styrene* ; Trichloroethylene*

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the diagnosis of leprosy. However, the fluorescent stain performs better and allows the detection of more positive smears. The limitation for its widespread use has been the high cost for fluorescent microscopes. Novel light-emitting diodes (LED) are inexpensive solutions for fluorescent microscopes, and thus fluorescent stain may be a cost-effective step to improve the diagnosis of leprosy in resource-poor countries. And the comparison of auramine and acridine orange for staining of acid-fast bacteria was showed significantly more acid-fast rods after using acridine orange and the number of "false positive" results was somewhat higher on auramine staining. So acridine orange offers a good alternative to auramine which is considered carcinogenic. This study evaluated the comparison of the Ziehl-Neelson's AFB stain and the acridine orange stain in the skin smear based on PCR. As PCR results were taken as gold standard, results of the study revealed that the sensitivity of Ziehl-Neelson's AFB stain was 50% and that of acridine orange stain was 92.2%. This study confirmed that the fluorescence stain method is more sensitive than the Ziehl-Neelsen's staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.
Acridine Orange* ; Bacteria ; Benzophenoneidum ; Diagnosis ; Fluorescence ; Humans ; Laboratory Personnel ; Leprosy ; Microscopy, Fluorescence ; Mycobacterium leprae* ; Mycobacterium* ; Polymerase Chain Reaction ; Skin

BACKGROUND: Autophagy is an important adaptive mechanism in normal development and in response to changing environmental stimuli in cancer. Previous papers have reported that different types of cancer underwent autophagy to obtain amino acids as energy source of dying cells in nutrient-deprived conditions. However, whether or not autophagy in the process of lung cancer causes death or survival is controversial. Therefore in this study, we investigated whether nutrient deprivation induces autophagy in human H460 lung cancer cells. METHODS: H460, lung cancer cells were incubated in RPMI 1640 medium, and the starved media, which are BME and RPMI media without serum, including 2-deoxyl-D-glucose according to time dependence. To evaluate the viability and find out the mechanism of cell death under nutrient-deprived conditions, the MTT assay and flow cytometry were done and analyzed the apoptotic and autophagic related proteins. It is also measured the development of acidic vascular organelles by acridine orange. RESULTS: The nutrient-deprived cancer cell is relatively sensitive to cell death rather than normal nutrition. Massive cytoplasmic vacuolization was seen under nutrient-deprived conditions. Autophagic vacuoles were visible at approximately 12 h and as time ran out, vacuoles became larger and denser with the increasing number of vacuoles. In addition, the proportion of acridine orange stain-positive cells increased according to time dependence. Localization of GFP-LC3 in cytoplasm and expression of LC-3II and Beclin 1 were increased according to time dependence on nutrient-deprived cells. CONCLUSION: Nutrient deprivation induces cell death through autophagy in H460 lung cancer cells.
Acridine Orange ; Amino Acids ; Autophagy ; Cell Death ; Cytoplasm ; Flow Cytometry ; Humans ; Lung Neoplasms ; Malnutrition ; Organelles ; Proteins ; Vacuoles