• 제목/요약/키워드: Crystal violet assay

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In vitro cytotoxicity of Acanthamoeba spp. isolated from contact lens containers in Korea by crystal violet staining and LDH release assay

  • Shin, Ho-Joon;Cho, Myung-Soo;Jung, Suk-Yul;Kim, Hyung-Il;Im, Kyung-Il
    • Parasites, Hosts and Diseases
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    • v.38 no.2
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    • pp.99-102
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    • 2000
  • In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthauloeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml Iysate treatments, respectively. Acanthamoeba cutbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. costellunii and A. hafchefti which showed 83.6% and 75.5% or cytotoxicity. Acanthamoeba roureba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml Iysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain.

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Photodynamic Therapy induced Cell Death using ALA and 632nm Diode Laser in A549 Lung Cancer Cells (A549 폐암세포주에서 ALA와 632nm Diode Laser를 이용한 광역학치료 유도성 세포사)

  • Kim, Youn Seup;Park, Jae Seuk;Jee, Young Koo;Lee, Kye Young
    • Tuberculosis and Respiratory Diseases
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    • v.56 no.2
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    • pp.178-186
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    • 2004
  • Background : Photodynamic therapy (PDT) is a new therapeutic method aimed at the selective destruction of cancer cells. The outcome is death of cancer cells through apoptosis or necrosis. The aim of this study was to investigate the characterization of PDT induced cell death in A549 lung cancer cells. Materials and methods : A549 cells were used as the lung cancer cell. 5 aminolevulinic acid (ALA) was used as the photosensitizer and a 632nm diode laser (Biolitec, Germany) as the light source. Cells were incubated with various concentrations of ALA. The 632nm diode laser was then administered for various laser irradiation times. The treated cells were incubated with 24, 48 and 72 hours. The cell viabilities were measured using the crystal violet assay and light microscopy. To observe the cell death mechanism after PDT, cells were observed under fluorescence microscopy after double staining with Hoechst 33342 and propium iodide after PDT. Results : In the crystal violet assay at 24 hours after PDT with a $3.2J/cm^2$ laser irradiation power, the cell viabilities were $89.56{\pm}4.11$, $87.67{\pm}5.48$, and $69.37{\pm}8.84$ with ALA concentrations of 10, 100, and $1mg/m{\ell}$, respectively. In crystal violet assay at 24 hours after PDT with $1mg/m{\ell}$ of ALA, the cell viabilities were $74{\pm}19.85$, $55{\pm}6.1$, and $49.06{\pm}16.64%$ with 1.6, 3.2 and $6.4J/cm^2$ laser irradiation powers, respectively. However, increasing the interval time after PDT did not change the cell viabilities. In the apoptosis assay, photodynamic therapy was inducing the apoptotic cell death. Conclusions : This study shows the apoptotic anticancer effect of photodynamic therapy in A549 lung cancer cells. However, further evaluations with other cancer cells and photosensitizers are necessary.

Persistence of Multidrug-Resistant Acinetobacter baumannii Isolates Harboring blaOXA-23 and bap for 5 Years

  • Sung, Ji Youn;Koo, Sun Hoe;Kim, Semi;Kwon, Gye Cheol
    • Journal of Microbiology and Biotechnology
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    • v.26 no.8
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    • pp.1481-1489
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    • 2016
  • The emergence and dissemination of carbapenemase-producing Acinetobacter baumannii isolates have been reported worldwide, and A. baumannii isolates harboring blaOXA-23 are often resistant to various antimicrobial agents. Antimicrobial resistance can be particularly strong for biofilm-forming A. baumannii isolates. We investigated the genetic basis for carbapenem resistance and biofilm-forming ability of multidrug-resistant (MDR) clinical isolates. Ninety-two MDR A. baumannii isolates were collected from one university hospital located in the Chungcheong area of Korea over a 5-year period. Multiplex PCR and DNA sequencing were performed to characterize carbapenemase and bap genes. Clonal characteristics were analyzed using REP-PCR. In addition, imaging and quantification of biofilms were performed using a crystal violet assay. All 92 MDR A. baumannii isolates involved in our study contained the blaOXA-23 and bap genes. The average absorbance of biomass in Bap-producing strains was much greater than that in non-Bap-producing strains. In our study, only three REP-PCR types were found, and the isolates showing type A or type B were found more than 60 times among unique patients during the 5 years of surveillance. These results suggest that the isolates have persisted and colonized for 5 years, and biofilm formation ability has been responsible for their persistence and colonization.

Paeonia lactiflora Inhibits Cell Wall Synthesis and Triggers Membrane Depolarization in Candida albicans

  • Lee, Heung-Shick;Kim, Younhee
    • Journal of Microbiology and Biotechnology
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    • v.27 no.2
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    • pp.395-404
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    • 2017
  • Fungal cell walls and cell membranes are the main targets of antifungals. In this study, we report on the antifungal activity of an ethanol extract from Paeonia lactiflora against Candida albicans, showing that the antifungal activity is associated with the synergistic actions of preventing cell wall synthesis, enabling membrane depolarization, and compromising permeability. First, it was shown that the ethanol extract from P. lactiflora was involved in damaging the integrity of cell walls in C. albicans. In isotonic media, cell bursts of C. albicans by the P. lactiflora ethanol extract could be restored, and the minimum inhibitory concentration (MIC) of the P. lactiflora ethanol extract against C. albicans cells increased 4-fold. In addition, synthesis of $(1,3)-{\beta}-{\small{D}}-glucan$ polymer was inhibited by 87% and 83% following treatment of C. albicans microsomes with the P. lactiflora ethanol extract at their $1{\times}MIC$ and $2{\times}MIC$, respectively. Second, the ethanol extract from P. lactiflora influenced the function of C. albicans cell membranes. C. albicans cells treated with the P. lactiflora ethanol extract formed red aggregates by staining with a membrane-impermeable dye, propidium iodide. Membrane depolarization manifested as increased fluorescence intensity by staining P. lactiflora-treated C. albicans cells with a membrane-potential marker, $DiBAC_4(3)$ ((bis-1,3-dibutylbarbituric acid) trimethine oxonol). Membrane permeability was assessed by crystal violet assay, and C. albicans cells treated with the P. lactiflora ethanol extract exhibited significant uptake of crystal violet in a concentration-dependent manner. The findings suggest that P. lactiflora ethanol extract is a viable and effective candidate for the development of new antifungal agents to treat Candida-associated diseases.

Inhibitory effects of tuberostemonine on Staphylococcus aureus biofilm (Tuberostemonine에 의한 Staphylococcus aureus의 생물막 억제 효과)

  • Yum, Su Jin;Kim, Seung Min;Kwon, Jun Hyeok;Jeong, Hee Gon
    • Korean Journal of Food Science and Technology
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    • v.54 no.2
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    • pp.241-246
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    • 2022
  • Antibiotic resistance is a serious problem to food safety as well as human healthcare. To avoid this, there are several approaches for a new class of antibiotic agents that target only production of virulence factors such as biofilm without bacterial growth defect. The objective of this study was to investigate the antibiofilm activity of tuberostemonine in Staphylococcus aureus. Tuberostemonine significantly reduced the biofilm formation (26.07-47.02%) in the crystal violet assay whereas there were no effect on S. aureus growth. The dispersion in preformed biofilm was also observed by confocal laser scanning microscopy (CLSM). Quantification real-time PCR revealed that the icaA and agrA expression having an important role in biofilm production of S. aureus were strongly affected with tuberostemonine. These results suggest that tuberostemonine has potential for controlling biofilm formation and dispersion by effect on virulence regulation of S. aureus.

The Pathogenicity of Aeromonas hydrophila Isolated from Freshwater fish and Human (내수 양식어와 인체에서 분리한 Aerornonas hydrophila의 병원성에 관한 연구)

  • 이명원;김호훈;이연태;맹은호
    • Journal of environmental and Sanitary engineering
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    • v.6 no.1
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    • pp.31-46
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    • 1991
  • Aeromonas hydrophila which bacause various diseases in human also infects fresh water fish, severly damaging the fishing industries. To prevent disease in humans and reduce damaging on the fishing industries, We have examined several characteristics of Aeromonas hydrophila and obtained the following results. All of the strains gave a posive voges-proskauer, methyl-red, salicin and esculin reaction. Seventeen(94.4%) A. hydrophila strains presented the phenotype SP-PAB- in autoagglut-ination test, but only strain AH 997 showed $SP^{+}PAB^{+}$. in autoagglut ination test, but only strain AH 997 $SP^{+}PAB^{+}$ All of the strains took up the censored to various degrees. Three of 18 strains showed positive reaction in crystal violet binding test. Hemolytic activity ranged from titers of 0 to 1/256. Seven of the 17(38.8%) A. hydrophila strains were positive in sucking mouse assay. Cytotoxin activity on vero and RK cells was displayed various titers.(1/2-1/1024)

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Biofilm-forming ability and adherence to poly-(methyl-methacrylate) acrylic resin materials of oral Candida albicans strains isolated from HIV positive subjects

  • Uzunoglu, Emel;Bicer, Arzu Zeynep Yildirim;Dolapci, Istar;Dogan, Arife
    • The Journal of Advanced Prosthodontics
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    • v.6 no.1
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    • pp.30-34
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    • 2014
  • PURPOSE. This study evaluated the adhesion to acrylic resin specimens and biofilm formation capability of Candida albicans strains isolated from HIV positive subjects' oral rinse solutions. MATERIALS AND METHODS. The material tested was a heat-cured acrylic resin (Acron Duo). Using the adhesion and crystal violet assays, 14 oral Candida albicans isolated from HIV-positive subjects and 2 references Candida strains (C. albicans ATCC 90028 and C. albicans ATCC 90128) were compared for their biofilm production and adhesion properties to acrylic surfaces in vitro. RESULTS. There were no significant differences in adhesion (P=.52) and biofilm formation assays (P=.42) by statistical analysis with Mann-Whitney test. CONCLUSION. Denture stomatitis and increased prevalence of candidal carriage in HIV infected patients is unlikely to be related to the biofilm formation and adhesion abilities of C. albicans to acrylic resin materials.

Honokiol as an Effective Antimicrobial Compound against Causative Agent of American foulbrood, Paenibacillus larvae

  • Song, Hyunchan;Kim, Ki-Young
    • Journal of Apiculture
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    • v.34 no.2
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    • pp.131-136
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    • 2019
  • Recently, number of honeybees (Apis mellifera) has visibly decreased because they are vulnerable to some diseases like American foulbrood disease. American foulbrood disease, which is caused by Paenibacillus larvae, is emerged as great cause of decrease in number of honeybees. After antibiotic-resistant strain emerged, it is now more difficult to treat those pathogens successfully. Researches on finding alternative antibacterial compound are ongoing. In this study, we examined the antibacterial effect of honokiol on P. larvae. Honokiol showed great antibacterial effect with minimum inhibitory concentration of 12.5 ㎍/mL and minimum bactericidal concentration of 50 ㎍/mL. An agar diffusion test also confirmed the anti-Paenibacillus larvae activity of honokiol with an inhibitory zone of 9±0.5 mm. Since honokiol is known to interact membrane of some bacteria, we measured 260 nm absorbing particles, which could be induced by leakage of cells, and confirmed that the leakage of P. larvae occurred in dose-dependent manners. However, result of crystal violet assay suggested that honokiol has only mild anti-biofilm formation effect on P. larvae, which means honokiol controls the bacteria by inducing the bursting of membrane. Finally, an additive effect of honokiol with tetracycline and terramycin was found using a checkerboard assay with a fractional inhibitory concentration index value of 0.5.

Molecular Characterization and Antimicrobial Susceptibility of Biofilm-forming Acinetobacter baumannii Clinical Isolates from Daejeon, Korea (대전지역에서 분리된 생물막 형성 Acinetobacter baumannii 임상분리주의 분자유전학적 특성과 항균제 감수성양상)

  • Sung, Ji Youn
    • Korean Journal of Clinical Laboratory Science
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    • v.50 no.2
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    • pp.100-109
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    • 2018
  • The emergence and dissemination of multidrug-resistant (MDR) Acinetobacter baumannii isolates have been reported worldwide, with most of these possessing the ability to form biofilms. Biofilm formation is an important virulence factor associated with the resistance to disinfection and desiccation. This study examined the genetic basis of antimicrobial resistance mechanisms of biofilm-forming A. baumannii clinical isolates. Imaging and quantification of biofilms were performed by a crystal violet assay and 46 biofilm-forming A. baumannii isolates were selected. Subsequently, 16 isolates belonging to different clones were identified using REP-PCR, and detection of the antimicrobial determinants in the isolates was carried out. The 16 isolates included 9 non-MDR and 7 MDR isolates. The mean biomass $OD_{560}$ values of the non-MDR (0.96) and MDR (1.05) isolates differed but this difference was not significant. In this study, most biofilm-forming MDR A. baumannii isolates contained various antimicrobial resistance determinants ($bla_{OXA-23}$, armA, and mutations of gyrA and parC). On the other hand, most biofilm-forming non-MDR A. baumannii isolates did not contain antimicrobial resistance determinants. These results suggest that there is little correlation between the biofilm-forming ability and antimicrobial susceptibility in A. baumannii isolates. In addition, the emergence of MDR A. baumannii clinical isolates is generally caused by mutations of the genes associated with antimicrobial resistance and/or the acquisition of various antimicrobial resistance determinants.

Characterization of Nitric Oxide (NO)-Induced Cell Death in Lung Epithelial Cells (폐상피세포에서 Nitric Oxide (NO)에 의한 세포사에 관한 연구)

  • Yong, Wha Shim;Kim, Youn Seup;Park, Jae Seuk;Jee, Young Koo;Lee, Kye Young
    • Tuberculosis and Respiratory Diseases
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    • v.56 no.2
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    • pp.187-197
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    • 2004
  • Background : Nitric Oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in other cell types. This study was performed to characterize NO-induced cell death in lung epithelial cells and to investigate the roles of cell death regulators including iron, bcl-2 and p53. Methods : A549 cells were used for lung epithelial cells. SNP (sodium nitroprusside) and SNAP (S-nitroso-N-acetyl- penicillamine) were used for NO donor. Cytoxicity assay was done by MTT assay and crystal violet assay. Apoptotic assay was done by fluorescent microscopy after double staining with propidium iodide and hoecst 33342. Iron inhibition study was done with RBCs and FeSO4. For bcl-2 study, bcl-2 overexpressing cells (A549-bcl-2) were used and for p53 study, Western blot analysis and p53 functionally knock-out cells (A549-E6) were used. Results : SNP and SNAP induced dose-dependent cell death in A549 cells and fluorescent microscopy revealed that SNAP induced apoptosis in low doses but necrosis in high doses while SNP induced exclusively necrotic cell death. Iron inhibition study using RBCs and FeSO4 significantly blocked SNAP-induced cell death. And also SNAP-induced cell death was blocked by bcl-2 overexpression. Finally, we found that SNAP activate p53 by Western blot analysis and that SNAP-induced cell death was decreased in the abscence of p53. Conclusion : In lung epithelial cells, NO can induce cell death, more precisely apoptosis in low doses and necrosis in high doses. And iron, bcl-2, and p53 play important roles in NO-induced cell death.