• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.101-111
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• 2017

The task of improving a fungal strain is highly time-consuming due to the requirement of a large number of flasks in order to obtain a library with enough diversity. In addition, fermentations (particularly those for fungal cells) are typically performed in high-volume (100-250 ml) shake-flasks. In this study, for large and rapid screening of itaconic acid (IA) high-yielding mutants of Aspergillus terreus, a miniaturized culture method was developed using 12-well and 24-well microtiter plates (MTPs, working volume = 1-2 ml). These miniaturized MTP fermentations were successful, only when highly filamentous forms were induced in the growth cultures. Under these conditions, loose-pelleted morphologies of optimum sizes (less than 0.5 mm in diameter) were casually induced in the MTP production cultures, which turned out to be the prerequisite for the active IA biosynthesis by the mutated strains in the miniaturized fermentations. Another crucial factor for successful MTP fermentation was to supply an optimal amount of dissolved oxygen into the fermentation broth through increasing the agitation speed (240 rpm) and reducing the working volume (1 ml) of each 24-well microtiter plate. Notably, almost identical fermentation physiologies resulted in the 250 ml shake-flasks, as well as in the 12-well and 24-well MTP cultures conducted under the respective optimum conditions, as expressed in terms of the distribution of IA productivity of each mutant. These results reveal that MTP cultures could be considered as viable alternatives for the labor-intensive shake-flask fermentations even for filamentous fungal cells, leading to the rapid development of IA high-yield mutant strains.

• Journal of Korean Ophthalmic Optics Society
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• v.1 no.1
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• pp.115-118
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• 1996

The study on the cytotoxicities of heavy metals, metal-metal and metal-chelator combinations was carried out to evaluate the cytotoxic effect of those on mouse L929 fibroblasts. The colorimetric assays (NR and MTT) were conveniently carried out in 96-well microtiter plates. The rank order was Cd > Zn Ni > Cr(III) for the heavy metals tested. Examination of the effect of metal-metal interaction on cytotoxicity showed a moderate reduction of cadmium toxicity by zinc. The colorimetric assays were also effectively used to investigate the effect of the chelators, ethylenediamine tetra acetic acid (EDTA) and chitosan. Reduction of heavy metal toxixity by chelator was efficient.

• KSBB Journal
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• v.24 no.1
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• pp.106-112
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• 2009

An optical sensing membrane has been fabricated to measure the concentration of dissolved oxygen(DO) and pH value simultaneously. It has employed HPTS as a pH sensitive dye and a ruthenium(II) complex as a DO sensitive dye. The sensing membrane has been applied to wells in a 24-well microtiter plate. Using the 24-well microtiter plate the concentrations of dissolved oxygen and pH values have been on-line monitored during the cultivations of E.coli DH5${\alpha}$, B.cereus 318 and P.pastoris X-33. On-line monitoring of DO and pH in microorganism cultivation processes showed good performance of the sensing membrane containing 5 mM HPTS and 2 or 5 mg/mL Rudpp.

• Applied Biological Chemistry
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• v.36 no.1
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• pp.7-10
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• 1993

ELISA method was applied for the screening of zearalenone producing strains. The developed ELISA was as follow: $125\;{\mu}l$ of diluted solution (1 : 500) of antibody was added to each microtiter well and incubated overnight at $40^{\circ}C$. For direct competitive ELISA, samples and zearalenone-peroxidase conjugate were mixed in a 1 : 1 ratio, and a $100\;{\mu}l$ of aliquot was then added to antisera-coated wells. Plates were incubated for 30 minutes at $37^{\circ}C$, and wells washed 6 times, and $100\;{\mu}l$ of ABTS substrates was added. Plates were incubated for antother 15 minutes at $37^{\circ}C$, and $100\;{\mu}l$ of stopping reagent was added to the wells and absorbance was recorded at 410nm on ELISA Reader. Among 19 strains showed zearalenone-producing ability by ELISA, 3 strains (R-5, C-46, S-134) produced more than 50 ng/ml of zearalenone.

• The Korean Journal of Pesticide Science
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• v.3 no.3
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• pp.37-44
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• 1999

Two sulphamide (dichlofluanid and tolylfluanid) and three dicarboximide fungicides (iprodione, vinclozolin, procymidone) were used to investigate the correlation between in vitro antifungal activities and in vivo disease controlling activities against Botrytis cinerea, a causal agent of tomato gray mold and to develop efficient in vitro assays. They controlled effectively the development of tomato gray mold disease in vivo and their controlling activities were similar one another. However, several in vitro assays revealed that their in vitro antifungal activities were quite different between sulphamide and dicarboximide fungicides; the formers showed stronger inhibition activities for spore germination than the latters, whereas the formers inhibited mycelial growth less severely than the latters. The results indicate that the fungicides having different modes of action can show different in vitro antifungal activities according to in vitro assays, even if they have similar in vivo disease controlling activities. On the other hand, two rapid and efficient in vitro assays named Microtiter plate methods I (MPM I) and II (MPM II) were developed for the evaluation of fungicides for inhibitory activities against spore germination and mycelial growth of B. cinerea, respectively. The antifungal activities of five fungicides of two chemical groups in MPM I and II were correlated with the inhibitory activities against spore germination and mycelial growth using solid media, respectively.

• The Korean Journal of Pesticide Science
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• v.11 no.1
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• pp.18-26
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• 2007

Fungicidal screening was performed to control soybean black root rot caused by Calonectria ilicicola through in vitro and greenhouse assays. In in vitro assay, 25 fungicides were assessed by an agar dilution method and a 96-well microtiter plate method. While protective fungicides including dithianon, dichlofluanid, mancozeb, and captan showed a very low activity against the mycelial growth C. ilicicola SC03-15 in the agar dilution method, they displayed potent inhibitory activity against spore germination in a 96-well microtiter plate method with $EC_{50}$ values of 4.65, 0.61, 4.64, and $0.29{\mu}g\;mL^{-1}$, respectively. Ergosterol biosynthesis-inhibiting (EBI) fungicides showed different antifungal activity against mycelial growth and spore germination according to molecules. Difenconazole displayed higher antifungal activity against spore germination rather than mycelial growth, and prochloraz inhibited potently both mycelial growth and spore germination with EC50 values less than $1.8{\mu}g\;ml^{-1}$. In contrast, the other EBI fungicides inhibited more highly mycelial growth than spore germination. Carbendazim+diethofencarb and dazomet also inhibited both mycelial growth and spore germination of C. ilicicola SC03-15 at very low concentrations. In greenhouse assay, carbendazim+diethofencarb effectively controlled a soybean black root rot by drenching 2 days before or after inoculation. In addition, tebuconazole showed potent curative activity against soybean black root rot.

• Journal of Food Hygiene and Safety
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• v.13 no.4
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• pp.419-424
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• 1998

An enzyme-linked immunosorbent assay (ELISA) was established for the detection of zearalenone by using monoclonal antibodies produced by Z-M-26 hybridoma cells when injected into a mouse and zearalenone-oxime-OV A conjugate. Zearalenone-oxime-OV A conjugates were diluted with carbonyl buffer, coated to 96 well microtiter plates at $4^{\circ}C$ overnight and blocked with 1% BSA overnight. One thousand times diluted antibody solution together with standard zearalenone or sample was added to 96-well microtiter plates and stood overnight. A secondary antibody conjugated with HRP was added and an hour later, enzyme substrate (TMBZ) solution was added for color develpment. Mter 30 minutes, coloring reaction was terminated by adding 2 N $H_2S0_4$ and the O.D. was measured at 450 nm. Detection range of this method was about 0.1~100 ppb. The established indirect competitive ELISA method was suitable for a rapid and effective analysis of zearalenone in agricultural products.

• Journal of dental hygiene science
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• v.18 no.2
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• pp.69-75
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• 2018

The purpose of our study is to compare the adhesion and biofilm formation abilities of isolates from water discharged from dental unit waterlines (DUWLs). Bacteria were isolated from a total of 15 DUWLs. Twelve isolates were selected for the experiment. To confirm the adhesion ability of the isolates, each isolate was attached to a glass coverslip using a 12-well plate. Plates were incubated at $26^{\circ}C$ for 7 days, and the degree of adhesion of each isolate was scored. To verify the biofilm formation ability of each isolate, biofilms were allowed to form on a 96-well polystyrene flat-bottom microtiter plate. The biofilm accumulations of all isolates formed at $26^{\circ}C$ for 7 days were identified and compared. A total of 56 strains were isolated from 15 water samples including 12 genera and 31 species. Of the 56 isolates, 12 isolates were selected according to the genus and used in the experiment. Sphingomonas echinoides, Methylobacterium aquaticum, and Cupriavidus pauculus had the highest adhesion ability scores of +3 among 12 isolates. Among these three isolates, the biofilm accumulation of C. pauculus was the highest and that of S. echinoides was the third-most abundant. The lowest biofilm accumulations were identified in Microbacterium testaceum and M. aquaticum. Most isolates with high adhesion ability also exhibited high biofilm formation ability. Analysis of adhesion and biofilm formation of the isolates from DUWLs can provide useful information to understand the mechanism of DUWL biofilm formation and development.

• Microbiology and Biotechnology Letters
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• v.25 no.3
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• pp.335-337
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• 1997

In this study, we show a convenient MTT assay for detect the susceptibility of yeast-like form of Trichosporon beigelii against antifungal agents. This assay was developed based on mitocondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan. Cells of T beigelii are seeded into 96-well microtiter plates, and antifungal agents, amphotericin B, magainin and CA-ME hybrid peptide were added with various concentrations. After 24 hr incubation, MTT was added, then incubations were continued for 4 hr. Formazan formation was quantified photometrically after extraction of the formazan with acid sodium dodesyl sulfate (SDS). From this assay, we could obtained MICs of antifungal agents against T. beigelii. The presented method can easily be used as an effective methods to assess the antiftingal action of various agents on yeasts with minimal amounts of antifungal agents.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.21-26
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• 2000

A microarrayer system was developed mainly for manufacturing DNA chips. The 3-axis robot was designed to automatically collect samples from 96-or 384-well microtiter plates using up to 16 simultaneously moving pens and to deposit them on a surface-modified slide glass. This is followed by a wash/dry operation in a clean station. The cycle is repeated with a new set of samples, This system can deposit cDNA or oligonucleotides with spot intervals of $150{\;}\mu\textrm{m}$ and the spot size of $80\mu\textrm{m}$, thus allowing a high density DNA chip containing about 5,000 spots per $\textrm{cm}^2$. The entire procedure is controlled by the Visual C++ program that was written in our laboratory by using a personal computer with Pentium 100 CPU.