• Journal of Microbiology
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• v.42 no.1
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• pp.25-31
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• 2004

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8${\times}$l0$\^$6/ cells/$m\ell$ in a 500$m\ell$ spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-l strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15 % of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-l strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-l stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-l strain rose from 1.0${\times}$l0$\^$5/ to 1.5${\times}$l0$\^$6/ pfu/$m\ell$. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-l strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-l strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

• Korean Journal of Microbiology
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• v.34 no.3
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• pp.126-131
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• 1998

Reduction of hexavalent chromium to its trivalent form by leachate microorganisms was studied in batch and bench-scale continuous stirred tank reactor. The inoculum was a culture of microorganisms in leachate and capable of providing up to 90% chromate reduction during 72 h batch assay with $20mg\;Cr(VI)\;L^{-1}$ in minimal media containing different levels of leachate (10 to 60%) and glucose (50 to 200 mM). Addition of glucose increased the efficiency of chromate reduction, but adverse effect was observed with increase of leachate probably due to the competitive inhibition between chromate and sulfate ions. The continuous culture experiment was conducted for 124 days using synthetic feed containing different levels of chromate (5 to $65mg\;L^{-1}$) at room temperature. With a hydraulic retention time of 36 h, chromate reduction efficiency was mostly 100% when Cr(VI) concentrations in the reactor were in the range of 5 to $50mg\;L^{-1}$ Specific rate of Cr(VI) removal was calculated as $3.492mg\;g^{-1}\;protein\;h^{-1}$ during the period of 101~124 days from the start-up which showed 81.2% of average reduction efficiency. The results indicate the potential application of using leachate microorganisms for detoxification of hexavalent chromium in various chromium-contaminated wastewater from landfill or tannery sites.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.31-39
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• 2007

This study has been carried out for investigating the relatedness of representative 135 Shigella sonnei strains isolated from 2000 to 2004 by using biotyping and antimicrobial resistance. All strains showed typical biochemical characterisics of Shigella strain. Among 135 strains,79 (58.5%) strains were biotype "g",54 (40.0%) strains were biotype "a" and 2 (1.5%) strains were biotype "e". The results of susceptibility test against 16 antimicrobial agents were like this. Most of strains were susceptible to AN, CIP, C and GM. 129 (95.6%) strains were resistant to SXT, 126 (93.3%) strains were resistant to TE and 122 (90.4%) strains were resistant to SM. One hundred thirty two (97.8%) strains were resistant to more than two antimicrobial agents. R28 type (antimicrobial resistance patterns 28: resistant to AM, SAM, TE, TIC, SXT, K, SM and AmC) were 42 strains (31.1%). The other strains were showed 33 kinds of R patterns. The results of $bla_{TEM}$, sulII, tetA and strA gene detection were coincided with phenotype of antimicrobial resistance by disk diffusion method. But some strains which had sulII and strA genes were not showed the resistance against SXT and SM.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.87-91
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• 2011

The aim of this study was to compare the performance of conventional culture and real-time PCR for detection of Cronobacter spp. in powdered foods. Infant formula, baby food and Misugaru inoculated with Cronobacter were enriched in distilled water as first enrichment step, followed by incubating in Enterobacteriaceae enrichment (EE) broth as second enrichment step. A loopful of enriched sample was streaked onto Druggan-Forsythe-Iversen agar, followed by incubating at $37^{\circ}C$ for 24 h. One milliliter of the enriched distilled water and EE broth were used in real-time PCR assay. No statistical differences were observed in the number of positive samples between culture method and real-time PCR (p>0.05) in all types of food samples. The number of positives of real-time PCR was higher in the first enrichment media (distilled water) than the second enrichment media (EE broth), though there was no significant difference (p>0.05). It appears that some components of the second enrichment broth, EE broth, inhibit the reaction of real-time PCR. These results show that real-time PCR using a single enrichment with distilled water could be useful as an effective screening method for detection of Cronobacter while saving much time and labor compared to conventional culture method.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.17-23
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• 2013

Candida biofilms are organized microbial communities growing on the surfaces of host tissues or indwelling medical devices, and the biofilms show enhanced resistance against the conventional antifungal agents. The roots of Coptidis chinensis have been widely used for medicinal purposes in East Asia. The present study was aimed to assess the effect of C. chinensis aqueous extract upon preformed biofilms of 10 clinical Candida albicans isolates and the antifungal activities which contribute to inhibit the C. albicans biofilm formation. Its effect on preformed biofilms was judged using XTT [2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)] reduction assay, and metabolic activity of all tested strains was reduced significantly ($57.3{\pm}14.7%$) at $98{\mu}g/ml$ of the C. chinensis extract. The extract damaged the cell membrane of C. albicans which was analyzed by fluorescein diacetate and propidium iodide staining. The anticandidal activity was fungicidal, and the extract obstructed the adhesion of C. albicans biofilms to polystyrene surfaces, arrested C. albicans cells at $G_o/G_1$ as well, and reduced the growth of biofilms or budding yeasts finally. The data suggest that C. chinensis has multiple antifungal effects on target fungi resulting in preventing the formation of biofilms. Therefore, C. chinensis holds great promise for exploring antifungal agents from natural products in treating and eliminating biofilm-associated Candida infection.

• Korean Journal of Microbiology
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• v.39 no.1
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• pp.36-39
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• 2003

The rapid and reliable 16S rDNA multiplex polymerase chain reaction (PCR) assay was established to characterize bacterial etiologies of middle ear effusion. These etiologies included Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumonia, which were detected in middle-ear effusion (MEE) samples taken from patient with otitis media. A total of 39 MEE samples were aspirated from 26 patients. DNA was extracted from MEE samples, and PCR was done with DNA extracts by using the common primers, which is localized at C4 region in the 16S rDNA gene of all bacterial species, and species-specific primers: (i) Haemophilus-specific primer, (ii) Moraxella- specific primer, and (iii) Streptococcus-specific primer. Among 39 samples tested, 24 (61.5%) were positive for H. influenzae, 10 (25.6%) were positive for M. catarrhalis, 3(7.7%) were positive for S. pneumonia, and 11 (28%) were negative for 165 rDNA multiplex PCR reaction. Nine samples (28.6%) exhibited a mixed infection and were positive for both H. infuenzae and M. catarrhalis. We suggested that 16S rDNA multiplex PCR is a useful method to identify rapidly for rapid identification of the pathogenic bacteria and characterization of bacterial etiologies of middle ear effusion.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.1-6
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• 2006

Saccharomyces cerevisiae Dna2 helicase/endonuclease plays an essential role in removing DNA primers during Okazaki fragment processing in eukaryotic DNA replication. Genome-wide scale co-immunoprecipitation experiments predicted that Dna2 interacts with a novel protein YHR122W (1). In this study, we observed that overexpression of YHR122W gene suppressed the temperature-sensitive phenotype of $dna2\Delta405N$ mutation. To investigate direct interaction between these two proteins, a histidine-tagged recombinant YHR122W protein was expressed and purified from E. coli. Physical interaction between the purified YHR122W and Dna2 proteins was detected by enzyme-linked immunosorbent assays. Further more, the complex formation was most efficient at physiological salt concentration, 150 mM NaCl. The genetic and physical interactions between YHR122W and Dna2 shown in this study suggest that the biological functions of these two proteins may be closely related each other.

• Journal of Environmental Health Sciences
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• v.37 no.5
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• pp.376-386
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• 2011

Objectives: The aim of this study was to evaluate the microbial hazards posed by food utensils and fixtures in food service operations at selected middle and high schools located in Seoul, Korea. Methods: We collected 200 samples of utensils and fixtures including cups, spoons/chopsticks, food trays and tables from five different schools in Seoul. Target microorganisms of this study were divided into two groups: total bacterial count and total coliform as indicators of microbial contamination and Bacillus cereus and Staphylococcus aureus as pathogens of food poisoning. We used selective media to quantify microbial concentration and 16S rRNA PCR assay for qualitative analysis. In addition, intensive interviews with nutritionists were conducted and observations were made to identify factors that may affect microbial contamination. Logistic regression analysis was employed to examine the relationship between the microbial concentration and operation characteristics of each operation. Results: The level of microbial concentration in school B and C were significantly lower than in school A, D and E (p<0.05). Some samples from school A, D and E showed over 3.4 log CFU/100 $cm^2$ (total bacterial count) and 1.0 log CFU/100 $cm^2$ (total coliform), which requires immediate hygienic action. The number of customers per staff member, periodicity of hygiene education for staff and daily operation time of sterilizers were also found to be important factors related with the microbial contamination of food service operations. Conclusions: These results suggested that not only a HACCP (Hazard Analysis and Critical Control Point) approach, but also efforts to assess internal risk factors within operations be needed to reduce the microbial contamination of food utensils and fixtures. This study is expected to provide preliminary data for assessing microbial hazards in food service operations.

• Journal of Life Science
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• v.17 no.8 s.88
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• pp.1141-1146
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• 2007

This study investigated the effects on the biological activities of germinated brown rice soaked in mycelial culture broth of Phellinus linteus. The level of free amino acid was higher in the GBRP extract than those of BR and GBR. The major free amino acids were alanine, valine, isoleucine and methionine in both extracts. The level of ${\gamma}$-aminobutyric acid (GABA) was also increased significantly in the GBR and GBRP. Antioxidant activities of methanol extract of BR, GBR and GBRP were measured by using DPPH radical scavenging and SOD-like activity. Antioxidant activities showed the highest level of 83% and 76% when 100 mg/ml GBR and GBRP, respectively. Stimulation of the macrophages RAW264.7 cells with lipopolysaccharide (LPS) resulted in increased production of nitric oxide (NO) in the medium. However, the methanol extract of GBR and GBRP showed marked inhibition of NO synthesis in a does-dependant manner. These results showed that GBR and GBRP were significant role for activation of immune system in the pathogenesis of inflammatory diseases.