• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.20 no.4
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• pp.274-281
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• 2010

The objectives of this study are (a) to investigate the distribution patterns and exposure concentrations of biological agents in sawmill industries and (b) to compare sampling methods of biological agents. The representative processes of 5 sawmills were selected to measure total airborne bacteria, fungi, endotoxin as well as dust. Airborne bacteria and fungi were measured with one stage impactor, six stage impactor and gelatin filteration methods. Endotoxin was collected with polycarbonate filters and analysed by kinetic chromogenic Limulus Amebocyte Lysate method. Geometric mean levels of airborne bacteria, fungi, endotoxin and dust were 1,864 CFU/$m^3$, 2,252 CFU/$m^3$, 31.5 EU/$m^3$ and 2.4 mg/$m^3$. The ratios of indoor/outdoor concentrations were 3.7 for bacteria, 4.1 for fungi, 3.3 for endotoxin and 9.7 for dust. The respiratory fractions of bacteria were 68.0, 50.9, 49.2 and 45.1% in band-saw, table-saw, rip-saw process and outdoor air. The respiratory fractions of fungi were 78.7, 90.8, 87.5 and 84.8% in band-saw, table-saw, rip-saw process and outdoor air, respectively. There was no significant differences in bacterial concentrations among single stage, six stage impaction and filteration methods. But, fungal concentrations measured with filtration methods were significantly higher than those with impaction methods. Geometric mean levels of airborne bacteria and fungi were higher than the OSHA guideline values of 1,000 CFU/$m^3$. The respiratory fractions of fungi were above 75%. The concentrations of biological agents were significantly different among culture-based sampling methods. In the exposure assessments of biological agents, further studies are needed for the comparisons of diverse sampling methods and the investigations of environmental factors.

• Microbiology and Biotechnology Letters
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• v.13 no.1
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• pp.51-57
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• 1985

Delta-endotoxin in Bacillus thuringiensis var. finitimus, HD-1, HD-9 and HD-73 strains were isolated by NaBr, CsCl and Renografin density gradients. The purity of the toxin was about 98%. The purified o-endotoxin was analyzed by SDS-PAGE, electron microscopic observation and bioassay. According to SDS-PAGE, the molecular weight of subunits of the o-endotoxin were about 66,000 and 130,000 daltons. The shapes of the crystal toxin observed by TEM except finitimus strain were bipyramidal. When the purified endotoxin was bioassayed against tobacco horn worm, the entomocidal activities ($1{\mu}g/ml$) of HD-1 and HD-73 strains were, respectively, 60% and 100% at nine days after treated. The molecular weights of the plasmids isolated from B. thuringiensis were various from 0.5 to 120 Kb. The numbers of plasmids in HD-1, HD-9 and HD-73 strains were 12, 3 and 11, respectively, but B. thuringiensis var. finitimus strain had no plasmid.

• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.242-248
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• 2013

Endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria and are continuously released during bacterial growth. Endotoxins typically induce severe sepsis and septic shock, which cause more than 50% of mortalities. Endotoxins are easily measured in the serum by the limulus amebocyte lysate (LAL) test. However, a nonspecific result is obtained, because the high concentration of serum proteins disturbs the enzyme reaction of the LAL test. In order to solve this problem, the LAL test was performed in this study after the centrifugation of the boiled serum samples to remove the impurities. As a result, among the various conditions examined, endotoxin measurement with the LAL test was the most accurate and repeatable after centrifugation of the boiled serum at $100^{\circ}C$. Moreover, the endotoxin was accurately and repeatedly measured from the prepared sera of mice that had been administered an intraperitoneal injection of purified lipopolysaccharides (LPS) or E. coli. Therefore, the application of centrifugation to remove impurities from boiled serum gives an accurate measurement of endotoxins in the sera of normal subjects or patients, and this will lead to the improved diagnosis and prevention of diseases caused by endotoxins. In addition, the centrifugation of boiled serum samples should be considered and included in the development of endotoxin test kits.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.29 no.3
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• pp.14-26
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• 2016

Objectives : This study was performed to investigate the protective effects and mechanisms of Salvia miltiorrhizae Radix extract (SME) on endotoxin shock.Methods : We used two models; LPS-induced sepsis model for in vivo model, and murine peritoneal macrophages responses for in vitro. SME was administrated orally to mice. After 1 hr, LPS was injected intraperitoneally. Survival rate was checked each time per 12 hr for 5 days. Mice were sacrificed 3 hr after LPS injection, then blood samples and organs were harvested. Cytokines secretion was measured by ELISA. Organs tissues were observed with microscope. Murine peritoneal macrophages were cultured for 1 hr either in a medium alone or in a medium that contained SME, as indicated. Then, the cells were treated with LPS for 24 hr. mRNA levels of cytokines were measured by real-time RT-PCR. Cytokine levels in the supernatants were measured by ELISA. The amount of nitrite was measured by using the Griess method to evaluate NO production. The cell lysates were analysed by Western blotting using antibodies for iNOS and β-actin was used as an internal control to monitor equal protein loading.Results : SME improverd the survival rate of mice model. SME inhibited the secretion of inflammatory cytokines and organs damages on Endotoxin Shock model. SME suppressed cytokine expression, cytokine secretion,NO production, iNOS expression in LPS-induced murine peritoneal macrophages.Conclusions : The results suggest that SME has protective effects on endotoxin shock through suppression of inflammatory cytokines, organ damages, NO production and so on.

• Korean Journal of Food Science and Technology
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• v.52 no.1
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• pp.109-112
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• 2020

To evaluate the safety of bacteriophages for application of sanitizer, endotoxin content and cell cytotoxicity of two Escherichia coli and four Staphylococcus aureus phages were determined. Endotoxin ratio was determined by the Limulus amebocyte lysate (LAL) assay as a test for representative biological endotoxin content. The average endotoxin average content of the 9 log PFU/mL lysate was 18.6 EU/mL and that of the 10 log PFU/mL lysate was 5.9 EU/mL, suggesting that the phage lysate was not suitable for clinical applications, but suitable for food pathogen control applications. To confirm the cell cytotoxicity of the phage lysates, MTT assay was performed using Raw 264.7 cells treated with 9 log PFU/mL phages. Results of the assay indicated that the phage lysates did not significantly decrease the cell viability (p>0.05). These results indicated that bacteriophages would be suitable as a food safety sanitizer.

• KSBB Journal
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• v.18 no.5
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• pp.429-433
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• 2003

LAL (Limulus amebocyte lysate) test to detect and quantity endotoxin is based on gellation reaction between endotoxin and LAL from a blood extract of Limulus polyphemus. The test is labor intensive requiring dedicated personnel, takes relatively long reaction time (approximately 1 hr), requires relatively large volume of samples and reagents, and its end-point detection method is rather subjective. To solve these problems, we attempted to develop a miniaturized LOC (lab-on-a-chip) prototype using PDMS and glass. Using the 62 mm (length) ${\times}$ 18 mm (width) prototype in which 2 mm (width) ${\times}$ 44.34 mm (length) ${\times}$ 100 $\mu\textrm{m}$ (depth) microfluidic channel was provided, we compared the various detection methods of gellation, turbidometric, and chromogenic assays to find the chromogenic method to be the most suitable for small volume assay. In this assay, kinetic point method was more accurate than end point method. We also found the PDMS chip thickness should be minimized to around 2 mm to allow sufficient light transmittance, which necessitated a glass slide bonding for chip rigidity. Through the miniaturization, the test time was reduced from 1 hr to less than 10 minutes, and the sample volume could be reduced from 100 ${\mu}\ell$ to 4.4 ${\mu}\ell$. In sum, this study revealed that the mini LOC could be an alternative for a semi-automated and reliable method for LAL test.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.22 no.4
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• pp.265-275
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• 2012

Objectives: The aim of this study was to investigate the distribution patterns and exposure concentrations of biological hazards in waste handling industries. Methods: We selected 3 recyclable waste sorting plants(RWS), 2 food recycling plants(FR), 1 landfill area(LA) and 1 waste incineration plant(WI). Total airborne bacteria and fungi were measured with single stage impactor and gelatin filters. Endotoxin and glucan were measured with polycarbonate filters in total and respirable dust. Results: The geometric mean of airborne bacterial concentration was the highest in FR($3,273CFU/m^3$), followed by LA, RWS, and WI as 1,334, 934, and $860CFU/m^3$. The fungal concentrations were 6,031, 5,052, 3,307, and $713CFU/m^3$ in RWS, WI, FR, and LA, respectively. By process, WI pit showed the highest concentrations of bacteria, fungi, and endotoxin, followed by inside of bulldozer in LA. The indoor to outdoor ratios of bacteria, fungi, endotoxin and glucan were 2.3, 4.0, 2.3, and 5.0 in RWS, 29.5, 4.9, 7.6, and 5.0 in FR, 5.3, 8.7, 26.8, and 9.5 in WI, respectively. Conclusions: We found that biological hazards, specifically bacteria in FR, fungi in RWS and endotoxin in WI pit and bulldozer at LA, should be controlled to prevent worker's respiratory diseases.

• Food Science of Animal Resources
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• v.28 no.5
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• pp.623-628
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• 2008

This study was undertaken to determine the concentrations of airborne bacteria, fungi, particles, and endotoxin in swine and chicken houses. Six swine buildings and seven chicken houses were randomly selected in southern Gyonggi Province, South Korea. The geometric mean concentrations of airborne bacteria in swine and chicken houses were $2.7{\times}10^5\;CFU/m^3$ and $5.6{\times}10^7\;CFU/m^3$, respectively. The airborne bacteria concentrations in chicken houses were significantly higher than those of swine houses (p<0.05). The geometric mean concentration of airborne fungi in swine houses was $4.9{\times}10^3\;CFU/m^3$, which was higher than the value of $2.1{\times}10^3\;CFU/m^3$ found in chicken houses. The mean concentrations of airborne particles and endotoxin in swine houses were $3.48\;mg/m^3$ and $943.1\;EU/m^3$, and they were $15.43\;mg/m^3$ and $1,430.5\;EU/m^3$ in chicken houses, respectively. A significant difference between swine and chicken houses was found for total dust (p<0.05), but not for endotoxin. In this study, the concentrations of endotoxin in both swine and chicken houses as well as particles in chicken houses were high, and in about 50% of the samples exceeded the worker health safety levels of $614\;EU/m^3$ suggested in previous studies. These results may indicate a considerable respiratory hazard for workers in these environments.

• Korean journal of applied entomology
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• v.25 no.2 s.67
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• pp.71-76
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• 1986

Bacillus thuringiensis var. thuringiensis H1 (BTT) strain was cultured in the 4 different fermentation media and then measured their growths and the productions of endotoxin crystals from the culture media. Out of the 4 media, the productions of the endotoxin crystals and spores were maximal in the pH9-M-3 medium. The wet weight of BTT cells grown in the 150ml culture was approximately 3.218g and the number of the viable spores was $3.3{\times}10^{10}/ml$, and the ratio of the endotoxin weight over total cell weight was 20.05%. The generation time of the BTT bacteria in the M-1 was about 47.6 minutes in the M-2, 132.9 minutes in the M-3 and 110.2 minutes in the M-4. The proper pH for the production of the endotoxin by BTT appeared to be pH 6.5.