• Journal of Microbiology
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• 제41권3호
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• pp.262-265
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• 2003

Although enumerating bacterial cells is a fundamental step in understanding microbial ecosystems in marine environments, substantial decrease in bacterial counts with increasing sample storage time hampers the accurate estimation of bacterial biomass. We compared the variations in bacterial cell numbers caused by freezing and thawing of sample bottles or slides. Bacterial counts of seawater samples frozen only once in a sampling bottle yielded approximately 95％ of the original numbers after 90 days, whereas 80％ of the original count was obtained for samples prepared on slides. Only 67％ and 58％ of the original counts were recovered in samples repeatedly frozen and thawed in bottles or on slides, respectively. The results indicated that freezing a seawater sample in a bottle increased the consistency of the epifluorescence microscopic enumeration of bacterial cells.

• Journal of Veterinary Science
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• 제22권3호
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• pp.40.1-40.12
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• 2021

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

• 한국식품위생안전성학회지
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• 제23권3호
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• pp.191-197
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• 2008

희아리 고춧가루에 특징적인 표지자를 찾기 위하여 GC/MASS분석, 고춧가루 표면 분석, 미생물 계수법을 수행하였다. 희아리 고춧가루는 정상 고춧가루에서는 발견되지 않은 분자량 204의 1,2,3,5,6,7,8,$8\alpha$-octahydro-1,$8\alpha$-dimethyl-7-(1-methylethenyl)-naphthalene과, 분자량 204의 2-isopropenyl-$4\alpha$,8-dimethyl-1,2,3,4,$4\alpha$,5,6,$8\alpha$-octahydronaphthalene을 함유하고 있었다. 그러나 희아리 시료간의 함량 차이가 10배 이상으로 컸고, 낮은 함량을 지닌 희아리로 불량고춧가루를 제조 시 검출 기준 이하로 떨어지는 문제점이 있었다. 희아리의 전자 현미경 사진은 정상고추의 매끈한 표면과는 다르게 가루 표면이 거칠고 수많은 미생물이 자라고 있음을 보였다. 한편 희아리는 10%(w/w) 희아리가 혼합된 불량고춧가루 경우에도 정상고춧가루와 확실히 구분되는 세균 및 효모/곰팡이 균수의 증가를 나타냈다. 결과적으로 이 3가지 기술에 의한 정성 정량적인 결과를 상호 보완적으로 분석하는 경우 불량고춧가루 판정은 가능하다고 생각된다.

• 한국물환경학회지
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• 제34권2호
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• pp.210-215
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• 2018

It is difficult to count colonial cyanobacteria Microcystis cells since the thickness of colonies is constrained by amorphous mucilage, making it impossible to estimate the number of cells. Disaggregation of Microcystis colonies into single cell is needed to improve the accuracy and precision of cell density estimation of naturally collected samples. Uultrasonic treatment method is commonly used owing to the simplicity and immediacy of the procedure. However, amplitude, frequency, and duration of ultrasonic treatment also cause cell loss during the experiment. Optimal ultrasonic treatment has not been standardized yet. Therefore, the objective of this study was to investigate optimal ultrasonic treatment by analyzing cell density and colony numbers. We collected colonial Microcystis from Changnyeong-Haman weir area in Nakdong River during harmful algal boom period from September to October in 2017. Ultrasonic treatment method was applied to disrupt colonies into single cells to enumerate cell density. Among treatment conditions, results from continuously treated for 100 seconds were found to be the optimum to reduce colonies to a suspension of single cell without cell losses under high and low density of Microcystis cells. Lugol iodine fixed cells followed by sonication showed less negative impact of cell damage within the optimal treatment time (100 seconds). Furthermore, disaggregated cells treated by sonication enables microscopic observation more easily since gas vacuoles were collapsed to facilitate sedimentation of cells under the counting chamber for quantitative enumeration of buoyant Microcystis cells.

• 한국산업보건학회지
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• 제27권4호
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• pp.324-332
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• 2017

Objectives: Much scientific evidence indicate a positive association between moldy environments and respiratory illnesses and/or symptoms. However, few comprehensive assessments of mold have been performed for such settings. Spore counts or microscopic enumeration only may not be sufficient for evaluating fungal exposure. Recently, Mold Specific QPCR technology developed by the US EPA (Environmental Relative Moldiness Index, ERMI) has been widely used worldwide and great performance for assessing fungal exposure has been shown. Methods: We aimed to develop a Korean version of ERMI suitable for the distribution of fungal flora in Korea. Thirty dwellings in the Seoul and Incheon area were selected for sampling, and each was classified as 'Flooded, 'Water-damaged' or 'Non-water-damaged'. Results: Dust on the floor and airborne sampling were collected using an MAS100 and a 'Dustream' collector. Samples were analyzed by quantitative polymerase chain reaction(QPCR) for the 36 molds belonging to ERMI. Student t-test and ANOVA tests were carried out using SAS software. The median ERMI values of flooded, water damaged, and non-water damaged dwellings were 8.24(range: -5.6 to 27.9), 5.47(-25. 4 to 32.7), and -15.30(-24.6 to 14.8), respectively. Significant differences were observed between flooded and non-water damaged dwellings (P=0.001) and between water-damaged and non-water damaged dwellings (P=0.032). Conclusion: Our findings indicate that ERMI values attributed to dust samples in Korea could be applicable for the identification of flooded or water damaged buildings. However, much data is needed for continuously developing the Korean version of ERMI values.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.321-334
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• 2017

Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus, were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.