• Title, Summary, Keyword: Cell counting method

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Colonial Cyanobacteria, Microcystis Cell Density Variations using Ultrasonic Treatment (초음파 처리 조건에 따른 집락형 유해남조류 Microcystis 세포수 변화 연구)

  • Lee, Hae-Jin;Park, Hae-Kyung;Heo, Jun;Lee, Hyeon-Je;Hong, Dong-Gyun
    • Journal of Korean Society on Water Environment
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    • v.34 no.2
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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.

Micro Cell Counter Using a Fixed Control Volume Between Double Electrical Sensing Zones (다수의 계수구역간의 검사체적을 이용한 소형 세포농도센서)

  • Lee Dong Woo;Yi Soyeon;Cho Young-Ho
    • Transactions of the Korean Society of Mechanical Engineers A
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    • v.29 no.12
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    • pp.1615-1620
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    • 2005
  • We present a novel flow-rate independent cell counter using a fixed control volume between double electrical sensing zones. The previous device based on the single electrical cell sensing in a given flow-rate requires an accurate fluid volume measurement or precision flow rate control. The present cell counter, however, offers the flow-rate independent method for the cell concentration measurement with counting cells in a fixed control volume of $22.9{\pm}0.98{\mu}{\ell}$. In the experimental study, using the RBC (Red Blood Cell), we have compared the measured RBC concentrations from the fabricated devices with those from Hemacytometer. The previous and present devices show the maximum errors of $20.3\%\;and\;16.1\%$, which are in the measurement error range of Hemacytometer (about $20\%$). The present device also shows the flow-rate independent performance at the constant flow-rates ($5{\mu}{\ell}/min$ and $10{\mu}{\ell}/min$) and the varying flow-rate (4, 2, and $4{\mu}{\ell}/min$). Therefore, we demonstrate that the present cell counter is a simple and automated method for the cell concentration measurement without requiring an accurate fluid measurement and precision flow-rate control.

Quantitation of Antigen-Antibody Reaction Condition for Development of Fluorescence Image-based CD4 Rapid Test (형광 영상 기반 CD4 신속 검사법 개발을 위한 항원-항체 반응 조건 정량화)

  • Kim, Subin;Kim, Jung Kyung
    • Journal of the Korean Society of Visualization
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    • v.13 no.1
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    • pp.35-42
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    • 2015
  • CD4+ T-cell count determines the effectiveness for antiretroviral therapy (ART) in patients with human immunodeficiency virus (HIV). Although ART slows the progression of HIV to AIDS, rapid counting of CD4+ T lymphocytes with a drop of patient's blood sample is urgently needed to ensure timely ART treatment in rural areas. Recently point-of-care CD4 testing devices have been developed by using non-flow based imaging cytometer incorporated with a sample cartridge where CD4+ T cells are reacted with fluorescently tagged specific antibodies. Here we conducted an experimental study using a conventional fluorescence microscope-based imaging system to quantitate the interaction of CD4 antibodies with CD4+ T cells at different reaction conditions. We demonstrated that a fast and affordable point-of-care CD4 test is feasible with a far less amount of antibodies and a shorter incubation time compared with a conventional sample preparation protocol for flow cytometry. We also proposed a general method to evaluate and compare the detection limit across different CD4 counting platforms by using fluorescently labelled microbeads for intensity calibration.

Treatment of E. coli B with two Antibiotics and their Influence on $T_3$ phage Absorption (대장균(E. coli B)의 항생제처리가 $T_3$ phage의 부착에 미치는 영향)

  • Chung, Sang-Jin;Kim, Woon-Soo
    • Applied Microscopy
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    • v.10 no.1_2
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    • pp.19-25
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    • 1980
  • E. coli B was treated with colistin and kanamycin and the influence of these antibiotics on the absorption of $T_3$ phage was studied using the plaque counting method and the electron microscope. E. coli B treated with colistin was sharply inhibited on phage absorption and cell walls were severely damaged showing some spiny appearance around the walls. No influence of kanamycin was noted on phage absorption. Bacterial cells treated with kanamycin showed wave form in the structure of walls and a profound change was noted in the cytoplasm where it was concentrated along the periphery of the inner wall leaving the center of the cell to appear almost empty.

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Microscopic Detection of Urinary Tract Infection in Nepalese Patients

  • Dhakal, Bijaya-Kumar;Pokhrel, Bharat-Mani;Joohong Ahnn
    • Journal of Microbiology
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    • v.40 no.4
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    • pp.267-273
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    • 2002
  • Urinary tract infection (UTI) is one of the most common domiciliary and nosocomial bacterial infections prevalent in both males and females. UTI is diagnosed on the basis of clinical symptoms, microscopy and culture of urine. In order to evaluate the efficacy of microscopic detection for presumptive diagnosis of UTI we analyzed urine samples of Nepalese patients. We have conducted Gram staining and counting of pus cells, red blood cells (RBC) and epithelial cells. We observed that RBC and epithelial cell counts were not sensitive enough to be used for presumptive diagnosis of UTI. However, pus cell counts as well as Gram stain are sensitive and significant enough to presume UTI. When the Gram stain result was compared with the culture result, it was statistically significant. From this, we suggest that Gram stain of centrifuged urine is a very sensitive screening method to detect bacteriuria. In addition, we found that E. coli was the most predominant microorganism causing UTI and nitrofurantoin was the most effective antibiotic against the isolated urinary pathogens.

AN EXPERIMENTAL STUDY ON FAT CELL VIABLITY ACCORDING TO DIFFERENT HARVESTING TECHNIQUES (지방 채취 방법에 따른 지방 세포의 생존성에 대한 연구)

  • Lee, Won-Deok;Choi, Jin-Young
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.30 no.1
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    • pp.22-29
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    • 2008
  • Purpose: The purpose of this study is to test the efficacy of various methods of fat harvesting in animal model by viability comparison with assay including cell counting, MTT assay, and histologic evaluation. Materials and methods: New Zealand white rabbits experiments were used. Groin fat pads were subjected to different harvest method varying ingredients of solution(Experiment 1: T1 solution= lidocaine 1000mg/L, epinephrine 1mg/L, sodium bicarbonate 10mgEq/L, Triamcinolone 10mgEq/L; T2 solution=lidocaine 1000mg/L, epinephrine 1mg/L, sodium bicarbonate 0mgEq/L, Triamcinolone 0mgEq/L) and pressure exerted on harvesting with Luer-Lock syringe connected to suction cannula.(Experiment 2: P1 group=3cc intermittent pressure; P2 group=10cc sustained pressure) Fat cell viability was assessed with cell counting with a hemocytometer, MTT assay, and histologic evaluation. Results: Experiment 1 Cell count: T1=2.4/3.4/4.2, T2=9.6/8.4/7.2($\times10^5$ per mL); MTT assay: T1=0.516/0.41/0.453/0.412/0.421, T2=0.925/0.765/0.54/0.634/0.614 in 21 days(absorbance); Histology: T1 showed elongated and, different in size and shape, and ruptured adipocytes with only a few normal adipocytes whereas T2 showed central core of fat with almost intact fat cells Experiment 2 Cell count: P1=1.2/3.2/4.2, P2=1.2/2.4/3.8($\times10^5$ per mL); MTT assay:P1=0.256/0.245/0.258/0.21/0.264, P2=0.12/0.231/0.245/0.313/0.281 in 21 days(absorbance); Histology: P1 showed somewhat evenly distributed normal-looking fat cells and P2 showed relatively irregular shape of fat cells with small blood vessel amongst adiopocytes. Conclusion: Viability was higher in ‘modified tumescent solution’without sodium bicarbonate and triamcinolone and we also found no significantly different viability between using intermittent pressure and using sustained pressure. But in terms of initial viability of fat cell, we can assume that lower intermittent pressure would make better clinical results.

Development of mcyB-specific Ultra-Rapid Real-time PCR for Quantitative Detection of Microcystis aeruginosa (Microcystis aeruginosa의 정량을 위한 mcyB 특이 초고속 실시간 유전자 증폭법의 개발)

  • Jung, Hyunchul;Yim, Byoungcheol;Lim, Sujin;Kim, Byounghee;Yoon, Byoungsu;Lee, Okmin
    • Journal of Korean Society on Water Environment
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    • v.34 no.1
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    • pp.46-56
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    • 2018
  • A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

SELECTIVE DETECTION OF VIABLE ENTEROCOCCUS FAECALIS USING PROPIDIUM MONOAZIDE IN COMBINATION WITH REAL-TIME PCR (Propidium monoazide와 real-time PCR을 이용한 살아있는 Enterococcus faecalis의 선택적인 검출)

  • Kim, Sin-Young;Lee, Seung-Jong;Kim, Eui-Seong;Seo, Deog-Gyu;Song, Yoon-Jung;Jung, Il-Young
    • Restorative Dentistry and Endodontics
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    • v.33 no.6
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    • pp.537-544
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    • 2008
  • Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.

Selective Cytotoxic Effects of Doenjang (Korean Soybean Paste) Fermented with Bacillus Strains on Human Liver Cell Lines

  • Choi, Myeong-Rak;Lim, Hyun-Soo;Chung, Yoon-Ju;Yoo, Eun-Jeong;Kim, Jong-Kyu
    • Journal of Microbiology and Biotechnology
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    • v.9 no.4
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    • pp.504-508
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    • 1999
  • This report compares the selective cytotoxic effects of Doenjang fermented by various Bacillus strains (Bacillus sp. SS9, SSA3, and PM3) on human liver cell lines with that of conventional Doenjang (DTY, DTG, and DTK) and commercial Doenjang (DCM, DCD, and DCS). To investigate selective cytotoxic effects of Doenjang extracts, the cell density of HepG2 (Hepatocellular carcinoma) and CCL-13 (cells derived from human normal liver) was estimated after addition of the extracts by using a viable cell counting method. The maximum selectivity ratio ($IC_{50}$value against CCL-13/$IC_{50}$ value aganist HepG2) was observed by PM3 (extracts of Doenjang fermented with Bacillus sp. PM3). As for morphological changes shown by the addition of PM3 into HepG2 and CCL-13 cultures, HepG2 was significantly disrupted, however, CCL-13 was not affected. Also, the growth rate of HepG2 was decreased significantly by the addition of PM3. Consequently, PM3 showed a more detrimental effect on HepG2 than that on CCL-13.

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The Glucosinolate and Sulforaphane Contents of Land Race Radish and Wild Race Radish Extracts and Their Inhibititory Effects on Cancer Cell Lines (재래종 무와 갯무 추출물의 암세포주 증식 저해 활성 및 Glucosinolate와 Sulforaphane의 함량)

  • Choi, Sun-Ju;Choi, A-Reum;Cho, Eun-Hye;Kim, So-Young;Lee, Gun-Soon;Lee, Soo-Seong;Chae, Hee-Jeong
    • Journal of the East Asian Society of Dietary Life
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    • v.19 no.4
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    • pp.558-563
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    • 2009
  • The inhibitory effects of land race radish (LRR) and wild race radish (WRR) extracts on cancer cell lines were investigated. A and their glucosinolate and sulforaphane contents were analyzed. The anticancer activitiesy of the LRR and WRR extracts on the breast cancer cell line MCF-7 were determined by a CCK (cell counting kit) assay, in which WWR showed higher inhibition rates than LRR. The sulforaphane content of WRR was higher than that of LRR. In the lung cancer cell line, A-549, WRR showed higher inhibition rates and a higher total glucosinolate content than LRR. The glucosinolate contents of the radishes were analyzed by the Pd-quicktest method, showing that WRR contained more glucosinolate than LRR in both the trunk and root. In conclusion, these results indicate that wild race radish could be used for the quality improvement of radishes.

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