• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.348-355
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• 2023

Epifluorescence microscopy with image analysis was evaluated as a biofilm quantification method (i.e., quantification of surface area colonized by biofilms), in comparison with crystal violet (CV) staining. We performed different experiments to generate multispecies biofilms with natural and artificial bacterial assemblages. First, four species were inoculated daily in 16 different sequences to form biofilms (surface colonization, 0.1%-56.6%). Second, a 9-species assemblage was allowed to form biofilms under 10 acylase treatment episodes (33.8%-55.6%). The two methods comparably measured the quantitative variation in biofilms, exhibiting a strong positive relationship (R² ≥ 0.7). Moreover, the two methods exhibited similar levels of variation coefficients. Finally, six synthetic and two natural consortia were allowed to form biofilms for 14 days, and their temporal dynamics were monitored. The two methods were comparable in quantifying four biofilms colonizing ≥18.7% (R² ≥ 0.64), but not for the other biofilms colonizing ≤ 3.7% (R² ≤ 0.25). In addition, the two methods exhibited comparable coefficients of variation in the four biofilms. Microscopy and CV staining comparably measured the quantitative variation of biofilms, exhibiting a strongly positive relationship, although microscopy cannot appropriately quantify the biofilms below the threshold colonization. Microscopy with image analysis is a promising approach for easily and rapidly estimating absolute quantity of multispecies biofilms.

• Korean Journal of Food Science and Technology
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• v.50 no.2
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• pp.244-247
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• 2018

Quantitative analysis for non-host infection bacteriophage was conducted for their enumeration. Flow cytometry and epifluorescence microscopy (EPM) were selected as counting methods. Correlation analysis was performed based on the plaque assay method on the existing host infection and consisted of Pearson correlation statistical analysis, regression analysis, and difference analysis. Analyses of 12 samples with flow cytometry and plaque assay methods showed that there was a correlation of 96.7% with Pearson correlation value r=0.967, $R^2$ 0.9352, and difference value of 1.063. Analyses of 12 samples with EPM and plaque assay methods showed that there was a correlation of 99.0% with Pearson correlation value r=0.990, $R^2$ 0.9811, and difference value of 1.605. Therefore, flow cytometry and epifluorescence microscopy would be effective for enumeration of Weissella cibaria bacteriophage with plaque assay.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.61-65
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• 2001

Epifluorescence microscopy was used to observe epiphytic bacteria directly on plant leaf surfaces as well as indirectly in the leaf liberating solution by staining with fluorochromes of 4',6-diamidino-2-phenylindole (DAPI) and acridine orange(AO). Epiphytic bacteria could not be well observed on the leaf surface by staining with AO due to an intrusive orange or red background fluorescence. However, DAPI gave us clear epifluorescent images of the bacteria on the leaf. On the contrary, epiphytic bacteria in the liberating leaf solution were well observed on filters stained by both types of fluorochrome, although DAPI showed better fluorescent images than AO and not necessarily required a washing step of the filters stained. The optimum conditions of the DAPI stains were 5 $\mu$g/ml for 5 min both for leaves and for filters of the liberating solution. It was confirmed that a critical step in the epifluorescence microscopy of leaf surfaces was to minimize release of water from the leaf. For this, the stained leaf samples were put on a filter paper, kept in a dry oven at $70^{\circ}C$ for 2 min instead of air-drying, and then immediately observed by epifluorescence microscopy. The established technique was applied to enumerate epiphytic bacteria on oak tree leaf surfaces.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.899-901
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• 2001

Comparison of the abundances of heterotrophic bacteria in the East and South China Seas by stanctard epifluorescence was miscounted as heterotrophic bacteria in DAPI stained samples. This could result in 5-31% oversestimations of heterotrophic bacterial abundance in the study areas.

• Proceedings of the Zoological Society Korea Conference
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• 2000.10a
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• pp.129.2-130
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• 2000

No Abstract, See Full Text

• Journal of the Korean Society of Visualization
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• v.21 no.3
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• pp.65-74
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• 2023

Compared to epifluorescence(EPI) microscopy which captures fluorescence from the entire depth of sample, total internal reflection fluorescence(TIRF) can selectively visualize only a single surface of it. TIRF uses a thin evanescent field generated by the total internal reflection of laser light on surface. However, conventional TIRF system are designed for total internal reflection to occur at the upper surface of sample, making them unsuitable for sessile droplet imaging. We designed a TIRF system suitable for a sessile droplet imaging by utilizing slide glass as a lightguide. We presented the details for constructing the TIRF system using a prism, slide glass, air slit, and optical trap. Then, we compared the TIRF with EPI by imaging the droplet with fluorescent particles during its drying process. As a result, TIRF allows us to distinctly visualize the drying pattern on the bottom surface of droplet.

• Journal of the Korean Society of Manufacturing Process Engineers
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• v.12 no.4
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• pp.29-33
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• 2013

Here we report a partially aminated micropattern via direct functionalization and examine eleven different solution-phase probe DNAs hybridizing with the same target DNA on both hydrogen and oxygen terminated diamond. The hybridization intensities determined by epifluorescence microscopy were compared and are influenced strongly by the negatively charged surface and mismatched position of its sequence with immobilized probe DNA.

• Korean Journal of Environmental Agriculture
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• v.1 no.1
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• pp.48-52
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• 1982

Methods for the measurement of aquatic bacteria can be divided into two groups. The first group of these methods is based on the 'replicon' concept that live bacterial cells, when diluted and transferred to a suitable medium, produce colonies. These methods distinguish living from dead bacteria, but they massively underestimate bacterial numbers. The second group of enumeration methods uses visual counting technique using specific apparatus such as a microscope. These methods are generally direct and simple, but it is very hard to distinguish between live and dead bacteria and between small particle and bacteria. Recently developed technique in staining methods has provided a reliable method of visual determination of aquatic bacteria. This uses epifluorescence microscopy to measure the total bacterial population. In order to present the fluorescence microscopy as a new methodology for the determination of bacterial numbers in water supplies, data were obtained from chlorine and monochloramine doses added to samples. Total counts by fluorescence microscopy were compared with standard plate count method. The total number of bacteria in water supplies can be determined with fluorescence microscopy. This technique allows better resolution of small bacteria and differentiation of particle from bacteria. Chloramine was found to persist longer in natural waters and prevent bacterial regrowth.

• The Korean Journal of Physiology
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• v.28 no.2
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• pp.203-214
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• 1994

Using the methods of stopped-flow and epifluorescence microscopy with entrapped fluorophore, membrane permeability of $NH_3$ and weak acids in liposomes, renal brush border (BBMV) and basolateral membrane vesicles (BLMV), and primary culture cells from renal proximal tubule was measured. Permeability coefficient (cm/sec) of $NH_3$ was $(2.9{\times}10^{-2}$ in phosphatidylcholine liposome $25^{\circ}C)$, $5.9{\times}10^{-2}$ in renal proximal tubule cell $(37^{\circ}C)$, $4.0{\times}10^{-2}\;and\;2.4{\times}10^{-2}$ in BBMV and BLMV $(25^{\circ}C)$, respectively. Formic acid has the highest permeability coefficient among the weak acids tested, which was $4.9{\times}10^{-3}$ in liposome, $5.0{\times}10^{-3}$ in renal proximal tubule cell, $9.1{\times}10^{-3}$ in BBMV and $3.8{\times}10^{-3}$ in BLMV. There was a linear relationship between external concentration of nonionized formic acid and initial rate of flux of formic acid in liposome, and the slope coincided with the value of permeability coefficient of formic acid measured in pH 7.0. These results show that techniques of stopped-flow and epifluorescence microscopy with entrapped fluorophore provide the precise method of measurement of very rapid transport of nonelectrolytes through membranes with the advantages of instantaneous mixing effect, good resolution time and easy manipulation.

• Korean Journal of Microbiology
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• v.28 no.4
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• pp.331-336
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• 1990

Annual distribution of hiterotrophic marine bacteria and seasonal characteristics were investigated in the intertidal waters and sediments in the vicinity of Kunsan of Yellow Sea, Korea. Annual distribution of heterotrophic marine bacteria ranged from $ 7.5*10^{2}$ to $1.1*10^{5}$ c.f.u./ml in water and from $1.6*10 ^{4}$ to $4.8*10^{6}$ c.f.u.per g dry sediments. As for the morphological distribution measured by epifluorescence microscopy, rod-form bacteria were distributed more than 74% of total observed bacteria during surveying periods. Average biovolume of sampled bacteria ranged from $3.19\;+-\;0.59*10^{-2}$ to $6.19\;+-\;0.76*10^{-2}$ $\mu$$m^{3}$ for coccus-form bacteria. Isolated bacteria showed various utilization of carbon sources such as glucose, maltose, lactose, xylose and arabinose, and showed tolerance at various range of salinity. It was isolated 82 genus in seawater and 114 genus in sediments. Dominant genera were Pseudomonas, Vibrio, Flavobacterium and Acinetobacter in seawater, and Pseudomonas, Acinetobacter, Vibrio, and Mycobacterium in sediments.