Flow cytometric detection of ?-D-glucuronidase gene in wild-type bacterial cells using in-situ PCR

2003 ◽  
Vol 82 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Ramaiah Sachidanandham ◽  
Karina Yew-Hoong Gin
Keyword(s):  
Bacterial Cells ◽  
Wild Type ◽  
In Situ Pcr ◽  
Flow Cytometric

scholarly journals Visualization and Direct Counting of Individual Denitrifying Bacterial Cells in Soil by nirK-Targeted Direct in situ PCR

2011 ◽  
Vol 26 (1) ◽  
pp. 74-80 ◽  
Author(s):  
Noriko Ryuda ◽  
Tomoyoshi Hashimoto ◽  
Daisuke Ueno ◽  
Koichi Inoue ◽  
Takashi Someya
Keyword(s):  
Bacterial Cells ◽  
In Situ Pcr ◽  
Direct Counting

Development of a Direct In Situ PCR Method for Detection of Specific Bacteria in Natural Environments

1998 ◽  
Vol 64 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Katsuji Tani ◽  
Ken Kurokawa ◽  
Masao Nasu
Keyword(s):  
Alkaline Phosphatase ◽  
Single Cell ◽  
Natural Environments ◽  
Single Cell Level ◽  
Bacterial Cells ◽  
Cell Level ◽  
In Situ Pcr ◽  
Glass Slides ◽  
Pcr Products

ABSTRACT We applied HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis,E. coli cells in polluted river water also were detected.

scholarly journals Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

2002 ◽  
Vol 68 (1) ◽  
pp. 412-416 ◽  
Author(s):  
Katsuji Tani ◽  
Masahiro Muneta ◽  
Kanji Nakamura ◽  
Katsutoshi Shibuya ◽  
Masao Nasu
Keyword(s):  
Ralstonia Eutropha ◽  
Total Concentration ◽  
Phenol Hydroxylase ◽  
Bacterial Suspension ◽  
Bacterial Cells ◽  
In Situ Pcr ◽  
Hydroxylase Gene ◽  
Pcr Targeting ◽  
Number Of Bacteria

ABSTRACT Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 105 cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 107 cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.

Природные материалы для сорбционной очистки хром- и кадмийсодержащих инфильтратов полигонов ТБО

2019 ◽  
pp. 13-19
Author(s):  
A. Safonov ◽  
N. Andriushchenko ◽  
N. Popova ◽  
K. Boldyrev
Keyword(s):  
Sorption Capacity ◽  
Electron Acceptors ◽  
Bacterial Cells ◽  
Maximum Sorption Capacity ◽  
Sorption Material ◽  
Maximum Sorption ◽  
Properties Of Materials ◽  
Solid Waste Landfills ◽  
Sorption Characteristics

Проведен анализ сорбционных характеристик природных материалов (вермикулит, керамзит, перлит, цеолит Трейд ) при очистке кадмий- и хромсодержащих сточных вод с высокой нагрузкой по ХПК. Установлено, что цеолит обладает максимальными сорбционными характеристиками для Cd и Cr и наименьшим биологическим обрастанием. При использовании вермикулита и керамзита или смесей на их основе можно ожидать увеличения сорбционной емкости для Cd и Сr при микробном обрастании, неизбежно происходящем в условиях контакта с водами, загрязненными органическими соединениями и биогенами. При этом биообрастание может повысить иммобилизационную способность материалов для редоксзависимых металлов за счет ферментативных ресурсов бактериальных клеток, использующих их в качестве акцепторов электронов. Эффект микробного обрастания разнонаправленно изменял параметры материалов: для Cr в большинстве случаев уменьшение и для Cd значительное увеличение. При этом дополнительным эффектом иммобилизации Cr является его биологическое восстановление биопленками. Варьируя состав сорбционного материала, можно подбирать смеси, оптимально подходящие для очистки вод инфильтратов с полигонов твердых бытовых отходов с высокой нагрузкой по ХПК и биогенным элементам как при использовании in situ, так и в системах на поверхности.The analysis of the sorption characteristics of natural materials (vermiculite, expanded clay, perlite, Trade zeolite) during the purification of cadmium and chromium-containing leachate with a high COD load was carried out. It was determined that zeolite had the maximum sorption capacity for Cd and Cr and the lowest biological fouling. When using vermiculite and expanded clay or mixtures on their basis, one can expect an increase in the sorption capacity for Cd and Cr during microbial fouling that inevitably occurs during contacting with water polluted with organic compounds and nutrients. In this case biofouling can increase the immobilization properties of materials for redox-dependent metals due to the enzymatic resources of bacterial cells that use them as electron acceptors. The effect of microbial fouling changed the parameters of materials in different directions: for Cr, in most cases, downward, and for Cd, significantly upward. Moreover, chromium biological recovery by biofilms is an additional effect of immobilization. Varying the composition of the sorption material provides for selecting mixtures that are optimally suitable for the purification of leachates from solid waste landfills with high COD and nutrients load, both when used in situ and in surface systems.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

823 CD4 T cells are essential for an anti-tumor effect in a B78 murine melanoma tumor model

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A873-A873
Author(s):  
Arika Feils ◽  
Mackenzie Heck ◽  
Anna Hoefges ◽  
Peter Carlson ◽  
Luke Zangl ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Cd4 T Cells ◽  
Tumor Response ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Mhc I ◽  
Mhc Ii ◽  
Flow Cytometric

BackgroundMice bearing B78 melanoma tumors can be cured using an in situ vaccine (ISV) regimen that includes radiation (RT) together with immunocytokine (tumor-targeting mAb conjugated to IL-2). B78 melanoma cells, derived from B16 cells, express minimal to no MHC-I but express MHC-II upon IFN-g/TNF-a stimulation. Although B78 cells are primarily MHC-I-deficient, an increased CD8 T cell infiltration into the tumor microenvironment (TME) has been shown following ISV.1 To further investigate the potential role of specific immune cell lineages in the B78 anti-tumor response to ISV, immune subset depletion studies and flow cytometric analyses were performed.MethodsC57BL/6 mice bearing B78 tumors were depleted of immune cell subsets with mAbs (anti-CD4, anti-CD8, anti-NK1.1, or Rat IgG control) for 3 weeks during the course of treatment. Treatment groups included no treatment, RT (12 Gy), or ISV (RT D0 and immunocytokine D5-D9). 6 mice/group (repeated three times) were followed for survival/tumor growth, and flow cytometry studies included 4 mice/group, sacrificed on D8 and D13 following the start of ISV.ResultsMice depleted of CD4 T cells during the course of ISV showed a significant reduction of anti-tumor effect as compared to mice treated with ISV/Rat IgG (pConclusionsThese studies suggest that CD4 T cells are essential for an anti-tumor response in the B78 melanoma model. In vivo depletion data show that CD4 T cells, but not CD8 or NK cells, are required for a decrease in tumor growth via ISV. Flow cytometric analyses suggest an interplay between CD4 and CD8 T cells as indicated by a decrease in CD8/IFN-g expression following ISV in the absence of CD4 T cells. The role that MHC-I and MHC-II expression plays in this CD4/CD8 T cell anti-tumor response is under investigation. In future studies, B78 melanoma may serve as a critical syngeneic model for development of more effective immunotherapy treatment regimens.Ethics ApprovalAll animal experiments were performed in accordance with protocols approved by Animal Care and Use Committees of the University of Wisconsin-Madison.ReferenceMorris Z, Guy E, Francis D, et al. In situ tumor vaccination by combining local radiation and tumor-specific antibody or immunocytokine treatments. Cancer Res 2016;76(13):3929-3941.

Preliminary Study on In Situ Realtime Quantitation of Target Bacteria on the Principle of Flow Cytometry

2017 ◽  
Vol 262 ◽  
pp. 224-227
Author(s):  
Gen Murakami ◽  
Yuichi Sugai ◽  
Kyuro Sasaki
Keyword(s):  
Flow Cytometry ◽  
Bacterial Culture ◽  
Scattered Light ◽  
Bacterial Suspension ◽  
Culture Solution ◽  
Bacterial Cells ◽  
Monitoring Wells ◽  
Measurement Principle ◽  
Preliminary Study

In-situ realtime method that can monitor the target bacteria should be used to determine the real situation of the bacteria in deep parts of heaps in heap bioleaching plants. This study suggest to apply flow cytometry technology to in-situ realtime monitoring of target bacteria. Flow cytometry is a method that can rapidly quantify the bacterial cells in bacterial suspension based on the detection of lights that are emitted from bacterial cells. In this study, we estimated the possibility of the application of flow cytometry to the selective detection of target bacteria. The bacterial culture solution that had been diluted by water including other bacteria was provided for fluorescence spectral analysis and scattered light analysis that were functions of flow cytometry. Our target bacteria could be selectively detected by those analyses in this study, therefore, it was shown that the flow cytometry could be useful for detecting target bacteria selectively. Because the measurement principle of flow cytometry is quite simple, it can be expected to be installed into deep heaps through the monitoring wells and determine the dominance of target bacteria in-situ and realtime in the future.

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