A Spore Counting Method and Cell Culture Model for Chlorine Disinfection Studies of Encephalitozoon syn.Septata intestinalis

ABSTRACT The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth ofEncephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID50) and a minimal infective dose (MID) for E. intestinalis. The TCID50 is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID50 have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25°C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log10 reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

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A High Throughput Approach Based on Dynamic High Pressure for the Encapsulation of Active Compounds in Exosomes for Precision Medicine

International Journal of Molecular Sciences ◽

10.3390/ijms22189896 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9896

Author(s):

Eugenia Romano ◽

Paolo Antonio Netti ◽

Enza Torino

Keyword(s):

Cell Culture ◽

High Pressure ◽

Culture Media ◽

Preliminary Test ◽

Bilayer Membrane ◽

Microfluidic System ◽

Culture Model ◽

Before And After ◽

Dynamic High Pressure

In recent decades, endogenous nanocarrier-exosomes have received considerable scientific interest as drug delivery systems. The unique proteo-lipid architecture allows the crossing of various natural barriers and protects exosomes cargo from degradation in the bloodstream. However, the presence of this bilayer membrane as well as their endogenous content make loading of exogenous molecules challenging. In the present work, we will investigate how to promote the manipulation of vesicles curvature by a high-pressure microfluidic system as a ground-breaking method for exosomes encapsulation. Exosomes isolated from Uppsala 87 Malignant Glioma (U87-MG) cell culture media were characterized before and after the treatment with high-pressure homogenization. Once their structural and biological stability were validated, we applied this novel method for the encapsulation in the lipidic exosomal bilayer of the chemotherapeutic Irinotecan HCl Trihydrate-CPT 11. Finally, we performed in vitro preliminary test to validate the nanobiointeraction of exosomes, uptake mechanisms, and cytotoxic effect in cell culture model.

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Identification and Characterization of Two Subpopulations of Encephalitozoon intestinalis

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4966-4970.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4966-4970 ◽

Cited By ~ 10

Author(s):

Rebecca M. Hoffman ◽

Marilyn M. Marshall ◽

David M. Polchert ◽

B. Helen Jost

Keyword(s):

Cell Culture ◽

Polyclonal Antibodies ◽

Fluorescein Isothiocyanate ◽

Spore Wall ◽

Rabbit Kidney ◽

Sytox Green ◽

Encephalitozoon Intestinalis ◽

Propagation Methods ◽

Small Subpopulation

ABSTRACT Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% ± 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included ≥25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4′,6′-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or β-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.

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Rapid melanization of bomirski amelanotic melanoma cells in cell culture

Bioscience Reports ◽

10.1007/bf01121951 ◽

1983 ◽

Vol 3 (2) ◽

pp. 189-194 ◽

Cited By ~ 16

Author(s):

A. Słominski

Keyword(s):

Cell Culture ◽

Primary Cell Culture ◽

Growth Conditions ◽

Melanoma Cells ◽

Amelanotic Melanoma ◽

Primary Cell ◽

In Vitro Growth

Transfer of Bomirski amelanotic melanoma ceils from in vivo to in vitro growth conditions results in occurrence of rapid melanization in their cytoplasm. The melanized ceils from primary cell culture initiate tumours in hamsters, which do not contain traces of melanin and resemble typical amelanotic melanoma.

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Observations during in vitro growth of cells derived from rabbit kidney

Experimental Cell Research ◽

10.1016/0014-4827(61)90198-7 ◽

1961 ◽

Vol 25 (3) ◽

pp. 671-686 ◽

Cited By ~ 4

Author(s):

Roslyn E. Wallace ◽

E.V. Orsi ◽

Hilda B. Ritter ◽

A.W. Moyer

Keyword(s):

Rabbit Kidney ◽

In Vitro Growth

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Articular cartilage mechanical and biochemical property relations before and after in vitro growth

Journal of Biomechanics ◽

10.1016/j.jbiomech.2007.06.005 ◽

2007 ◽

Vol 40 (16) ◽

pp. 3607-3614 ◽

Cited By ~ 43

Author(s):

Timothy Ficklin ◽

Gregory Thomas ◽

James C. Barthel ◽

Anna Asanbaeva ◽

Eugene J. Thonar ◽

...

Keyword(s):

Articular Cartilage ◽

Biochemical Property ◽

In Vitro Growth ◽

Property Relations ◽

Before And After

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Bobel-24 Activity against Cryptosporidium parvum in Cell Culture and in a SCID Mouse Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01019-07 ◽

2007 ◽

Vol 52 (3) ◽

pp. 1150-1152 ◽

Cited By ~ 4

Author(s):

Cristina Rueda ◽

Soledad Fenoy ◽

Fernando Simón ◽

Carmen del Aguila

Keyword(s):

Cell Culture ◽

Mouse Model ◽

Cryptosporidium Parvum ◽

Scid Mouse ◽

In Vitro Growth ◽

Scid Mouse Model ◽

First Time

ABSTRACT The anticryptosporidial activity of Bobel-24 (2,4,6-triiodophenol) was studied for the first time, resulting in a reduction of the in vitro growth of Cryptosporidium of up to 99.6%. In a SCID mouse model of chronic cryptosporidiosis, significant differences (P < 0.05) in oocyst shedding were observed in animals treated with 125 mg/kg/day. These results merit further investigation of Bobel-24 as a chemotherapeutic option for cryptosporidiosis.

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Hydrogen Peroxide Vapor Decontamination in a Patient Room Using Feline Calicivirus and Murine Norovirus as Surrogate Markers for Human Norovirus

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2016.15 ◽

2016 ◽

Vol 37 (5) ◽

pp. 561-566 ◽

Cited By ~ 9

Author(s):

Torsten Holmdahl ◽

Mats Walder ◽

Nathalie Uzcátegui ◽

Inga Odenholt ◽

Peter Lanbeck ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Culture ◽

Tissue Culture ◽

Feline Calicivirus ◽

Murine Norovirus ◽

Patient Room ◽

Human Norovirus ◽

Log Reduction ◽

Infective Dose ◽

Hydrogen Peroxide Vapor

OBJECTIVETo determine whether hydrogen peroxide vapor (HPV) could be used to decontaminate caliciviruses from surfaces in a patient room.DESIGNFeline calicivirus (FCV) and murine norovirus (MNV) were used as surrogate viability markers to mimic the noncultivable human norovirus. Cell culture supernatants of FCV and MNV were dried in triplicate 35-mm wells of 6-well plastic plates. These plates were placed in various positions in a nonoccupied patient room that was subsequently exposed to HPV. Control plates were positioned in a similar room but were never exposed to HPV.METHODSVirucidal activity was measured in cell culture by reduction in 50% tissue culture infective dose titer for FCV and by both 50% tissue culture infective dose titer and plaque reduction for MNV.RESULTSNeither viable FCV nor viable MNV could be detected in the test room after HPV treatment. At least 3.65 log reduction for FCV and at least 3.67 log reduction for MNV were found by 50% tissue culture infective dose. With plaque assay, measurable reduction for MNV was at least 2.85 log units.CONCLUSIONSThe successful inactivation of both surrogate viruses indicates that HPV could be a useful tool for surface decontamination of a patient room contaminated by norovirus. Hence nosocomial spread to subsequent patients can be avoided.Infect Control Hosp Epidemiol 2016;37:561–566

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The Potential of CO2 Incubator "Sriwijaya CO2 Incubator" Against Cell Culture Proliferation In Invitro Study As Smart Controlling-Based CO2 Incubator For Cell Culture

BioScientia Medicina ◽

10.32539/bsm.v5i2.207 ◽

2020 ◽

Vol 5 (2) ◽

pp. 196-199

Author(s):

Wardiansyah ◽

Rachmat Hidayat ◽

Msy Rulan Adnindya

Keyword(s):

Cell Culture ◽

Primary Culture ◽

Cultured Cells ◽

In Vitro Study ◽

Fibroblast Cell ◽

Fibroblast Cells ◽

Culture Cells ◽

Before And After ◽

Post Test

A B S T R A C TIntroduction. A CO 2 incubator is an essential tool for the initiation of theproliferation of primary culture cells or cell lines. In principle, this tool works bykeeping the sample cell line at an optimum temperature of 37 o C and 5% carbondioxide supply. The ability of the CO 2 incubator to maintain temperature and supplyof 5% carbon dioxide are essential points in the development of the CO 2 incubator.This study is an attempt to convince the potential of Sriwijaya CO 2 Incubator inmaintaining the proliferation ability of cultured cells in an in vitro study. Methods.This study is an experimental pre-post test that explores the percentage of viabilityof primary culture cells (fibroblasts) before and after incubation in CO 2 incubators.The object of this study was fibroblast cells obtained from the prepuce of patientswho performed circumcision. Results. Fibroblast cell proliferation in CO 2 incubatorsshows an increase in the number of fibroblast proliferation which can be seen withthe increasing number of cells visualized by inverted microscopy. Conclusion.Sriwijaya CO 2 incubator has the potential to be used in in vitro research to triggerthe growth and proliferation of fibroblast cells.

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Effect of endotoxin administration on the growth of Candida albicans in sera from various species

Canadian Journal of Microbiology ◽

10.1139/m73-105 ◽

1973 ◽

Vol 19 (5) ◽

pp. 639-641 ◽

Cited By ~ 2

Author(s):

Ronald J. Elin ◽

Sheldon M. Wolff

Keyword(s):

Candida Albicans ◽

Guinea Pig ◽

Animal Species ◽

Specific Resistance ◽

Bacterial Endotoxin ◽

In Vitro Growth ◽

Before And After ◽

Resistance To Infection

The ability of sera from five different animal species to support the growth of C. albicans before and after the animals were inoculated with bacterial endotoxin was studied. A significant diminution was found of the in vitro growth of C. albicans in sera from four (man, rabbit, guinea pig, and mouse) of the five animal species inoculated with bacterial endotoxin 24 h before bleeding when compared with control sera. These findings suggest that the previously reported in vivo non-specific resistance to infection induced by bacterial endotoxin as measured by lethality in the mouse is probably present in man, the rabbit, and the guinea pig. No decrease in the growth of C. albicans was found in sera from dogs inoculated with bacterial endotoxin.

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