Increased mitochondrial distribution in early-cleaving embryos indicate successful pre-implantation development

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development.

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Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽

10.1530/rep.1.01212 ◽

2006 ◽

Vol 132 (6) ◽

pp. 859-867 ◽

Cited By ~ 13

Author(s):

Xiao-Qian Meng ◽

Ke-Gang Zheng ◽

Yong Yang ◽

Man-Xi Jiang ◽

Yan-Ling Zhang ◽

...

Keyword(s):

Oocyte Maturation ◽

Actin Filaments ◽

Laser Scanning ◽

Polar Body ◽

Laser Scanning Microscopy ◽

Meiotic Spindle ◽

Confocal Laser ◽

Cell Cortex ◽

First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

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286SPERM-OOCYTE INTERACTION DURING IN VITRO FERTILIZATION IN THE HORSE

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab286 ◽

2004 ◽

Vol 16 (2) ◽

pp. 263

Author(s):

J.L. Tremoleda ◽

T.A.E. Stout ◽

B.M. Gadella ◽

B. Colenbrander

Keyword(s):

In Vitro Fertilization ◽

Laser Scanning ◽

Transvaginal Ultrasound ◽

Polar Body ◽

Molecular Probes ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Vitro Fertilization

In vitro fertilization (IVF) has proven to be a surprisingly unsuccessful way of producing horse embryos. The aim of this study was to investigate the interaction between sperm and the cumulus oocyte complex (COC) during IVF. In experiment 1, three IVF conditions were tested: (A) COCs recovered from slaughtered mares were categorized with respect to cumulus morphology (C: compact, n=86, or E: expanded, n=55) and matured in TCM199 containing 0.01IU/mL porcine FSH and equine LH (IVM); after IVM, the oocytes were denuded and those with a visible polar body were incubated with sperm (IVF) in the presence or absence of 150ng/mL progesterone (P4) to induce the acrosome reaction (AR); (B) IVM oocytes from C-COCS were denuded (n=52) or not (n=67) before IVF in the presence of P4;; (C) in vivo-matured oocytes (n=15) recovered by transvaginal ultrasound-guided aspiration from preovulatory follicles 32h after the donor mare was treated with hCG, were fertilized in vitro in the presence of P4. In all cases, IVF was performed with frozen-thawed, Percoll-selected sperm from a single stallion, at a final concentration of 1×106spermatozoa/ml in fertil-TALP for 20h (Parrish et al., 1988 Biol. Reprod. 38, 1171–1180). In experiment 2, the possibility that semen cryopreservation or stallion critically influenced IVF was examined by incubating denuded IVM oocytes with fresh or frozen/thawed sperm from the same (fresh;; n=17 for both C- and E-COCs and frozen-thawed; n=12 and 21 for C and E-COCs, respectively) or one other stallion (Fresh;; n=12 and 19 and frozen-thawed; n=12 and 19 for C and E-COCs, respectively), in the presence of P4 for 20h. In both experiments, the resulting sperm-oocyte complexes were fixed, permeabilized and labelled with fluorescein-conjugated peanut agglutinin (EY Laboratores, San Mateo, CA, USA) and ethidium homodimer (Molecular Probes, Eugene, OR, USA) to stain the acrosomal membrane and DNA, respectively, so that membrane status and position of the sperm within the oocyte investments could be detected by confocal laser scanning microscopy. The total number of sperm bound per oocyte was compared between treatments using one-way ANOVA with pair-wise multiple comparison (Bonferroni t-test). Despite binding to the zona pellucida (ZP), neither fresh nor frozen/thawed sperm from either stallion acrosome-reacted or penetrated any oocytes, irrespective of cumulus morphology at the onset of IVM, denudation prior to IVF or the presence of P4. However, more sperm bound to the ZP of cumulus-denuded IVM oocytes (65±32 and 62±28 [mean±sd] for C and E-COCs, respectively), than cumulus-intact IVM (5±4) or in vivo-matured oocytes (23±17: P<0.001). None of the other factors investigated affected bound sperm numbers. In all cases, ZP-bound sperm failed to AR in the classical fashion, and all oocytes remained arrested at the MII stage. In summary, fertilization failed because sperm did not acrosome-react after binding to the ZP. It is concluded that failure to adequately activate stallion sperm is an important obstacle to successful IVF in horses.

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104 3-D VISUALIZATION OF THE FIRST CLEAVAGE AND ABERRATIONS OF BOVINE ZYGOTES BY CONFOCAL LASER SCANNING MICROSCOPY

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab104 ◽

2013 ◽

Vol 25 (1) ◽

pp. 199

Author(s):

A. A. Gratao ◽

A. Beck ◽

M. Reichenbach ◽

H. D. Reichenbach ◽

E. Wolf ◽

...

Keyword(s):

Mitotic Spindle ◽

Laser Scanning ◽

Likelihood Estimation ◽

Mitotic Division ◽

Blastocyst Stage ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Real Point ◽

Step Size

A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA = 1.3). Details were recorded with high spatial sampling densities (pixel size 50 × 50 nm, z-step size of 200 nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos. This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

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Blastomere movement correlates with ploidy and mosaicism in early-stage human embryos after in vitro fertilization

Zygote ◽

10.1017/s0967199420000817 ◽

2021 ◽

pp. 1-15

Author(s):

Xiaodong Zhang ◽

Jingwei Yang ◽

Wei Han ◽

Chong Li ◽

Guoning Huang

Keyword(s):

Laser Scanning ◽

Early Stage ◽

Screening Method ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Human Embryos ◽

Cell Stage ◽

Preimplantation Genetic Testing ◽

Vitro Fertilization

Summary Embryos undergo chaotic division and decrease in quality on day 3 with a reduction in the rates of subsequent blastocyst formation. Disordered cleavage causes a deterioration in embryonic quality, here we assessed the relationship between an cleavage model in first mitosis and the chromosomal status of human embryos, and discuss the potential biological and clinical implications for the cleavage model as a single parameter that can be used to assess embryonic quality. Thirty-two infertile couples, with normal karyotypes and who underwent their first IVF cycle were recruited to donate one normal two-cell-stage embryo each for this study between 2019 and 2020. Twenty-eight two-cell embryos underwent preimplantation genetic testing of each blastomere, and four chaotic-division embryos were stained with Hoechst and cultured in a confocal laser-scanning microscopy incubator system. This system showed high specificity and PPV but low sensitivity and NPV using the CM in the prediction of euploidy, indicating that CM could be considered a screening method for embryo selection; additional observational studies using the CM to select transferable embryos are needed before it can be used in clinical practice.

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182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab182 ◽

2014 ◽

Vol 26 (1) ◽

pp. 205

Author(s):

Y. Nagao ◽

H. Yamamoto ◽

B. Sarentonglaga ◽

K. Ogata ◽

M. Yamaguchi ◽

...

Keyword(s):

Embryo Development ◽

Intracytoplasmic Sperm Injection ◽

Laser Scanning ◽

Blastocyst Stage ◽

Early Embryo ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Mitochondrial Activity ◽

Early Embryo Development

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P < 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P < 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P < 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.

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290 3-DIMENSIONAL VISUALIZATION OF BOVINE OOCYTE FERTILIZATION BY CONFOCAL LASER SCANNING MICROSCOPY

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab290 ◽

2015 ◽

Vol 27 (1) ◽

pp. 234

Author(s):

E. De Monte ◽

M. Reichenbach ◽

H. Reichenbach ◽

E. Wolf ◽

F. Habermann

Keyword(s):

High Speed ◽

Laser Scanning ◽

Polar Body ◽

Three Dimensional ◽

Reproductive Technologies ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Step Size ◽

3 Dimensional

Cattle can serve as a model organism to resolve central questions in mammalian reproduction that cannot be clarified in the mouse model due to notable species-specific peculiarities of early mouse embryogenesis. As part of a project on structural, molecular, and functional deficiencies of bovine oocytes, we started to systematically investigate fertilization and the onset of embryo development in vitro by 3-dimensional multicolor fluorescence microscopy. We are using 3D visualisation as key approach to clarify the multiple parallel and sequential processes and events of fertilization as well as to identify and classify errors and failures. Moreover, we aim to gain insights into the mechanisms of aberrations by linking processes at the cellular and the molecular level. We studied class I and II oocytes collected from slaughterhouse ovaries and matured for 23 h in vitro. Oocytes were fixed at different times from 4 to 12 h postinsemination with formaldehyde in a microtubule-stabilising buffer containing taxol in such a way that the 3-dimensional cell architecture was maintained, and were stained for DNA, microtubules, and f-actin microfilaments. In addition, serine 10-phosphorylated histone H3 was used as a marker for chromosome condensation and the spindle midbody. For 3-dimensional imaging of the oocytes in toto, confocal serial sections were captured at 1-µm intervals using a 40× objective (NA = 1.3). For imaging details, we used a high spatial sampling density (pixel size: 50 × 50 nm, z-step size: 200 nm) close to the Nyquist criterion and image restoration by maximum likelihood estimation (MLE) deconvolution. A series of more than 500 three-dimensional snapshots of fertilized oocytes at different points in time gives a first detailed view on the spatial and temporal course of the sperm entry, the formation of the paternal pronucleus and the sperm aster, completion of oocyte meiosis and the formation of the maternal pronucleus, as well as dynamic changes of the cytoskeleton. Moreover, we can document a spectrum of abnormalities including spontaneous parthenogenetic oocyte activation, polyspermy, and aberrations of meiosis I and II. The latter include irregular spindle formation and chromosome segregation, the occurrence of chromatin bridges and abnormal spindle positioning and rotation (e.g. leading to nonextrusion of a first or a second polar body or the extrusion of two second polar bodies). Our microscopic investigation in the bovine system contributes to unraveling the origins of irregular cleavage, aneuploidy, and mosaicism in mammals. Three-dimensional high-speed microscopy of oocytes and zygotes in affordable timeframes could be of great value in improving the differential diagnosis of oocyte and sperm dysfunction, as well as in identifying and dissecting problems, limitations, and potential risks of reproductive technologies (ART).This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

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Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158388 ◽

1990 ◽

Vol 48 (3) ◽

pp. 168-169

Author(s):

M. H. Chestnut ◽

C. E. Catrenich

Keyword(s):

Acridine Orange ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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Effect of dissolution rate and subsequent ion release on cytocompatibility properties of borophosphate glasses

Biomedical glasses ◽

10.1515/bglass-2019-0008 ◽

2019 ◽

Vol 5 (1) ◽

pp. 85-97

Author(s):

Nusrat Sharmin ◽

Mohammad S. Hasan ◽

Md. Towhidul Islam ◽

Chengheng Pang ◽

Fu Gu ◽

...

Keyword(s):

Dissolution Rate ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ion Release ◽

Scanning Calorimetry ◽

Cell Culture Study ◽

Borophosphate Glasses ◽

Mg63 Cells

AbstractPresent work explores the relationship between the composition, dissolution rate, ion release and cytocompatibility of a series of borophosphate glasses. While, the base glass was selected to be 40mol%P2O5-16mol%CaO-24mol%MgO-20mol%Na2O, three B2O3 modified glass compositions were formulated by replacing Na2O with 1, 5 and 10 mol% B2O3. Ion release study was conducted using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The thermal scans of the glasses as determined by differential scanning calorimetry (DSC) revealed an increment in the thermal properties with increasing B2O3 content in the glasses. On the other hand, the dissolution rate of the glasses decreased with increasing B2O3 content. To identify the effect of boron ion release on the cytocompatibility properties of the glasses, MG63 cells were cultured on the surface of the glass discs. The in vitro cell culture study suggested that glasses with 5 mol% B2O3 (P40B5) showed better cell proliferation and metabolic activity as compares to the glasses with 10 mol% (P40B10) or with no B2O3 (P40B0). The confocal laser scanning microscopy (CLSM) images of live/dead stained MG63 cells attached to the surface of the glasses also revealed that the number of dead cells attached to P40B5 glasses were significantly lower than both P40B0 and P40B10 glasses.

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Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz378 ◽

2019 ◽

Vol 75 (1) ◽

pp. 117-125 ◽

Cited By ~ 8

Author(s):

Odel Soren ◽

Ardeshir Rineh ◽

Diogo G Silva ◽

Yuming Cai ◽

Robert P Howlin ◽

...

Keyword(s):

Nitric Oxide ◽

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Clinical Isolates ◽

Nitric Oxide Donor ◽

Confocal Laser ◽

Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

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Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

Scientific Reports ◽

10.1038/s41598-021-87922-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Beatriz H. D. Panariello ◽

Justin K. Kindler ◽

Kenneth J. Spolnik ◽

Ygal Ehrlich ◽

George J. Eckert ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Root Canal ◽

Laser Scanning ◽

Confocal Imaging ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Microscopy Imaging ◽

Electromagnetic Stimulation ◽

Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

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