In vitro Maturation of the Exoerythrocytic Stage of Plasmodium knowlesi Observed under Phase Contrast Microscopy

1990 ◽  
Vol 76 (6) ◽  
pp. 923 ◽  
Author(s):  
Pascal Millet ◽  
William E. Collins ◽  
Claude E. Monken ◽  
Bobby G. Brown
Keyword(s):  
Phase Contrast ◽  
In Vitro Maturation ◽  
Plasmodium Knowlesi ◽  
Phase Contrast Microscopy ◽  
Contrast Microscopy

scholarly journals 209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 226 ◽  
Author(s):  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
L. Anel ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Phase Contrast ◽  
Egg Yolk ◽  
Phase Contrast Microscopy ◽  
White Rhinoceros ◽  
Contrast Microscopy ◽  
Before And After ◽  
Epididymal Spermatozoa

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.

Microtubules and parental genome organisation during abnormal fertilisation in humans

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 223-228 ◽  
Author(s):  
Vanesa Yanina Rawe ◽  
Santiago Brugo Olmedo ◽  
Florencia Noemí Nodar ◽  
Alfredo Daniel Vitullo
Keyword(s):  
Phase Contrast ◽  
Polar Body ◽  
Phase Contrast Microscopy ◽  
Genome Organisation ◽  
Contrast Microscopy ◽  
Chromatin Configuration ◽  
Second Polar Body ◽  
Main Pattern ◽  
Human Zygotes

We analysed the distribution of β-tubulins, acetylated α-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, β-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.

scholarly journals Comparative Morphologic and Morphometric Study on the Developmental Aspects of In Vitro and In Vivo Reared Echino-coccus granulosus Sensu Stricto Using Differential Interference Contrast (DIC)/Nomarski and Phase Contrast Microscopy

2019 ◽  
Author(s):  
Seyedeh Faezeh SADJJADI ◽  
Mina MOTAMEDI ◽  
Tahereh MOHAMMADZADEH ◽  
Seyed Mahmoud SADJJADI
Keyword(s):  
Phase Contrast ◽  
Sensu Stricto ◽  
Whole Body ◽  
Phase Contrast Microscopy ◽  
Differential Interference Contrast ◽  
Morphometric Characters ◽  
Reproductive Structures ◽  
Contrast Microscopy

Background: Echinococcus granulosus is a zoonotic parasite with worldwide distribution. The present study focused on comparative morphologic and morphometric observations on the developmental aspects of whole body, more special the reproductive structures of in vitro reared adult worms (RAW) and in vivo reared adult worms in definitive host (AWIDH) using differential interference contrast (DIC)/Nomarski, phase contrast and routine optical microscopy. Methods: A total number of 10 in vitro and 10 in vivo reared adult worms of E. granulosus sensu stricto, G1 strain were selected. The worms were processed by Formaldehyde-Alcohol-Azocarmine-Lactophenol (FAAL). The details of morphological factors and reproductive structures of each worm including 25 biometrical parameters were studied by routine optical, phase contrast and Nomarski microscopy. The details of the samples were photographed, measured and analyzed. The fine structures of the parasite including the details of cirrus sac and developmental stages in different strobila were more obvious observing by Nomarski microscopy.  Results: The morphometric characters in the RAW and AWIDH showed that length of immature proglottid, length of mature proglottid, length of suckers are larger in RAW than AWIDH worms with statistical difference. Characters in E. granulosus of RAW and AWIDH showed that total number of segments, number of mature segments and the total number of testes were greater in RAW than AWIDH worms; while only the number of mature segments was statistically different is two groups. Conclusion: Application of DIC/Nomarski and phase contrast microscopy together with morphometric criteria are useful means for comparing the developmental aspects of in vitro and in vivo reared adults of E. granulosus.

scholarly journals Evaluation of Roflumilast (BCRP inhibitor) and Methotrexate (BCRP substrate) on Viability of Primary Squamous Cell Carcinoma – An in vitro Study

2019 ◽  
Vol 12 (2) ◽  
pp. 683-687
Author(s):  
Miss Pamila ◽  
Ramya Sugumar ◽  
Darling Chellathai David
Keyword(s):  
Cell Cycle ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Line ◽  
Phase Contrast ◽  
Phase Contrast Microscopy ◽  
Primary Squamous Cell Carcinoma ◽  
Cycle Arrest ◽  
Contrast Microscopy

In this study we evaluated the possible beneficial drug- interaction between Roflumilast (BCRP inhibitor) and Methotrexate (BCRP substrate) on viability of primary squamous cell carcinoma cell line using an in vitro technique. The KB cell line was treated with Roflumilast and Methotrexate to evaluate its anticancer activity using MTT assay. Image analysis under phase contrast microscopy was performed and flow-cytometry was done to see for cell cycle arrest as a result of drug treatment. Cell viability gradually decreased with the increasing concentrations of roflumilast, methotrexate and the cytotoxic effect with the combination of roflumilast and methotrexate also increased proportionally. Phase contrast microscopy indicated characteristic features of apoptosis which was confirmed in flow cytomtery and indicated cell cycle arrest in M phase. Efflux pump mediated multidrug resistance being a common feature among all cancers, the results of our study evidence the use of combined methotrexate and roflumilast to overcome drug resistance by exploiting the fact that the former is a BCRP substrate and latter a BCRP inhibitor. By combining the two drugs, it allows optimization of therapy by dose reduction of methotrexate and roflumilast and thereby resulting in better efficacy.

scholarly journals Pixantrone, a new anticancer drug with the same old cardiac problems? An in vitro study with differentiated and non-differentiated H9c2 cells

2018 ◽  
Vol 11 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Ana Reis-Mendes ◽  
Marisa Alves ◽  
Félix Carvalho ◽  
Fernando Remião ◽  
Maria Lourdes Bastos ◽  
...  
Keyword(s):  
Anticancer Drug ◽  
Phase Contrast ◽  
Published Data ◽  
Phase Contrast Microscopy ◽  
H9c2 Cells ◽  
Nuclear Condensation ◽  
Reduction Assay ◽  
Contrast Microscopy ◽  
Mtt Reduction

Abstract Pixantrone (PIX) is an anticancer drug approved for the treatment of multiple relapsed or refractory aggressive B-cell non-Hodgkin’s lymphoma. It is an aza-anthracenedione synthesized to have the same anticancer activity as its predecessors, anthracyclines (e.g. doxorubicin) and anthracenediones (e.g. mitoxantrone), with lower cardiotoxicity. However, published data regarding its possible cardiotoxicity are scarce. Therefore, this work aimed to assess the potential cytotoxicity of PIX, at clinically relevant concentrations (0.1; 1; and 10 µM) in both non-differentiated and 7-day differentiated H9c2 cells. Cells were exposed to PIX for 48 h and cytotoxicity was evaluated through phase contrast microscopy, Hoescht staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytotoxicity was observed in differentiated and non-differentiated H9c2 cells, with detached cells and round cells evidenced by phase contrast microscopy, mainly at the highest concentration tested (10 µM). In the Hoechst staining, PIX 10 µM showed a marked decrease in the number of cells when compared to control but with no signs of nuclear condensation. Furthermore, significant concentration-dependent mitochondrial dysfunction was observed through the MTT reduction assay. The NR assay showed similar results to those obtained in the MTT reduction assay in both differentiated and non-differentiated H9c2 cells. The differentiation state of the cells was not crucial to PIX effects, although PIX toxicity was slightly higher in differentiated H9c2 cells. To the best of our knowledge, this was the first in vitro study performed with PIX in H9c2 cells and it discloses worrying cytotoxicity at clinically relevant concentrations.

scholarly journals Rough Dental Implant Surfaces and Peri-Implantitis: Role of Phase-Contrast Microscopy, Laser Protocols, and Modified Home Oral Hygiene in Maintenance. A 10-Year Retrospective Study

2021 ◽  
Vol 11 (11) ◽  
pp. 4985
Author(s):  
Gianluigi Caccianiga ◽  
Gérard Rey ◽  
Paolo Caccianiga ◽  
Alessandro Leonida ◽  
Marco Baldoni ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Phase Contrast ◽  
Vertical Movement ◽  
Subgingival Plaque ◽  
Phase Contrast Microscopy ◽  
Implant Surface ◽  
Total Percentage ◽  
Contrast Microscopy

The aim of this study was to evaluate two different kinds of rough implant surface and to assess their tendency to peri-implantitis disease, with a follow-up of more than 10 years. Data were obtained from a cluster of 500 implants with Ti-Unite surface and 1000 implants with Ossean surface, with a minimum follow-up of 10 years. Implants had been inserted both in pristine bone and regenerated bone. We registered incidence of peri-implantitis and other causes of implant loss. All patients agreed with the following maintenance protocol: sonic brush with vertical movement (Broxo), interdental brushes, and oral irrigators (Broxo) at least two times every day. For all patients with implants, we evaluated subgingival plaque samples by phase-contrast microscopy every 4 months for a period of more than 10-years. Ti-Unite surface implants underwent peri-implantitis in 1.6% of the total number of implants inserted and Ossean surface implants showed peri-implantitis in 1.5% of the total number of implants. The total percentage of implant lost was 4% for Ti-Unite surfaces and 3.6% for Ossean surfaces. Strict control of implants leads to low percentage of peri-implantitis even for rough surfaces dental implants.

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