PCR-DGGE Analysis of Bacterial Communities Structure in Babylonia areolata Culture Systems of The Subtidal Zone and The Pond Mulched Plastic Film and Sand in Bottom

To know the bacterial communities structure in Babylonia areolata culture systems and to research and optimize the management pattem of Babylonia areola-ta culture systems of the pond mulched plastic film and sand in bottom, the bacte- rial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom were analyzed at molecular level by adopting the denaturing gradient gel electrophoresis （DGGE）. The results indicated that the dominant bacterial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom, which were built on the basis of the seawater in East-island of Zhanjiang, included Proteobac- teda Chloroflexi, Cyanobacteria and Actinobacteria. The dominant bacterial groups in the above pond culture system were Garnmaproteobacteria, Alphaproteobacteria, Deltaprotecbacteda, Epsilonproteobacteda, Anaerolineae, Cyanobacteria and Acti- nobacteda. The dominant bacterial communities in the subtidal zone culture system were Gammaprotecbacteda, Alphaproteobacteria, Deltaproteobacteria, Anaerolineae and Cyanobacteda, and there were less Epsilonproteobacteria and Actinobacteria in the culture system. The higher diversity was detected in the above two culture sys- tems. The results of unweighted pair group method with arithmetic average （UPG- MA） showed that the bacterial communities of the sediment samples S1 and S2 in the above two culture systems were a cluster, the similarity of bacterial communities was 54.5%. The bacterial communities of seawater samples S3 and S4 in the above culture systems were in clusters, and the similarity of the bacterial communi- ties was 84.0%. The results showed that the microorganism ecological level in the Babylonia areolata culture systems of the pond mulched plastic film and sand in bottom could be similar to the sub-tidal zone culture systems through changing the pond seawater and monitoring the microbial population.

Design and Research on Artificial Rumen Continuous Culture System

[Objective] The paper was to study the dynamic changes of forage nutrient substance fermentation in rumen, and a set of continuous culture system of artificial rumen was designed. [Method] With in vivo as control, the simulating rumen fer- mentation effect in vitro culture system was evaluated. [Result] The simulation rumen fermentation test needed adaptive phase of 2-3 d, and the fermentation state was relatively stable within 3-9 d, with good effects. The test showed certain regularity variation with index value of rumen in vivo. [Conclusion] The continuous culture sys- tem of artificial rumen could be used as the ideal model to study the rumen fermen- tation in vivo.

COMPARATIVE STUDIES ON NITROGEN BUDGETS OF CLOSED SHRIMP POLYCULTURE SYSTEMS

April to October, 1997 comparative studies on the nitrogen budgets of closed shrimp polyculture systems showed that, in all the studied polyculture systems, nitrogen from feeds and fertilizers were the main input items, which comprised 70.7%-83.9% of the total input nitrogen, 3.2%-7.4% of which was provided by nitrogen fixation. It was in monoculture enclosures (Y 4, Y 11 and Y 12) that the percentage reached the maximum value. The output nitrogen in harvested products comprised 10.8%-24.6% of total input nitrogen, and the highest percentage, 24.6%, was found in shrimp fish tagelus polyculture systems. In shrimp monoculture and shrimp fish polyculture systems, they were 19.1% and 21.9%, respectively. The nitrogen utilization efficiency was different and varied from 12.2% to 20.1%. The highest, 20.1%, was found in shrimp fish tagelus polyculture systems, and the average of 20.0% was found in shrimp tagelus polyculture systems. The lowest, 12.2%, was found in shrimp monoculture systems. All the nitrogen utilization efficiencies in shrimp fish systems or shrimp scallop systems seemed to be higher than that of the monoculture system, but they showed little statistical difference. The main outputs of nitrogen were found in sediment mud, and comprised 48.2%-60.8% of the total input, the lowest percentage was found in shrimp fish tagelus polyculture systems, and the highest percentage in shrimp scallop systems. During the experiment, nitrogen lost through denitrification and ammonia volatilization comprised 1.9%-6.2%, averaged 2.8%, of the total input, and the loss through seepage comprised 5.9%-8.9% of the total. The estimated nitrogen attached to the enclosure wall comprised 3.7%-13.3% of the total, and was highest in shrimp monoculture systems. Compared with the classic shrimp farming industry, the closed shrimp polyculture systems may improve the nitrogen utilization efficiency, and hence reduce the environmental impacts on coastal waters. The nitrogen discharging rates for all the studied polyculture systems ranged from 3.0% to 6.0% of total input nitrogen.

Development of an in vitro culture system of Rosa spp.Plants

[Objective] This study was to develop an in vitro tissue culture system of Rosa spp.[Method] Using ten species of Rosa spp.plants as experimental materials,different combinations of hormones were designed to establish their in vitro tissue culture system with the stem segments as explants.[Result] All ten tested varieties germinated when the nodal segment explants were cultured on the sprouting medium MS＋ 0.5 mg/L BA ＋0.01 mg/L NAA and grew vigorous shoots,and the sprouting rate was up to 70%.Of the ten tested rose varieties,each has a respective optimal proliferation medium,and the multiplication rates for all the varieties reached 3.0%.The axillary buds were vigorous and normal in leaf color.The optimal medium for rooting and acclimation was 1/2MS medium containing 0.1 mg/L or 0.2 mg/L NAA,in which the rooting frequency reached 90%-100% and the root system was developed.After acclimation and transplant,the survival rate was as high as 95%.[Conclusion] An in vitro tissue culture system of Rosa spp.has been established in this study,which lays foundation for the molecular breeding of Rosa spp.

MORPHOLOGICAL MODEL FOR RHEOLOGICAL PROPERTIES OF PLANT CELL SUSPENSION CULTURE SYSTEM

This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheological properties of the system as the effect of the flow behavior index on plant cell concentration are interpreted correctly and the mechanism of the rheological properties of the system is further understood.Therefore the model can be applied in the technological design and optimum conditions of the system and the reformation,evaluation and scale up of reactors.

Effects of Different Culture Systems on In Vitro Fertilization of Oocyte in Bovine

[ Objective] To investigate the optimal culture system for in vitro fertilization （IVF） of oocyte in bovine. [ Method] The IVF of mature bovine oocytes was conducted and the fertilized eggs were cultured in vitro with different culture systems. In the traditional culture system, the TALP medium and BO medium were used as fertilization medium, respectively; and M-199 medium supplemented with 5% FCS was used as fertilized egg culture medium. In the transitional culture system, the TALP medium and BO medium were used as fertilization medium; 60% TALP and 40% M-199 supplemented with 5% FCS and 60% BO and 40% M-199 supplemented with 5% FCS were used as fertilized egg culture medium, respectively. The effects of different culture systems on fertilization rate and cleavage rate were observed. E Result] In the transitional culture system （with TALP medium as fertilization medium and with 60% TALP and 40% M-199 supplemented with 5% FCS as fertilized egg culture medium）, the fertilization rate and cleavage rate were increased to 77.8% and 55.6%, respectively. [ Conclusion] The transitional culture system （with TALP medium as fertilization medium and with 60% TALP and 40% M-199 supplemented with 5% FCS as fertilized egg culture medium） is the optimal culture system for IVF of bovine oocyte.

Characteristics and Value of Traditional Lotus Culture System in Guangchang,Jiangxi Province

Jiangxi Guangchang traditional lotus culture system was selected into the fourth batch of China's important agricultural cultural heritage in 2017.The system consists of three parts:lotus,lotus cultivation techniques and lotus culture.It has notable characteristics of ancient origin,regional distribution,system complexity,biodiversity,advanced technology and extensive influence,and has ten kinds of value,i.e.,edible value,medical value,economic value,social value,ecological value,cultural value,tourism value,popular science value,scientific research value and educational value.

Sustainable Development of Wannian Rice Cultivation Culture System

Based on the research on the relevant background and significance of the sustainable development of agricultural cultural heritage,taking Wannian rice cultivation culture system as an example and its natural environment,traditional agricultural production activities,unique cultural patterns,socio-economic development and protection,and protection and development approach as the basis for evaluation,an evaluation index system for the sustainable development of Wannian rice cultivation culture system was constructed.Each index was quantified and standardized,and their weight was determined through the analytic hierarchy process to obtain the comprehensive evaluation value of the sustainable development of Wannian rice cultivation culture system.The evaluation value was analyzed to propose the main issues in the sustainable development of Wannian rice cultivation culture.

Differentiation of murine male germ cells to spermatozoa n a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system （SACS）, which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum （FCS）. The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia （Vasa, Dazl, OCT-4, C-Kit, GFR- a-l, CD9 and a-6-integrin）, meiotic cells （LDH, Crem-1 and Boule） and post-meiotic cells （Protamine-1, Acrosin and SP-IO）. Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3D)culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures.In cancer and stem cell research,the natural cell characteristics and architectures are closely mimicked by the 3D cell models.Thus,the 3D cell cultures are promising and suitable systems for various proposes,ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives.This review provides a comprehensive compendium of recent advancements in culturing cells,in particular cancer and stem cells,using 3D culture techniques.The major approaches highlighted here include cell spheroids,hydrogel embedding,bioreactors,scaffolds,and bioprinting.In addition,the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed,and the prominent studies of 3D cell culture systems were discussed.

Effect of Lamb Age and in vitro Culture System of Oocytes on JIVET Technology

[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 us, with a blastocyst development rate of (20.12 ± 8.18) % (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse (P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35) % ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) % , P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 + 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79, P<0.01). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2(5%CO2: 7%O2:88%N2) also showed no significant difference from those in high O2(5%CO2 in air) [(20.78 ± 8. 80)% and 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, P>0.05]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. METHODS: Mouse embryonic stem cells(ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional(3-D) self-assembling scaffolds and compared with traditional two-dimentional(2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate(PEG-4-Acr) and thiolfunctionalized dextran(Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoB lue(PB) assays. Genetic expression of pluripotency markers(Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D cultureconditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining(Oct4 and Nanog) and western blot analysis(Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH(1:1 v/v) to a final concentration of 5%(w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels(P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury(mesoderm), NCAM(ectoderm), and GATA4(endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.

Bacterial Blood Isolates in Children: Conventional vs. Bactec Automated Blood Culture System in a Tertiary Health Centre in Gombe, North East Nigeria

Background/Aim: Blood culture is critical in the diagnosis and treatment of blood stream infections (BSIs) especially in children. BSIs are among the most common cause of morbidity/mortality and blood culture has remained the gold standard for diagnosis. We sought to compare Blood Culture Isolates (BCI) from conventional and Bactec automated blood culture system (ABCS) among paediatric patients at the Federal Teaching Hospital Gombe (FTHG) Nigeria. Methods: BCI in children (0 - 18 years) by conventional method from 2008-2012 and Bactec Automated culture system from 2015-2020 were retrieved from the clinical microbiology laboratory register. Information analyzed included, age, sex, month, and year and blood culture isolates. Results: There were 5276 (56.9% males, 43.1% females) and 1169 (54% males, 46% females) Blood Culture Isolates by CM and ABCS respectively. Overall positive culture isolates were 9.7% (515/5276) in CM and 45.9% (536/1169) in ABCS (p = 0.01). Positivity rate in newborn was 13.3% (282/2114) by CM and 40.9% (219/263) by ABCS p = 0.01;under-5 was 10.5% (448/4253) vs. 37% (359/873) (p = 0.01);Gram positive 32.6% (172) vs. 65% (759) (p = 0.01;Gram negative 55% (2910) vs. 34% (397) (p = 0.01). Staph aureus 22% (114/515) by CM vs. 61.9% (332/536)) by ABCS (p = 0.01);Klebsiella 24.9% (128/515) by CM vs. 7.5% (40/536) p = 0.01) in ABCS, E. coli 8.9% (46/515) vs. 2.1% (11/536) p = 0.01;Proteus vs. 1.1% (6/515) by ABCS, Pseudomonas 3.3% (17/515) vs. 5.6% (30/536) p = 0.05, Alkaligenes 1% (5/515) vs. 8.2% (44/536) p = 0.01 and Citrobacter 1% (5/515) vs. 8.4% (45/536) p = 0.01. Conclusion: Blood culture yield was five times higher with Bactec compared with Conventional method.

The famous versus the inconvenient-or the dawn and the rise of 3D-culture systems

One of the greatest impacts on in vitro cell biology was the introduction of three-dimensional(3D)culture systems more than six decades ago and this era may be called the dawn of 3D-tissue culture.Although the advantages were obvious,this field of research was a "sleeping beauty"until the 1970s when multicellular spheroids were discovered as ideal tumor models.With this rebirth,organotypical culture systems became valu-able tools and this trend continues to increase.While in the beginning,simple approaches,such as aggregation culture techniques,were favored due to their simplicity and convenience,now more sophisticated systems are used and are still being developed.One of the boosts in the development of new culture techniques arises from elaborate manufacturing and surface modification tech-niques,especially micro and nano system technologies that have either improved dramatically or have evolved very recently.With the help of these tools,it will soon be possible to generate even more sophisticated and more organotypic-like culture systems.Since 3D per-fused or superfused systems are much more complex to set up and maintain compared to use of petri dishes and culture flasks,the added value of 3D approaches still needs to be demonstrated.

The Effect of Three Culture Methods on Intensive Culture System of Pacific White Shrimp (Litopenaeus vannamei)

Different culture methods may affect the intensive culture system of Pacific white shrimp(Litopenaeus vannamei) regarding water quality and growth and economic performance.This study evaluated the potential effects of three culture methods through cultivation of juvenile shrimps under consistent tank management conditions for 84 d.The three methods involved shrimp cultivation in different tanks,i.e.,outdoor tanks with cement bottom(mode-C),greenhouse tanks with cement bottom(mode-G) and outdoor tanks with mud-substrate(mode-M).Results showed that water temperature was significantly higher in mode-G than that in mode-C(P < 0.05).In contrast to the other two treatments,mode-M had stable pH after 50 d cultivation of shrimps.In the mid-late period,the average concentrations of TAN,NO2-N,DIP and COD were significantly lower in mode-M and mode-G compared with those in mode-C(P < 0.05).Despite lack of differences in the final shrimp weight among different treatments(P > 0.05),mode-M had significantly higher shrimp yield,survival rate and feed conversion rate(P < 0.05) than other modes.There were significant differences in revenue and net return among different treatments(P < 0.05).These demonstrated that the treatments of mode-G and mode-M were conductive to the intensive culture system of L.vannamei.

An Improved Culture System for Virus Isolation and Detection

Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.

Tissue Culture System for Different Hybrid of Indica Rice

The effect of varieties and media compositions on callus induction from rice anther and subsequent plant regeneration was studied. The results showed that the callus induction was not significantly different in different media, but mostly depended on genotypes; mean frequency of callus induction on F1 hybrid varieties showed that the medium supplemented with 100 mg·L^-1 concentration of the proline was useful for callus induction; when the anther derived callus sub-cultured on MS medium supplemented with BAP （0.5 mg·L^-1）, KT （1.0 mg·L^-1）, IAA （0.25mg·L^-1） and NAA （0.5 mg·L^-1） for plant regeneration, the frequency of regeneration ranged from 0 to 54.17%, and the highest differentiation index reached 30.5 after sub-cultured for three times; the plants were rooted after transferred to 1/2MS medium with NAA （0.5 mg·L^-1） and IAA （0.5mg·L^-1）.