Screening and Characterization of Nitrite-Degrading Bacterial Isolates Using a Novel Culture Medium

In this study,a novel culture medium that simulates shrimp pond conditions was established to screen nitrite-degrading isolates.The medium was supplemented with nitrite as a nitrogen source and shrimp feed as the major carbon source,to achieve the high nitrogen and low carbon nutritional status found in shrimp farming ponds.Screening using this medium identified potent denitrifying Bacillus isolates,among which Bacillus subtilis M7-1 was considered best.M7-1 was able to completely degrade nitrite-N in 24 h without much consumption of dissolved oxygen.Efficient denitrification activity took place in liquid cultures within a set of non-stringent ranges of pH(5.0–9.0),salinity(0–30)and temperature(25–35℃).The denitrifying enzyme gene was amplified,sequenced and further identified as nirS type.In biosecurity assessments,M7-1 had no negative effects on shrimps at a dose of 106 cfu mL−1.M7-1 could therefore be used in aquaculture to reduce and control the nitrogen concentration,and to promote the development of sustainable and healthy culture systems.

Evaluation of Endotoxin in Culture Medium for Human in vitro Fertilization

To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.

COMPARISON OF POLYSACCHARIDES ISOLATED FROM THE MYCELIA OF A CULTIVATED STRAIN OF PORIA COCOS GROWN IN DIFFERENT LIQUID CULTURE MEDIA

Mycelium of a cultivated strain of Poria cocos was grown by submerged fermentation in a liquid mediumcontaining corn steep liquor with orbital shaking. Six polysaccharides coded as ac-PCM1, ac-PCM2, ac-PCM3-Ⅰ andⅡ, ac-PCM4-Ⅰand Ⅱ were isolated from the myelium by extracting with 0.9% NaCl aqueous solution, hot water, 0.5 mol/L NaOHaqueous solution and 88% formic acid. Exo-polysaccharide was obtained from the culture medium and coded as ac-PCM0.The monosaccharide composition and molecular weights of these polysaccharides were characterized by using infraredspectroscopy, gas chromaography, elemental analysis, ^(13)C-NMR, viscometry and light scattering. The results indicated thatac-PCM0, ac-PCM1 and ac-PCM2 are heteropolysaccharides containing glucose, galactose, mannose and fucose, and ac-PCM3-Ⅰ and ac-PCM3-Ⅱ mainly consist of D-glucose. The content of the glucose in the polysaccharides increased with theisolation progress. Remarkably, α-glucan and β-glucan coexisted in the extract by NaOH aqueous solution (ac-PCM3), andcould be separated by chemical methods. The protein in the ac-PCM polysaccharides cultured from the medium containingcorn steep liquor was higher than that in the ab-PCM from the medium with bran extract. Therefore, the polysaccharidesfrom Poria cocos mycelia cultured in different media have different chemical composition, molecular weights and conformations.

Relationship Between Differential Expression of Bax and Bcl-2 Genes and Developmental Differences of Porcine Parthenotes Cultured in PZM-3 and NCSU-23

The developmental competence of porcine parthenotes cultured in porcine zygote medium-3 （PZM-3） and North Carolina State University-23 （NCSU-23） media was investigated. After in vitro maturation oocytes were electro-activated, and the subsequent developmental competence, rates of apoptotic, fragmented and arrested embryos from the two media were evaluated. Also, the ratio of mRNA expression of Bcl-2 and Bax gene was determined. Results demonstrated that cleavage, blastocyst, hatched blastocyst rates, and blastocyst cell numbers were significantly higher in PZM-3 than in NCSU-23. The rate of fragmented embryos in PZM-3 was lower than in NCSU-23 on d 1 and 3 （P〈0.05） while the percentage of arrested embryo was lower in PZM-3 than in NCSU-23 on d 4 and 5 （P〈0.05）. TUNEL positive signals were higher in NCSU-23 than in PZM-3 from d 3 to 7 （P〈0.05）. The ratios of Bcl-2 and Bax mRNA expression in embryos were higher on d 5 than on d 3 and 1 in PZM-3 （P〈0.05）. In contrast, the ratios of Bcl-2 and Bax mRNA expression in embryos on d 1 were higher than on d 3 and 5 in NCSU-23 （P〈0.05）. Additionally, the ratios of Bcl-2 and Bax mRNA expression in embryos in PZM-3 were higher than in NCSU-23 on d 3 and 5 （P〈0.05）. In conclusion, lower apoptotic embryo rates and down-regulating Bax together with up-regulating expression of Bcl-2 transcripts may be responsible for the better developmental competence of porcine parthenotes in PZM-3.

Effects of different culture media on the growth of Indian sandalwood(Santalum album L.) seedlings in Zhanjiang,Guangdong,southern China

We studied the effects of different culture media on the growth of India sandalwood （Santalum album L.） seedlings in Zhanjiang, Guangdong Province in southern China. Five different growth substrates, lateritic subsoil, burnt soil, agricultural soil, peaty soil and coconut dust, were used as the basic culture materials and seven different treatments of composition were used as potting media. Kuhnia rosmarinifolia Vent. was used as a primary host plant for all treatments. Statistically significant differences were found between treatments in respects of survival rate （p 〈 0.001）, height （p 〈 0.001）, ground diameter （p 〈 0.001） and biomass （p = 0.002）, as well as for quality index （p 〈 0.001） of S. album seedlings after 6-month growth in containers with different culture media. Among all treatments, the treatment combining burnt soil, peat and coconut dust in a weight ratio of 1：1：1 plus 2% calcium super-phosphate as basal manure achieved the best performance for most of the seedling growth parameters, including survival rate （98%）, height （35.81 cm）, ground diameter （0.56 cm）, biomass （4.46 g） and quality index （0.65）, followed by the treatment using only burnt soil plus 2% calcium super-phosphate as the culture medium （survival rate 86%, height 29.23 cm, ground diameter 0.48 cm, biomass 3.36 g and quality index 0.52）, while the treatment using only lateritic subsoil plus the basal manure as the medium obtained the poorest results in survival rate （38%）, height （12.04 cm）, ground diameter （0.19 cm）, biomass （0.26 g） and quality index （0.043）. Increasing the proportion of lateritic subsoil in the medium when mixed with peat and coconut dust did not show statistically significant differences in survival, height, ground diameter, biomass nor in quality index. In consideration of cost, using burnt soil plus 2% calcium super-phosphate as basal manure may be the optimum culture medium for large-scale production of Indian sandal- wood seedlings in Guangdong, southern China.

Culture Media Influenced Laboratory Outcomes But Not Neonatal Birth Weight in Assisted Reproductive Technology

Summary： Whether the type of culture media utilized in assisted reproductive technology has impacts on laboratory outcomes and birth weight of newborns in in-vitro fertilization （IVF）/intracytoplasmic sperm injection （ICSI） was investigated. A total of 673 patients undergoing IVF/ICSI and giving birth to live singletons after fresh embryo transfer on day 3 from Jan. 1, 2010 to Dec. 31, 2012 were included. Three types of culture media were used during this period： Quinn＇s Advantage （QA）, Single Step Medium （SSM）, and Continuous Single Culture medium （CSC）. Fertilization rate （FR）, normal fertilization rate （NFR）, cleavage rate （CR）, normal cleavage rate （NCR）, good-quality embryo rate （GQER） and neonatal birth weight were compared using one-way ANOVA and Z2 tests. Multiple linear regression analysis was performed to determine the impact of culture media on laboratory outcomes and birth weight. In IVF cycles, GQER was significantly decreased in SSM medium group as compared with QA or CSC media groups （63.6% vs. 69.0% in QA; vs. 71.3% in CSC, P=0.011）. In ICSI cycles, FR, NFR and CR were significantly lower in CSC medium group than in other two media groups. No significant difference was observed in neonatal birthweight among the three groups （P=0.759）. Multiple linear regression analyses confirmed that the type of culture medium was correlated with FR, NFR, CR and GQER, but not with neonatal birth weight. The type of culture media had potential influences on laboratory outcomes but did not exhibit an impact on the birth weight of singletons in ART.

Isolation and Screening of Oenococcus oeni and Its Medium Optimization

[Objectives] This study was conducted to further improve the application of Oenococcus oeni in the wine brewing industry. [Methods] O. oeni was isolated, screened, and identified, and medium optimization was performed to determine the best medium, temperature and oxygen conditions. [Results] O. oeni grew better in mFT80 medium, and the culture conditions were 25 ℃ and an anaerobic condition. [Conclusions] This study provides a reference for the development of China’s wine industry.

A Preliminary Study on Tissue Culture and Rapid Propagation of Virus-free Plantlets of Benihoppe Strawberry

[ Objective] This study aimed to investigate the tissue culture and rapid propagation techniques of Benihoppe strawberry. [ Method] Shoot tips of new stolons of Benihoppe strawberry were used as experimental materials to analyze the effects of media type, cytokinin type and concentration, carbon source type and concentration, and light intensity on tissue culture and propagation of Benihoppe strawberry. [ Result] MS was the optimal media for plantlet propagation. In the media containing 1.2 ing/L BA （with addition of 0.1 mg/L NAA）, fresh weight, dry weight and propagation coefficient of strawberry plantlets reached the maxi- mum, which were 2.259 g, 0. 221 g and 12.4, respectively. The optimal carbon source was 30 g/L sucrose, and the optimal light intensity was 1 600 lx. [ Conclu- sion] The best media for tissue culture and rapid propagation of Benihoppe strawberry was MS ＋ BA 1.2 mg/L ＋ NAA 0. 1 mg/L ＋ sucrose 30 g/L ＋ agar 8 g/L. This study provided scientific basis for large-scale production of Benihoppe strawberry.

Study on Tissue Culture and Adventitious Bud Induction of Eucheuma striatum

This study aimed to investigate the aseptic treatment and disinfection of E. striatum explants and the effects of three liquid culture medium （ PES, PESI, f/2） and plant hormones on adventitious bud induction from explants. The results showed that the vast majority of explants disinfected by combined treatment exhib- ited good growth and normal color with an average mortality rate of only 9%. E. striatum explants exhibited the best growth in PES liquid medium and the survival rate reached 66.67% at 40 d after culture. Under different concentrations （0.5, 2, 5 mg/L） of 6-BA, the induction rate ofE. striatum explants was significantly higher than that in control group, but no remarkable differences were found in the induction of adventitious buds from E. striatum explants. In PES liquid medium containing 6-BA and 2,4-D, the adventitious bud induction rate was 90% and the average number of induced adventitious buds was 6.63.

Study on Optimization of Culture Conditions for Wild Isaria cicadae Miquel Liquid Strain

[Objectives] To study the effect of different culture conditions on the growth of wild I. cicadae Miq. liquid strain. [Methods]The I. cicadae Miq. strain was inoculated into the liquid culture medium with different carbon sources,nitrogen sources,micronutrients and p H,cultured in the constant temperature shaking incubator with rotating speed of 120 r/min at 19℃,and the mycelium pellet diameter,density and weight were compared between different treatments. [Results] The results showed that the optimum components of I. cicadae Miq.strain liquid included soluble starch,milk powder and vitamin B_(12),and the optimum p H was 5. 0-6. 0. [Conclusions] Soluble starch was the most suitable carbon source for the culture medium of I. cicadae Miq. liquid strain; milk powder was the most suitable nitrogen source for the culture medium of I. cicadae Miq. liquid strain; the most suitable p H was 5. 0-9. 0 for the mycelial growth of I. cicadae Miq.; the formula and mixture ratio of the optimum culture medium for the growth of liquid strain were determined.

Effect of Different Culture Media on the Proliferation of Avian Influenza Virus H9 Subtypes in MDCK Cells

[Objective] To screen the best culture media for the proliferation of avian influenza virus （AIV） H9 subtypes in MDCK cells. [Method] The DMEM containing 10% （V/V） newborn calf serum, low-serum containing medium （ MEM-MD-611 ） and serum-free medium （SFE4Mega） were used to culture the MDCK monolayer ceils, which were then inoculated with different dilutions of AIV H9 subtypes, and the 3 kinds of media were al- so used as the maintenance solution to culture the virus. The cytopathic changes were observed at every 24 h, and the HA titers of the culture su- pernatants were also determined. [ Result] After culturing for 72 -96 h, the HA titers of the serum-free media were higher than that of low-serum culture media, while the HA titers were higher in the low-serum media than in the serum containing media. [ Conclusion] The 3 kinds of media can all used for the proliferation of AIV_ but the low-serum culture medium （MEM-MD-611 ） and serum-free medium （SFE4Meaa3 are preferred.

Screening of Optimal Differentiation Medium to Lonicera edulis

An excellent Lonicera edulis strain, L1-8 that was bred by Northeast Institute of Geography and Agro-ecology, Chinese Academy of Sciences, was used as material in this research. The axillary buds of its dormant branches were used as explants. A fourfactor and four-level orthogonal test was designed in order to choose the best differentiation medium for providing the technical support of Lonicera edulis micropropagation. The results showed that the culture medium and concentration of 6-BA were the main factors, and the best differentiation condition was MS culture medium containing 2.0 mg · L-1 6-BA, 0.3 mg · L-1 IBA and 1.5 mg · L-1 GA3.

Preliminary Study on in Vitro Germination of Yangmei Pollen

[Objectives]To explore the conditions for in vitro culture of Yangmei pollen.[Methods]Experiments were conducted on the germination characteristics of three types of Yangmei pollen using in vitro culture and germination method.[Results]The suitable medium ratio for the germination of Yangmei pollen was 10%sucrose+0.01%borax+1%agar;the cultivation temperature of 30℃was more suitable for the germination of Yangmei pollen than 25℃;through analysis of variance,among the three types of Yangmei pollen,pollen of male 1 had the strongest viability,and it was the better pollination type.[Conclusions]The research could provide certain basis for introduction and cultivation of Yangmei and improving hybrid breeding effect.

Micropropagation of Eucalyptus grandis×E.urophylla AEC 224 clone

Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis x E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 μM BA and 0.5 μM NAA for another 30 days. In these media, the explant oxidation rate was high （95 %）. Thus, in order to reduce oxidation, different ;culture media were compared： WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-D were tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25 μM TDZ and 0.1μM NAA during 30 days for callus induction and then with 5.0 μM BA and 0.5 μM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA （2.46, 4.90 and 7.35μM）. The highest rooting percentage （35 %） was obtained on medium containing 2.46μM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %.

Characteristics of a kHz helium atmospheric pressure plasma jet interacting with two kinds of targets

This study investigates the influence of two types of target,skin tissue and cell culture medium,with different permittivities on a k Hz helium atmospheric pressure plasma jet(APPJ)during its application for wound healing.The basic optical-electrical characteristics,the initiation and propagation and the emission spectra of the He APPJ under different working conditions are explored.The experimental results show that,compared with a jet freely expanding in air,the diameter and intensity of the plasma plume outside the nozzle increase when it interacts with the pigskin and cell culture medium targets,and the mean velocity of the plasma bullet from the tube nozzle to a distance of 15 mm is also significantly increased.There are also multiple increases in the relative intensity of OH(A^(2)Σ→X^(2)Π)and O(3p^(5)S-3s^(5)S)at a position 15 mm away from nozzle when the He APPJ interacts with cell culture medium compared with the air and pigskin targets.Taking the surface charging of the low permittivity material capacitance and the strengthened electric field intensity into account,they make the various characteristics of He APPJ interacting with two different targets together.

Character of cell-free genomic DNA in embryo culture medium and the prospect of its clinical application in preimplantation genetic testing

There is increasing evidence that cell-free DNA (cfDNA) in spent culture media (SCM) can be amplified for genetic testing. Therefore, this paper reviews the characteristics of cfDNA, including its fragment size, amount, origin, as well as some factors affecting the success rate of its amplification, together to provide researchers with a more comprehensive perspective on embryonic cfDNA. The origin of cfDNA in SCM is complicated and poses challenges to the interpretation of genetic test results. Advanced molecular techniques should distinguish between embryonic and contaminated DNA to maximize the success rate of amplification and analysis. Recent data showed that the type of culture medium, assisted hatching or not, the type of amplification kit, and fresh or thawed embryos were not related to the success rate of amplification, but the length of culture time might affect the success rate. The longer culture time, the more cfDNA is available in the SCM. Then we focused on the concordance between trophectoderm (TE), inner cell mass, whole embryo, and embryonic cfDNA. Despite successful amplification, the concordance between TE and embryonic cfDNA was low. In summary, non-invasive genetic testing using SCM could represent a major advance in future single embryo selection, however, contamination and timing for media collection are key factors affecting the results, and current non-invasive cfDNA testing should not be directly applied to clinical practice. Further research is needed to improve the methods used for testing techniques and genetic analysis to achieve greater accuracy and trace its origins before it can be used in the clinics.

In Vitro Propagation of Grapevine Cultivars with Potential for South of Brazil

The micropropation is an important biotechnological tool for obtaining and maintaining mother vine plants with high quality plant health. The objective was to evaluate the establishment and multiplication in vitro and ex vitro acclimatization of grape genotypes with potential for Southern Brazil. Vine nodal segments were cultured in five culture medium formulations without adding growth regulators. It was evaluated the number of leaves and roots, length of roots and shoots, replication rate, relative chlorophyll index, percentage of regenerated and rooted plants, dry biomass of shoot, root and total plants grown in vitro and after acclimatization. In vitro propagation of IAC 571-6 rootstock and cv. Poloskei Muskotaly through nodal segments provided high rates of regeneration and rooting. High survival rates were obtained in the acclimatization of IAC 571-6 and P?l?skei Muskotaly. Considering all the variables, the culture medium Roubelakis showed the best growth rates and development for shoots and roots, and in vitro multiplication rate for IAC 571-6 and Poloskei Muskotaly grape varieties.

From nature to nurture:Essence and methods to isolate robust methanotrophic bacteria

Methanotrophic bacteria are entities with innate biocatalytic potential to biofilter and oxidize methane into simpler compounds concomitantly conserving energy,which can contribute to copious industrial applications.The future and efficacy of such industrial applications relies upon acquiring and/or securing robust methanotrophs with taxonomic and phenotypic diversity.Despite several dramatic advances,isolation of robust methanotrophs is still a long-way challenging task with several lacunae to be filled in sequentially.Methanotrophs with high tolerance to methane can be isolated and cultivated by mimicking natural environs,and adopting strategies like adaptive metabolic evolution.This review summarizes existent and innovative methods for methanotrophic isolation and purification,and their respective applications.A comprehensive description of new insights shedding light upon how to isolate and concomitantly augment robust methanotrophic metabolism in an orchestrated fashion follows.

Methods for extracellular vesicle isolation from cancer cells

Cells are known to release different types of vesicles such as small extracellular vesicles(sEVs)and large extracellular vesicles(LEVs).sEVs and LEVs play important roles in intercellular communication,pre-metastatic niche formation,and disease progression;both can be detected cell culture media and biological fluids.sEVs and LEVs contain a variety of protein and RNA cargo,and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins.It has recently been established that standard isolation techniques,such as differential ultracentrifugation,yield a mixed population of EVs.However,density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris.Here,we describe the most common methods used to isolate sEVs from cell culture medium,mouse and human plasma,and a new technique for isolating sEVs from tissues as well.This article also provides detailed procedures to isolate LEVs.