Culture Media Options for Growth and Morphological Characterisation of Cercospora coffeicola Affecting Coffee in Zimbabwe

Cercospora leaf spot is fast turning into a critically important disease in Zimbabwe.The disease is caused by Cercospora coffeicola which significantly reduces productivity and quality of coffee.Disturbingly,optimum sporulation of Cercospora coffeicola in culture remains a limiting factor for microbial analysis and quantitative studies of Cercospora leaf spot.Faced with this challenge,an in-vitro study was conducted at Coffee Research Institute,Manicaland,Zimbabwe to examine growth of Cercospora coffeicola in different nutrient media and to determine the best media for Cercospora coffeicola analysis.Six nutrient media were assessed(corn meal agar,oat meal agar,Czapek Dox agar,malt extract agar,yeast extract agar and potato dextrose agar)for the growth of Cercospora coffeicola.The laboratory-based experiment was duplicated,laid out in a Completely Randomized Design,replicated three times and based on Cercospora coffeicola nutrient inoculation.Data were collected on radial growth,colour and texture of mycelium at 3 and 6 days after inoculation.There were significant differences(p<0.05)in the growth of Cercospora coffeicola in media after 3 and 6 days.Malt extract agar had the greatest radial growth(34 mm and 32 mm)of Cercospora coffeicola for trials 1 and 2 respectively,whilst the least growth was in the oat meal agar(14.2 mm and 15.7 mm)for trials 1 and 2 respectively.There were variations in colour and texture of mycelium with malt extract agar,potato dextrose agar and oat meal agar associated with darker colours and rough texture while smooth white mycelia were found in corn meal agar.After considering all nutrient media,malt extract agar was found to be the best media for the growth of Cercospora coffeicola in-vitro.On the basis of our findings,the authors recommend the use of malt extract agar as the primary media for identification and characterisation of Cercospora coffeicola.

Are Early Embryo Cleavage Kinetics Affected by Energy Substrates in Different Culture Media?

Objective This study aimed to investigate the influence of different culture media on early embryonic cleavage kinetics using time-lapse analysis and to determine the possible relationships between energy substrates in culture media and the cleavage kinetics.Methods A total of 10021 embryos from 1310 couples were cultured in time-lapse incubators.Embryos cultured in Vitrolife media were allocated to group I,and those in COOK media to group II.Embryo cleavage time points up to the 8-cell stage(t2–t8)were observed after pronuclei fading.Results The baseline demographic features,in vitro fertilization indications,ovarian stimulation protocol,oocyte-cumulus complexes,fertilization rate,together with pregnancy and perinatal outcomes were similar(P>0.05)between groups I and II.According to the time-lapse analysis,all embryos in group I showed significantly faster cleavage speed than those in group II(P<0.05).Furthermore,there was better synchrony in division(s3)and a longer length of the third cell cycle duration(cc3)in group II.Interestingly,implanted embryos in group II showed faster cleavage speed than those in group I,especially at t4 and t7.The glucose contents and multiple major amino acids were similar between the two groups.Lactic and pyruvic acid contents were generally higher in group I than those in group II.Conclusion Because different commercial culture media may influence cleavage kinetics of embryos,it is essential for embryologists to take culture media into consideration in selecting a potential embryo when using a time-lapse system before implantation.

Coscinium fenestratum: Callus and Suspension Cell Culture of the Endangered Medicinal Plant Using Vermicompost Extract and Coelomic Fluid as Plant Tissue Culture Media

In vitro tissue culture of hard woody, endangered, medicinal plant Coscinium fenestratum is most challenging to plant tissue culturists. In the present study, petiole and leaf explants of Coscinium fenestratum were induced to form callus when cultured on vermicompost extract media along with coelomic fluid. Suspension medium was developed using vermicompost extract and coelomic fluid in 3:1 ratio. Phytochemical analysis of the alkaloid berberine was confirmed from callus, suspension cell culture and suspension medium by Thin Layer Chromatography and High Performance Liquid Chromatography. Vermicompost and its extracts with coelomic fluid have shown maximum (100 per cent) response of callus induction. Callus mass enlarged with increasing concentration of coelomic fluid and callus growth was assessed from the biomass. Incubation of culture tubes in dark supported callus development significantly. The Rf value of 0.36 confirmed the presence of berberine by Thin Layer Chromatography. Qualitative analysis confirmed the presence of alkaloid berberine with the retention time of 2.8 minutes similar to that of standard reference sample from Sigma chemicals, USA. The suspension medium turned deep yellow because of the release of the alkaloid. Vermicompost and its extracts along with coelomic fluid have shown the economical approach for micropropagation of economically and medicinally important plants.

Effect of agar concentration in media on green plantlet regeneration frequency in rice anther culture

We studied the effect of agar concentration inmedia on callus induction rate and green plant-let regeneration frequency in rice.Materialswere Fgeneration of indica/indica or indica/japonica,which were 96E76(Hei’e/Zhaiye- qing 8),96E80[(IR 24/Guanglu＇ai 4//Zhongnan’ai)/Yifengzao],96E86(Zhong- munong 9/Zhaiyeqing 8).The induction mediaused was M8+2mg/L 2,4-D,and agar con-centrations were 0.6％,0.8％,and 1.0％,respectively.Regeneration media was MS+2mg/L KT+0.5mg/L IAA+0.5mg/LNAA,and agar concentrations were 0.6％ and1.0％.Results indicated that the calli induc-

The effects of different strength of MS media in solid and liquid media on in vitro growth of Typhonium flagelliforme

Objective: To determine the effects of different strength of Murashige and Skoog(MS)media(full,1/2and1/4) in solid and liquid media on in vitro growth of Typhonium flagelliforme(T. flagelliforme), whereby an optimum media composition can be provided for mass propagation of T. flagelliforme.Methods: Rhizome bud of T. flagelliforme was obtained from the axenic in vitro established T. flagelliforme plantlets in Plant Tissue Culture Laboratory, Universiti Teknologi MARA, Shah Alam. Rhizome bud was used as explant and cultured onto shoot proliferation medium under different strength of MS media(full,1/2,1/4) in solid and liquid culture media.Results: After 6 weeks of culture, the number of shoot, number of leaf, number of root,height of shoot, fresh weight, dry weight and chlorophyll content of T. flagelliforme were analyzed. A comparison was made between liquid and solid culture media. The results revealed that the liquid culture media were more effective for all the growth parameters(shoot height, shoot number, leaf number, root number, fresh weight, dry weight, chlorophyll a and chlorophyll b content) compared to solid culture media. Apart from that,this study revealed the positive relationship between strength of MS media and type of culture media(solid and liquid media) to the growth of T. flagelliforme. Growth of T. flagelliforme was improved when MS strength was increased in liquid media. In contrast, growth of T. flagelliforme was improved when MS strength was decreased in solid media.Conclusions: Through this study, an optimum media composition for mass propagation of T. flagelliforme had been established by observing effects of MS media strength and type of culture media(solid and liquid media) on the growth of T. flagelliforme.

An Antimony Oxide pH Electrode for Tissue Culture Medium

A new approach named“caterpillar melt method”was developed to prepare wire type antimony oxide electrode for pH measurement in agar medium for tissue culture.A micro antimony wire was prepared from melt of the metal with the help of a glass capillary and the surface of the wire was oxidized in nitrate melt to obtain an antimony oxide electrode. Characterization results showed that the oxide layer is dense and uniform,with high physical and chemical stability.The electrode has a fast and stable response toward pH change for aqueous solutions.The potential of the antimony electrode has a linear relationship with the pH of the solution (R^2=1.00) with a sensitivity of 54.1mV/pH.The electrode works well and is more stable in agar medium during tissue culture for pH monitoring.

Morphology and Molecular Identification of Dry Fish Fungus Cunninghamella blakesleeana from Small Indigenous Fish “Kachki” Corica soborna (Hamilton 1822) in Bangladesh

The present experiment was conducted to investigate a dry fish fungus, Cunnighamella blakesleeana, which was identified from the infected part of the Corica soborna, locally named as Kachki fish. Mycelium was hyaline, often with granular content, and conidiophores were erected, with verticillate or solitary branches. Zygospores were globose, tuberculate, suspensors equal, smooth, hyaline and heterothallic. Using ITS4 and ITS5 primers, the 740 bp-long ITS region was amplified and sequenced. The ITS region sequences had reciprocal homologies of 98% to 100%. The findings showed that several species of C. blakesleeana fall into the same cluster. It has been determined by molecular data that the fungus we had studied was C. blakesleeana. The maximum mycelial growth (95.33 mm) was observed in the PDA medium, followed by the PSA medium, and the lowest growth (65.50 mm) was measured in the HPA medium in the study of the impact of culture media on the mycelial growth of C. blakesleeana. The influence of temperature on the radial mycelial growth of C. blakesleeana on PDA medium was investigated through five different temperatures. Although pH is a crucial factor in understanding the ecology of spoilage fungus, the highest mycelial growth of C. blakesleeana (88.25 mm) was seen at pH 7, followed by pH 8 and pH 6, while pH 9 was revealed to have the lowest mycelial growth. The outcome suggested that C. blakesleeana thrived in neutral environments.

Methods for the extraction and RNA profiling of exosomes

AIM:To develop protocols for isolation of exosomes and characterization of their RNA content.METHODS:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from cell culture media)reagent,and Total exosome isolation(from serum)reagent respectively.Identity and purity of the exosomes was confirmed by Nanosight?analysis,electron microscopy,and Western blots for CD63 marker.Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit.Finally,RNA was profiled using Bioanalyzer and quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR)methodology.RESULTS:Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum,with subsequent isolation and analysis of RNA residing within these vesicles.The isolation procedure is completed in a fraction of the time,compared to the current standard protocols utilizing ultracentrifugation,and allows to recover fully intact exosomes in higher yields.Exosomes were found tocontain a very diverse RNA cargo,primarily short sequences 20-200 nt(such as mi RNA and fragments of m RNA),however longer RNA species were detected as well,including full-length 18S and 28S r RNA.CONCLUSION:We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes,followed by isolation of RNA and its analysis by q RT-PCR and other techniques.

First Report on Rhizome Rot Disease of Curcuma longa Caused by Fusarium solani in Bangladesh

Turmeric (Curcuma longa L.) is a valuable medicinal plant as well as spice crop in Bangladesh. The rhizome rot disease is a severe danger to turmeric cultivation. The current study sought to identify the fungal pathogen linked to turmeric rhizome rot disease. Rhizome of turmeric with distinct rotted symptoms was collected from the experimental site of the Botanical Garden, Jahangirnagar University, Bangladesh. The sample was screened to isolate the causative fungal pathogen through the tissue planting technique. Macro and micro-morphological characterization based on colony appearance, mycelial and conidial characteristics primarily identified the fungus as Fusarium sp. The ITS sequence of rDNA of the fungus exhibited 99 to 100 percent similarity with the other F. solani species formerly deposited in the NCBI database which confirmed the fungal identity as F. solani. An in vitro pathogenicity test validated the pathogenic nature of the fungus. Growth behaviors of the fungus were evaluated on different solid culture media viz., Potato dextrose agar, Potato sucrose agar, Sabouraud dextrose agar and Hansen’s agar;temperature conditions (10?C, 15?C, 20?C, 25?C, 30?C and 35?C) and pH levels (pH 4, pH 5, pH 6, pH 7 and pH 8). Maximum mycelial growth was obtained on PSA medium at 30?C temperature and pH 7 conditions. Current findings also conclude that F. solani favors a wide range of temperature and pH levels. To the best of our search, the present investigation revealed the relationship of F. solani with the rhizome rot disease of turmeric for the first time in Bangladesh.

Pathway for interferon-gamma to promote the differentiation of cholinergic neurons in rat embryonic basal forebrain/septal nuclei

BACKGROUND： The supernatant of interferon-gamma （IFNγ） co-cultured with neonatal rat cortical glia can promote the cells in embryonic basal forebrain/septal nuclei to differentiate into cholinergic neurons, but the mechanism is still unclear. OBJECTIVE： To analyze the pathways for IFNγ to promote the differentiation of primarily cultured cholinergic neurons in rat embryonic basal forebrain/septal nuclei through culture in different conditioned medium. DESIGN： A controlled experiment taking cells as the observational target. SETTINGS： Department of Biochemistry and Molecular Biology, Youjiang Medical College for Nationalities; Department of Cell Biology, Beijing University Health Science Center. MATERIALS： Sixty-four pregnant Wistar rats for 16 days （250-350 g） and 84 Wistar rats （either male or female, 5-7 g） of 0-1 day after birth were provided by the experimental animal department of Beijing University Health Science Center. Rat IFNγ were provided by Gibco Company; Glial fibrillary acidic protein by Huamei Company. METHODS： The experiments were carried out in the Department of Cell Biology, Beijing University Health Science Center and Daheng Image Company of Chinese Academy of Science from July 1995 to December 2002. ① Interventions： The nerve cells in the basal forebrain/septal nuclei of the pregnant Wistar rats for 16 days were primarily cultured, and then divided into four groups： Blank control group （not any supernatant and medium was added）; Control group （added by mixed glial cell or astrocyte conditioned medium）; IFNγ group （added by mixed glial cell or astrocyte conditioned medium＋IFNγ）. Antibody group （added by mixed glial cell or astrocyte conditioned medium＋IFNγ＋Ab-IFNγ）. Mixed glial cell or astrocyte conditioned medium was prepared using cerebral cortex of Wistar rats of 0-1 day after birth. ② Evaluation： The immunohistochemical method was used to perform the choline acetyltransferase （ChAT） staining of cholinergic neurons. The ChAT positive cells were counted. MAIN OUTCOME MEASURES： Comparison of ChAT positive cells in rat basal forebrain and septal nuclei in different conditioned medium. RESULTS： ① ChAT positive cells in mixed glial cell conditioned medium： The ChAT positive cells in the IFNγ group and antibody group were significantly more than those in the control group （P 〈 0.01）. ② ChAT positive cells in astrocyte conditioned medium： The ChAT positive cells in the IFNγ group were significantly more than those in the control group, but there was no significant difference between the antibody group and control group （P 〉 0.05）. CONCLUSION： IFNγ cannot directly promote the differentiation of cholinergic neurons, but plays a role through activating glial cells （except astrocytes） to produce IFNγ like molecules.

Selective Lactococcus Enumeration in Raw Milk

The Lactococcus diversity in cow and goat raw milk was investigated. To do so, a protocol had to be established for the specific enumeration of lactococci. Eight agar media and one control medium were analysed to compare their proficiency in evaluating the Lactococcus population in raw milk: M17 Nal, Elliker, modified Elliker, PCA + milk, modified KCA, modified Chalmers, Turner, FSDA. The M17 medium was used as reference. Eighteen pure strains were tested on these media for their selectivity towards lactococci: six Lactococcus species or subspecies, three Leuconostoc, three Enterococcus, two Lactobacillus, one Streptococcus thermophilus, one Pseudomonas fluorescens, one Escherichia coli and one Staphylococcus aureus. All these bacteria were chosen for their regular presence in raw milk. The KCA medium proved to be the most selective towards lactococci, on condition that 1) we discriminated the colonies using the catalase test and 2) we subtracted the Enterococcus population counted on BEA. However, it was not possible to separate the Streptococcus from the Lactococcus colonies on KCA. The “Lactococcus-like” population including these two genera was estimated at a mean level of 3.18 log(cfu)/mL and 4.14 log(cfu)/mL in cow and goat raw milk respectively. This is consistent with the data already published.

Modernity and Postmodernity： The Characteristics of Postmodern Cultural Media

From the academic frontier of modernity and postmodernity, the author aims at exploring the fission between modernity and postmodernity and also the characteristics of postmodern cultural media from a philosophical vantage point. This paper illustrates three aspects of Western modernity： individual modernity, social modernity, and instrumental modernity, and it also clarifies the issues in modernity, starting by explaining three forms of the cultural fission： avant-garde, modernism, and postmodemism. The paper then demonstrates that the rise of postmodemity represents a new transformation and new characteristics of contemporary Western spirit, namely, the collision and compatibility of various concepts, in which popular culture and high culture, mass culture and elite culture, fashion and games as well as noise and silence have constituted an uncanny landscape of cultural media. This eerie landscape displays the indeterminacy of language, culture, art, consciousness, and aesthetics. From the perspective of theoretical innovation, the author proposes that the postmodem cultural media always displays its commodity and instrumentality, plays and entertainment, anti-culture and anti-art, replication and fabrication logically and practically as an outcome of post-industrial society. In conclusion, three critical issues are addressed： personal spiritual belief, the development of mass culture, and aesthetic principles. The postmodern cultural media has deeply influenced traditional culture, aesthetics, and how they are evaluated, resulting in cultural conflicts and a humanistic dilemma in the world of contemporary capitalism.

Intermediality in the Contemporary Brazilian Literature

Literature adapts following the changes of its objects,as do all arts.However,while fictional characters and context can be said to have equivalents in people and society,the same cannot be said about the literature form itself.Especially with the advent of the internet,this transformations in literature seem more visible:it has become spread in the cloud,surpassing its boundaries and mixing with the profusion of voices of the cyberspace.Intermediality,in addition to its theories about the intermedial phenomena,has become a methodology or even a pathway to understand arts.This paper presents part of an extensive analysis of a corpus formed by contemporary Brazilian literature,in order to describe different manifestations on how the"mediatized society"appears through the literature forms,from the Media Culture to today.

Noninvasive preimplantation genetic testing in assisted reproductive technology:current state and future perspectives

Invasive genetic screening of pre-implantation embryos via biopsied trophectoderm(TE)cells has been in use for more than 20 years,while its benefits in selecting euploid embryos remain controversial.Recent advances in the ability to process embryonic cell-free DNA(cfDNA)from blastocoel fluid(BF)and spent culture media(SCM)of blastocysts in a manner similar to that of a biopsied TE sample provide a potential alternative holding great promise for obtaining cytogenetic information of the embryos without intrusive biopsy of traditional biopsy-based pre-implantation genetic testing(PGT).Several studies have reported even higher diagnostic accuracy in non-invasive PGT(ni-PGT)than conventional PGT.However,there are still several technical challenges to be overcome before ni-PGT can be accepted as a reliable genomic information source of embryo.In this review,we have summarized the emergence and current state of ni-PGT,and discussed our own perspectives on their limitations and future prospect.There is still a long way to go before truly wide clinical application of ni-PGT.

Effect of the P1 Medium and the ECM Medium on Embryo Quality in IVF

Objective To investigate the effect of the glucose-free preimplantation stage one （P1） medium and the ECM medium on embryo development quality in IVF. Methods The patients with ≥4 zygotes of 2PN were studied. A total of 201 retrieval cycles were included in a prospective randomized study. Each patient was herself control. Half of zygotes of 2PN were transferred into ECM medium （group A） and half into P1 medium （group B）for further culture. Embryo development was evaluated on the day of embryo transfer. The efficacy of ECM was compared with P1 as culture medium for the development of preimplantation embryos. Results No statistically significant differences were noted between the two groups regarding embryo-cleavage rate （97.13% vs 97.55%） and rate of normal-cleaving embryos （58.29% and 58.37%）. The rate of top-quality embryos was statistically higher in group A than in group B （27.59% vs 19.75%, P〈O.05）. Embryo quality, as assessed by morphological parameters （the amount of detached anuclear fragments 〉30%）, was better in group A than in group B （19.86% vs 21.75%）, however, there was no statistically significance. Both the rate of good-quality embryos （47.95% vs 46.17%） and available embryos （63.22% vs 61.19%） were higher in group A than in group B, but there was also no statistically significance. Conclusion The ECM medium may be associated with a better embryo quality compared with the P1 medium.

Comprehensive evaluation and selection of the potential complex medium for industrial glutamate decarboxylase (GAD) production by Escherichia coli

To address the need for producing large quantities of an enzyme,glutamate decarboxylase(GAD),this study evaluated the performance of the currently used complex and defined culture media in terms of biomass production,GAD yield,and byproducts accumulation,and selected the candidate(s)that could maximize the production of GAD by Escherichia coli(AS 1.505,the China General Microbiological Culture Collection Center,Beijing,China).The highest yield of the enzyme per biomass in the final broth(4431 U/g)corresponded to the Luria Bertani medium(LBM),which was 1.16 times the second highest from the Terrific Broth medium(TBM),while the highest biomass production(4.5 g/L)was achieved in the cultivation with TBM.For the byproducts,the profiles of all the media except the TK medium displayed a distinct exponential phase after six hour cultivation with a rapid byproducts generation,which could be attributed to the mixed acid fermentation caused by oxygen limitation.In addition,the subsequent re-assimilation of acetate could reduce the loss of GAD accumulated by stabilizing the media pH.The comprehensive analysis showed that although the highest biomass production was achieved by TBM,the fastest intracellular accumulation of GAD occurred in the cultivation with LBM(184.64 U/(g·h)),which also was the cheapest medium(0.56$/103 U).Therefore,LBM was considered the potentially competitive candidate medium for large-scale production of GAD by E.coli.

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy;however, PGT relies on embryo biopsy, that is, polar body, embryo cells, or trophectoderm biopsy, to obtain embryonic DNA, increase its technical limitations. Studies have indicated that biopsy may have an influence on the quality and development of embryos, and increase the chance of abnormal epigenetic modifications. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) has gradually become a hot research topic in the field of assisted reproduction. Studies showed cfDNA could be detected in blastocyst fluid and spent culture medium (SCM) in vitro cultured embryos. The cfDNA collection requires less skill and makes lower risk to embryos. Some studies have been conducted to evaluate the feasibility of SCM-based niPGT approaches. When comparing the ploidy consistency of cfDNA in SCM, its consistency to the conventional PGT for aneuploidies results fluctuated widely, it is critical to recognize the factors influencing accuracy. These contradictory results may be related to factors such as the difference in SCM sampling methods and sampling time, and the definition of consistency. In this review, we aimed to comprehensively summarize how researchers use embryonic cfDNA to conduct niPGT detection. It also systematically reviews the factors affecting the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a basis for future niPGT research and a useful reference for the standardized operation of niPGT that can be widely applied in clinical practice.