Subculture Method Research Articles

Exploring the Potential of Turmeric as a Natural Antibacterial Agent: In Vitro Evaluation of Crude Paste and Aqueous Extract

Background: There is growing concern in the use of natural antimicrobial compounds due to the emergence of antibiotic resistance. One such contender is turmeric (Curcuma longa), an herb known for its usage in medicine for centuries; nowadays, however, its prospects in pharmacology are being actively discussed. Since access and utilization of modern health care is scarce in Bangladesh, such natural cures are worth researching. Objective: To assess the in vitro antibacterial activity of turmeric’s crude paste and aqueous extract against Staphylococcus aureus and Escherichia coli. Methods: The study was carried out in the Department of Pharmacology with the collaboration of Department of Microbiology, Mymensingh Medical College, Bangladesh, over a period of one year from July 2010 to June 2011, and it consisted of three experiments: Effects of crude turmeric paste incorporated in nutrient agar media, Minimum Inhibitory Concentration of Aqueous Turmeric Extract and Amikacin by broth dilution method, and Subculture method to confirm bactericidal effect. Results: CTP demonstrated effective growth of S. aureus at 10% concentration and E. coli at 30% concentration. The MIC of ATE was found to be 800 μg/ml for S. aureus and 2000 μg/ml for E. coli. The MIC for amikacin was 10 μg/ml for both organisms. These effects have been further substantiated in subculture studies regarding their bactericidal properties. Conclusion: From the findings of the current study, it is evident that both turmeric and its crude paste had profound antibacterial effects against S. aureus and E. coli, though the effect of crude paste was observed to be stronger than that of the aqueous extract. Although not as strong as Amikacin, its organic nature and possibility of side effects make it worth pursuing. Hence, this work adds to the literature on the fact that turmeric has health benefits as highlighted by traditional medical practice and points to the need to find new natural sources of .............

Three Species of Stingless Bee (Meliponini) Honey from Sudan: A Contribution to the Nest Components, Proximate Composition, and Antimicrobial Properties

There is historical traditional use of stingless bee honey (SBH). However, no information concerning composition and antimicrobial properties of SBHs from Sudan was reported. Thus, the paper was aimed to focus on the components of the nests and honey characteristics of three species of stingless bee. Proximate analysis showed similar composition, except protein and ash were significantly (p > 0.01) different. Meliplebeia beccarii honey contained more protein 5.31% and slightly more ash 1.01% than the other species. Antibacterial activity was determined by cup-plate agar diffusion; Hypotrigona squamuligera and M. beccarii were more active against Staphylococcus aureus. None of the Studied SBHs showed activity against Escherichia coli, Apergillus niger, and Candida albicans. Anti-parasite activity as determined by sub-culture method showed that “almost all” SBHs have similar activities against trophozoites of Giardia lamblia and Entamoeba histolytica with statistically significant differences when compared to the untreated control. The amount of honey produced by M. beccarii varied from 100 to 150 ml per nest while honey produced by Hypotrigona ruspolii and H. squamuligera varied from 500 to 1000 ml/colony/year. Results also showed different nest architecture and components (entrance, brood areas, honey pots, and pollen pots) as indication for existence of many taxa of Meliponini in Sudan.

A newly improved method of primary cell culture: Tissue block with continuous adhesion subculture in skin fibroblast

BackgroundFibroblasts (FBs) have been widely used as a typical in vitro cell model for investigating the biological processes and cell pathophysiological mechanisms. However, FBs are prone to senescence in cell culture process after several passages. Thus, a new approach to cell culture is quite required to enhance the viability of cells. ObjectiveTo explore a novel method of cell culture based on skin FBs. MethodsDermal tissue blocks were obtained from BALB/c neonatal mice and randomly divided into experimental group and control group. The experimental group received the newly improved culture method, namely, continuous adherence subculture of tissue block (CASTB) method; while the traditional subculture method was applied in the control group. Cells at 1st, 5th and 10th passages were collected and identified by using histological/immunohistochemical and western blot analysis. Cellular viability, proliferation, senescence and apoptosis were analyzed through application of cell growth curve, CCK-8 assay, Ki67 assay, PCNA protein analysis, β-galactosidase staining, flow cytometry and western blot analysis. ResultsCells under two culture patterns exhibited spindle/irregular shape and vimentin positive expression. With the increase of passage times, the cellular growth rate in the control group gradually decreased, but no alterations emerged from the experimental group. CASTB method remarkably promoted cell growth and proliferation. Moreover, a greatly lower apoptosis and senescence tendency appeared in the experimental group than the control group with passages increasing. ConclusionThe method of CASTB is superior to traditional subculture, offering a large number of primary FBs with higher efficiency and success rate and being worth of further popularization and application.

Optimization of Biomass and Secondary Metabolite Production in Gynura procumbens (Lour.) Merr. Adventitious Roots Culture by Using the Method of Subculture and Fed-batch Cultivation in a Bioreactor

The valuable extract of bioactive compounds from Gynura procumbens has been widely manufactured into various health products. The demand for these compounds is continuously increasing, but production through conventional farming methods is insufficient due to limited agricultural land and environmental stresses. An alternative to producing plant biomass is in vitro cultivation methods. This method requires less space and enables biomass propagation in a controlled condition that can facilitate stable and efficient production of plant secondary metabolites. This study evaluated the effect of inoculum subculture periods and culture methods on G. procumbens biomass and secondary metabolite production in a bioreactor. The 3-L airlift balloon type-bubble bioreactors was modified in this study to adopt the treatment of 1st-5th subculture periods and fed- and batch-cultivation strategies. We found the G. procumbens adventitious root culture was optimally derived from the 1st subculture produced biomass of 148.02±1.45 g FW and 8.59±0.12 g DW, and TPC (14.48±1.08 mg GAE/g DW) and TFC (116.89±0.44 mg KE/g DW and 33.97±0.13 mg QE/g DW). Additionally, the fed method after 28 days of culture using double distilled water replenishment improved adventitious root biomass (213.75±35.00 g FW and 11.21±0.18 g DW), while nutrient replenishment improved TFC (52.14±0.44 mg KE/g DW and 14.54±0.13 mg QE/g DW). These results can be used to optimize the cultivation of G. procumbens adventitious roots in a large-scale bioreactor.

Confirmation of presumptive Legionella colonies on culture media according to ISO 11731:2017: principles, problems, and practice.

The ISO 11731 norm, published in 2017, describes a method to identify and enumerate Legionella based exclusively on the confirmation of presumptive colonies by subculturing them on BCYE and BCYE-cys agar (BCYE agar without L-cysteine). Despite this recommendation, our laboratory has kept confirming all presumptive Legionella colonies by combining the subculture method with the latex agglutination and polymerase chain reaction (PCR) assays. Here, we show that the ISO 11731:2017 method adequately performs in our laboratory according to ISO 13843:2017. We compared the performance of the ISO method in detecting Legionella in typical and atypical colonies (n=7156) from health care facilities (HCFs) water samples to that of our combined protocol, and we found a 2.1% false positive rate (FPR), underscoring the importance of combining agglutination test and PCR with subculture to achieve optimal confirmation. Lastly, we estimated the water system disinfection cost for HCFs (n=7), which due to false positive results, would display Legionella values exceeding the threshold of risk acceptance established by the Italian guidelines. Overall, this large-scale study indicates that the ISO 11731:2017 confirmation method is error-prone, leading to significant FPRs, and higher costs for HCFs due to remedial actions on their water systems.

Role of Indonesian Indigenous Cyanobacteria Culture Collection as An Ex-situ Conservation Effort and Microalgae Biodiversity Study Material

Exploratory research has been carried out on Indonesian indigenous Cyanobacteria in an effort to collect these isolates to be used as learning materials for microalgae biodiversity. Cyanobacteria are known as photosynthetic bacteria and prokaryotic microalgae. The members of the cyanobacteria found were isolated, cultured, identified, and collected. The location for sampling cyanobacteria is the UI Depok campus area which consists of water from small lakes (situ) and soil. The results showed that 22 strains of Cyanobacteria were isolated and cultured properly, and consisted of 12 genera/species, namely Merismopedia sp. (1 isolate), Synechococcus sp. (1 isolate), Limnothrix redekei (1 isolate), Oscillatoria sp.1 (1 isolate), Oscillatoria sp.2 (1 isolate), Pseudanabaena sp. (3 isolates), Nostoc sp. (4 isolates), Anabaena sp. (1 isolate), Scytonema sp. (1 isolate), Fischerella sp. (1 isolate), Stigonema sp. (1 isolate), and Hapalosiphon sp. (6 isolates). Twenty-two (22) strains were then collected and treated by subculture method. Of the 11 genera found, there are 3 genera that have been known as genera that have potential as sources of biobased materials, biofuels, biofertilizers, and others, namely Synechococcus, Nostoc, and Anabaena

Direct inoculation method for identification and antimicrobial susceptibility testing using matrix-assisted laser desorption ionization-time of flight mass spectrometry and both the Vitek 2 and MicroScan Walkaway 96 Plus systems

The aim of our study was to evaluate a protocol utilizing serum separator tubes (SST) to facilitate a faster, cost-effective, direct method for rapid sensitivity testing and identification of positive blood cultures. Spiked cultures were inoculated into either Becton Dickinson (BD) BACTECTM Aerobic Plus or Anaerobic/F bottles containing sterile human blood. Bottles were immediately processed when positive. A parallel study using patient isolates was used in which bacteria were pelleted by SST from positive blood cultures. For identification, a portion of the pellet was tested by matrix-assisted laser desorption/ionization as described by the manufacturer. MicroScan panels and Vitek 2 results were compared. Categorical agreement was used as comparison to standard subculture and/or polymerase chain reaction methods. No discordant identifications were observed, and 86% generated a successful identification when compared to subculture methods. For the Vitek 2, we observed a 99% essential agreement when compared to the subculture method. For the MicroScan Walkaway, we observed 94.9%, 97.4%, and 100% categorical agreement for MIC panels 53, 38, and MICroSTREP Plus 2, respectively. Turnaround times were reduced from 4 hours for identification and 11 hours for antimicrobial sensitivity testing. We conclude that the SST method results in timelier, actionable results for antimicrobial stewardship initiatives.

Science and Technology in Manufacture of Oyster Mushroom Seedlings

Oyster mushrooms (Pleurotus ostreatus) currently are in high demand because of their good taste and very high protein content. This nutritious and healthy food source make oyster mushrooms a preferable commodity in meeting food needs. It can be marked by the growing demand for oyster mushrooms while oyster mushroom production has not been able to meet the needs of the market, especially mushroom farmers in North Sumatra began to go from just farming by starting from a baglog that was ready to harvest, to providing its own seedlings. Farmers are expected to be able to produce initial seeds directly from fresh mushrooms that have commercial values. This program provides knowledge to farmers on how to develop seeds from fresh mushrooms to produce F-0, F-1, F-2 F-3 and finally baglog. Socialization, live demonstrations and interviews have been conducted by the team. Techniques for obtaining good seedlings by working with aseptic culture techniques and sterilization of growing media have also been given briefly to farmers. This program is expected that farmers have skills in producing seeds with subculture methods, as well as gain knowledge on using sterilization and aseptic techniques to be able to produce good and quality seeds.

Identification of micro-organism from positive blood cultures: comparison of three different short culturing methods to the Rapid Sepsityper workflow.

Sepsis is one of the leading causes of death worldwide. The rapid identification (ID) of the causative micro-organisms is crucial for the patients' clinical outcome. MALDI-TOF MS has been widely investigated to speed up the time-to-report for ID from positive blood cultures, and many different procedures and protocols were developed, all of them attributable either to the direct separation of microbial cells from the blood cells, or to a short subculture approach. In this study, the Rapid Sepsityper workflow (MBT Sepsityper IVD Kit, Bruker Daltonics GmbH and Co. KG, Bremen, Germany) was compared to three different short subculturing methods, established into the routine practice of three different clinical microbiology laboratories. A total of N=503 routine samples were included in this study and tested in parallel with the two approaches. Results of the rapid procedures were finally compared to routine proceedings with Gram-staining and overnight subculture. Among monomicrobial samples, the Rapid Sepsityper workflow enabled overall the correct identification of 388/443 (87.6 %) micro-organisms, while the short subculturing methods of 267/435 (61.8 %). Except for the performance with Streptococcus pneumoniae, in each one of the three sites the Rapid Sepsityper workflow proved to be superior to the short subculture method, regardless of the protocol applied, and it delivered a result from 1 to 5 h earlier.

Evaluation of the antifungal activity of α, β, and δ-damascone and inclusion complexes in β-cyclodextrin against Candida spp.

Due to the increase in fungal resistance to existing drugs, a need exists to search for new antifungals. This study aimed to evaluate the antifungal activity of α, β, and δ-damascone and inclusion complexes with β-cyclodextrin against different Candida spp. The inclusion complex of β-damascone was prepared by the co-evaporation method using three molar proportions (1:1; 2:1; 3:1 (βDA-βCD)) and analyzed using Fourier transform infrared spectroscopy (FTIR). Standard Candida albicans (CA INCQS 40,006), Candida krusei (CK INCQS 40,095), and Candida tropicalis (CT INCQS 40,042) strains were used to evaluate antifungal activity. The substances were tested individually or in association with fluconazole (FCZ). The IC50 and cell viability curve constructions were performed using the microdilution method. The minimum fungicidal concentration (MFC) was determined by the subculture method in a solid medium. The α, β, and δ-DA isolated or in combination with fluconazole (FCZ) showed significant antifungal activity. β-damascone showed effective complexation in the three molar proportions assayed; however, none of the inclusion complexes was demonstrated clinically significant effects against the fungal tested. Then, all compounds have shown promising antifungal activities; however, in vivo assays are necessary to have therapeutical application in the future.

Porcine Intestinal Apical-Out Organoid Model for Gut Function Study.

Simple SummaryPigs have been used in various animal model studies on the gastrointestinal tract (GIT) across both animal science and biomedical science fields. Recently, intestinal organoids have been used as a research tool for the GIT, and they have also been applied to farm animals, including pigs. However, to our knowledge, no functional studies of the porcine intestine using intestinal organoids have been conducted to date. In the present study, we developed two porcine intestinal organoid models (basal-out and apical-out organoids) and compared their characteristics. We also confirmed the possibility of conducting research related to intestinal functions, such as nutrient uptake and gut barrier function. The present study suggests that porcine intestinal organoids can be used as potential models for future GIT mechanism studies, such as host–microbe interactions, harmful ingredient tests, and nutritional research.Pig models provide valuable research information on farm animals, veterinary, and biomedical sciences. Experimental pig gut models are used in studies on physiology, nutrition, and diseases. Intestinal organoids are powerful tools for investigating intestinal functions such as nutrient uptake and gut barrier function. However, organoids have a basal-out structure and need to grow in the extracellular matrix, which causes difficulties in research on the intestinal apical membrane. We established porcine intestinal organoids from jejunum tissues and developed basal-out and apical-out organoids using different sub-culture methods. Staining and quantitative real-time PCR showed the difference in axis change of the membrane and gene expression of epithelial cell marker genes. To consider the possibility of using apical-out organoids for intestinal function, studies involving fatty acid uptake and disruption of the epithelial barrier were undertaken. Fluorescence fatty acid was more readily absorbed in apical-out organoids than in basal-out organoids within the same time. To determine whether apical-out organoids form a functional barrier, a fluorescent dextran diffusion assay was performed. Hence, we successfully developed porcine intestinal organoid culture systems and showed that the porcine apical-out organoid model is ideal for the investigation of the intestinal environment. It can be used in future studies related to the intestine across various research fields.

Theoretical Base for the Application of the DiVSaL Method to Bacterial Spores to Evaluate Injured Populations Occurring After Exposure to Lethal Stress.

To assess injury in bacterial spore populations exposed to lethal stress, we proposed a theoretical basis for applying the DiVSaL method, which has already been reported for general microorganisms as a double subculture method. We constructed a mathematical model in which both injuries to the germination system and the spore body were taken into the theory. In this theory, we reasonably assumed that the viable and germinable spore count is constant before the subsequent vegetative growth and that the delay of germination and outgrowth can be included in the concept of λ injury previously reported as the growth-independent injury. By introducing these assumptions, the double subculture method can be considered to apply to spores as well. As examples of the application of this theory, the growth delays of Bacillus subtilis spores treated with heat and UV irradiation were analyzed and the numbers of injured spores were evaluated. Based on the results obtained, heat is indicated to have a higher injury generation ability than UV irradiation. The applicability of the DiVSaL method as a tool for food preservation and sanitation designs is presented.

English

BACKGROUND Azadirachta indica, commonly known as neem, neem tree or Indian lilac, Limbo, Nim, Nimba, Medusa and Vempu. It is typically grown in tropical and subtropical regions. Neem belongs to family meliaceae. Neem is a native tree of India. It is also called “village pharmacy of south Asia” (India) because of its enormous medicinal properties and people use it most of the time without knowing its multiple advantages. This study was done to know the efficacy of medicinal plant named Azadirachta indica (Neem) against pathogenic microorganisms and about its utility as disinfectant and floor cleaner. METHODS Agar well diffusion method, Culture plate method, inoculum preparation & its subculture methods were used to determine the antimicrobial activity of the neem leaf extract. Different concentrations of neem leaf extracts in culture plates were used for the study. In this method inhibited zones were measured. RESULTS Neem leaf extracts showed considerable antimicrobial activity against four target pathogens. In agar well diffusion method it showed maximum antimicrobial activity against Enterococcus and Staphylococcus aureus. Growth of inhibition was increased as the concentration of the neem extract in agar media increased and was maximum for Staphylococcus aureus. CONCLUSIONS The extract of A. indica, has antimicrobial activity against target pathogens Enterococcus, Staphylococcus aureus, Pseudomonas, and E.coli. With the old medicinal knowledge on neem, better economic and therapeutic utilization can be done by using modern approaches of drug development. KEY WORDS Neem Leaf extract, Antimicrobial Activity, Agar Well Diffusion Test, Zone of Inhibition

Evaluation of purity, viability, stability and biochemical characteristics of clinical fungal isolates preserved by three different methods

Introduction: In era of organ transplants and immunocompromised patients, systemic mycosis has emerged as a major cause of morbidity and mortality among these patients. Research on these clinical fungal isolates is the key to the development of antifungal diagnostics and therapeutic agents. Mycology laboratories has to preserve these pathogenic fungal strains for various experiments. These laboratories requires a preservation method that ensures purity, viability, stability and biochemical characteristics of the fungal isolates during the storage period. Objectives: To determining the purity, viability, stability and biochemical characteristics of clinical fungal isolates subjected to three different, but simple and inexpensive preservation methods. Materials and Methods: A total of 50 clinical isolates of yeast and mold were subjected to different preservation Methods: All the isolates were evaluated for purity, viability, stability and biochemical characteristics before storage and after 6 months and one year. Stability testing was based on antifungal susceptibility testing as recommended by CLSI. Results: All the three preservation methods, ensured survival of all the fungal strains for one year duration. Periodic subculture method on agar slope demonstrated mild colony morphological changes in 2 molds and high contamination rate. Water culture technique and oil overlay technique, demonstrated protection of purity, viability, stability and biochemical characteristics of all the fungal isolates up to one year of storage. Conclusion: Comparison of different fungal storage methods suggested that both water technique and oil overlay technique can be utilized as a simple, effective and inexpensive methods for long term preservation of clinical fungal isolates. Keywords: Preservation, Storage, Yeast, Molds, CLSI.

ANALISIS METODE PRESERVASI SUBKULTUR BERKALA DAN DENGAN AIR STERIL UNTUK JAMUR Aspergillus sp. DAN Candida albicans

Pendahuluan: Preservasi yang baik dan tepat dapat mempertahankan koleksi kultur dengan baik. Teknik preservasi subkultur berkala merupakan teknik lama yang kurang dapat menjamin sifat-sifat genetik jamur dalam jangka waktu lama. Air steril adalah salah satu metode alternatif dalam berbagai penelitian dalam menjamin sifat-sifat genetik jamur dalam waktu lama dan dapat dilakukan pada konsep laboratorium sederhana. Penelitian ini bertujuan untuk melihat metode preservasi berkala dan dengan air steril.
 Metode: Penelitian ini merupakan penelitian deskriptif dengan desain eksperimental laboratorium pada jamur Aspergillus sp. dan Candida albicans.Penelitian ini melihat metode subkultur berkala dan dengan air steril dalam menjaga viabilitas, tingkat kontaminasi, morfologi dan resistensi terhadap antijamur setelah enam bulan.
 Hasil: Jamur dapat hidup setelah dipreservasi dengan subkultur berkala tanpa kontaminasi, perubahan morfologi dan resistensi. Aspergillus sp. dan Candida albicans juga hidup setelah dipreservasi dengan air steril selama enam bulan dengan morfologi yang sama dan tanpa kontaminasi. Zona inhibisi Aspergillus sp. mengalami penurunan namun pada Candida albicans tetap sama.
 Simpulan: Preservasi jamur dengan subkultur berkala dan dengan air steril dapat diterapkan pada Aspergillus sp. dan Candida albicans.

Development of Alternative Technology for the Long-term Storage of Microalgal Stock Culture in Fish Hatcheries

Maintenance of algal culture faces several problems associated with climate change, contaminations, equipment failures, power failures, unexplained crashes, poor lab facilities. Microalgae are traditionally preserved by serial subculture methods that are laborious, costly, and high risk of culture contamination. While unique characteristics may not be durably maintained with general subculture, cryopreservation methods better prevent alterations from desired characteristics. Because of the cost-effective in replacing of liquid nitrogen at every interval of time and technical person needed for the preservation. The objective is to find out an alternative and a low-cost technology for the preservation of microalgae Stock culture. The microalgae Nannochloropsis salina, Chlorella volutis, Cheatoceros gracilis, Dunaliella sp. and Amphora sp., were preserved using common cryoprotectants (methanol, DMSO, ethylene glycol and glycerol) for 6 months at –196°C and –20°C. The viabilities of the microalgae were assessed after thawing, and cell counts were measured. The preserved algae Nannochloropsis salina, Chlorella volutis, Dunaliella sp. and Amphora sp. evoked good responses with negligible changes in their survivability in 6 months incubation period when preserved at –20°C and – 196°C, while the Cheatoceros gracilis regenerate only in –196°C but not restored in –20°C. In this study, an alternative method for liquid nitrogen preservation has been standardized and this new method can be a boon for small level fish hatcheries and microalgal stock holders.

A Modified Double Subculture Method for the Two-Mode Injuries Evaluation in a Stressed Fungal Spore Population.

In order to evaluate injury of a stressed fungal spore population, a modification of formerly presented double subculture method, which consists of both the conventional plate count method and the growth delay analysis method, was proposed. In this method, an apparent logarithmic growth kinetics was assumed and the previous kinetic model was improved to be able to estimate injured subpopulations in two different modes containing early occurring growth-independent and late occurring growth-dependent injuries, called the λ and μ injuries, respectively. Based on the kinetic theory developed here, this novel method was applied to heat-treated conidia of Cladosporium cladosporioides and these two mode injuries were evaluated.

Effects of Different Hormone Concentrations on Callus Induction of Astragalus mongholicus Bunge

A method of callus induction and subculture of Astragalus mongholicus was established. Different methods were used to sterilize seeds to induce sterile seedlings. The different parts of plants and Astragalus aseptic seedlings were used for the explants, different concentrations of auxin NAA and Cytokinin BAP were used in MS medium to induce callus, and then the callus with better growth condition was selected for secondary culture. Results showed that the browning phenomenon in the secondary culture was inhibited and the best disinfectant was mercuric chloride. The germination rate of seeds treated with 75% ethanol for 40 s, 1.5% NaCl for 20 minutes, and 0.1% HgCl2 for 3 minutes can reach up to 66.2%. The hypocotyls of sterile seedlings as explants were the best way to induce callus. The induction rate was close to 100% when 0.5 mg/L BAP and 0.15 mg/L of NAA and 0.01 mg/L of nano-TiO2 were added to MS medium. Besides, Nano-TiO2 can effectively prevent bacterial contamination of MS medium. Cotyledons, induced by cotyledon as explants, showed the most severe browning. Therefore, the best hormone combination of Astragalus subculture was 1.0 mg/L BAP, 1.5 mg/L NAA and 0.01 mg/L nano-TiO2 .

Diagnostic performance of direct latex agglutination, post-enrichment latex agglutination and culture methods in screening of group B streptococci in late pregnancy: a comparative study.

BackgroundGroup B streptococcus (GBS) is one of the main causes of neonatal sepsis.PurposeEvaluation of the diagnostic performance of direct latex agglutination test (DLA), post-enrichment latex agglutination (LA) test, and direct culture on chromogenic media in rapid identification of GBS carrier in pregnant women in comparison with the conventional post-enrichment CDC-recommended culture method and further to estimate GBS carriage prevalence and its antimicrobial susceptibility.MethodsTwo hundred pregnant women at gestational age (35–37 weeks) were enrolled. Three low vaginal swabs were obtained from each participant. One swab was directly inoculated into Strep B Select (SBS) agar. The second swab was inoculated in enrichment Lim broth for immunological antigen detection by post-enrichment latex agglutination (5 h and 24 h) and subculture for bacteriological detection. The third swab was used for immunological detection of GBS antigen by direct latex agglutination. The isolated GBS was subjected to antimicrobial susceptibility testing.ResultsAmong 200 pregnant women, 47 (23.5%) were GBS carriers. Considering post-enrichment subculture on SBS medium as a gold standard, the sensitivities for post-enrichment 5 h and 24 h LA were 66% and 95.7%, respectively. However, direct cultivation of the vaginal swabs on SBS medium and DLA recorded 83% and 4.3%, respectively, for sensitivity. All GBS isolates (100%) were sensitive to penicillin G, ampicillin, ceftriaxone, and vancomycin. In contrast, 21.3% and 12.8% of isolated GBS were resistant to erythromycin and clindamycin, respectively.ConclusionGroup B streptococcal antigen detection by latex agglutination after 5 h enrichment is a reliable, easy, and relatively rapid method for screening of GBS carriage in pregnant woman not in labor. Latex agglutination after 18–24 h enrichment can be used alternative to standard subculture method for screening GBS carriage.

Development of a primary culture system for haematopoietic tissue cells from Cherax quadricarinatus and an exploration of transfection methods

Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2′-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells.