The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 microg/ml (MSC-4) and 6 microg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests ($0.70 and $1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.
Box-Wilson design (BWD) model was applied to determine the optimum values of influencing parameters in anaerobic fermentation to produce hydrogen using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). The main focus of the study was to find the optimal relationship between the hydrogen yield and three variables including initial substrate concentration, initial medium pH and reaction temperature. Microbial growth kinetic parameters for hydrogen production under anaerobic conditions were determined using the Monod model with incorporation of a substrate inhibition term. The values of micro(max) (maximum specific growth rate) and K, (saturation constant) were 0.398 h(-1) and 5.509 g L(-1), respectively, using glucose as the substrate. The experimental substrate and biomass-concentration profiles were in good agreement with those obtained by the kinetic-model predictions. By varying the conditions of the initial substrate concentration (1-40 g L(-1)), reaction temperature (25-40 degrees C) and initial medium pH (4-8), the model predicted a maximum hydrogen yield of 3.24 mol H2 (mol glucose)(-1). The experimental data collected utilising this design was successfully fitted to a second-order polynomial model. An optimum operating condition of 10 g L(-1) initial substrate concentration, 37 degrees C reaction temperature and 6.0 +/- 0.2 initial medium pH gave 80% of the predicted maximum yield of hydrogen where as the experimental yield obtained in this study was 77.75% exhibiting a close accuracy between estimated and experimental values. This is the first report to predict bio-hydrogen yield by applying Box-Wilson Design in anaerobic fermentation while optimizing the effects of environmental factors prevailing there by investigating the effects of environmental factors.
Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.
The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.
The aim of this study was to identify the foodborne pathogens mainly, Aspergillus sp. colonizing rice grains using cultural and microscopic methods. Four differential media (Czapek Dox Agar (CZA), Czapek Yeast Agar (CYA), Malt Extract Agar (MEA) and Czapek yeast 20% sucrose agar (CYA20S)) were used for differentiation of five Aspergillus sp., colonizing rice grains comparing with standard cultures. We studied macroscopic (colony color and diameter, conidia color, exudates, sclerotia and colony texture) and microscopic (conidiophore color, length and breadth, conidia size, shape and surface texture, vesicle diameter and phialides length and breadth) characteristics for identification of 110 isolates of Aspergillus sp. isolated from 65 rice grain samples collected from various countries in South Asia (Cambodia, India, Indonesia, Malaysia and Thailand). According to morphological characters, all these isolates were belonging to Aspergillus flavus (45), A. fumigatus (8), A. ochraceus (7), A. niger (42) and A. tamarii (8). This is the first report on identification of large number of Aspergillus strains isolated from rice grains in South Asia.
An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.
An efficient in vitro plant regeneration system was established for elite, recalcitrant Malaysian indica rice, Oryza sativa L. CV. MR 219 using mature seeds as explant on Murashige and Skoog and Chu N6 media containing 2,4-dichlorophenoxy acetic acid and kinetin either alone or in different combinations. L-proline, casein hydrolysate and L-glutamine were added to callus induction media for enhancement of embryogenic callus induction. The highest frequency of friable callus induction (84%) was observed in N6 medium containing 2.5 mg l(-1) 2,4-dichlorophenoxy acetic acid, 0.2 mg l(-1) kinetin, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate, 20 mg l(-1) L-glutamine and 30 g l(-1) sucrose under culture in continuous lighting conditions. The maximum regeneration frequency (71%) was observed, when 30-day-old N6 friable calli were cultured on MS medium supplemented with 3 mg l(-1) 6-benzyl aminopurine, 1 mg l(-1) naphthalene acetic acid, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate and 3% maltose. Developed shoots were rooted in half strength MS medium supplemented with 2% sucrose and were successfully transplanted to soil with 95% survival. This protocol may be used for other recalcitrant indica rice genotypes and to transfer desirable genes in to Malaysian indica rice cultivar MR219 for crop improvement.
Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.
Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and 'chondrocytes-fibrin' construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM+10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM+2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM+10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs>FD2GFs>FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and 'chondrocytes-fibrin' constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation.
The cytotoxicity of cell-free culture filtrates of 31 isolates of Vibrio cholerae O1 and O139, 5 reference strains and 26 clinical isolates, was tested on Madin Darby Bovine Kidney (MDBK) cells and Vero cells. The 3-[4,5-dimethylthiazol-2-y]-2, 5-diphenyltetrazolium bromide (MTT) test was used to detect the effect of the filtrates on the proliferation and viability of cultured cell populations. The filtrates were prepared from serial ten-fold dilutions of inoculated AKI and APW broth media with and without the addition of polymyxin B. The APW culture filtrates of both V. cholerae O1 and O139 with and without added polymyxin B showed greater toxicity to MDBK cells as compared to AKI filtrates. The cytotoxicity of AKI-grown V. cholerae O139 to MDBK cells was greater than that of V. cholerae O1 grown in the same medium. The cytotoxicity of APW filtrates on Vero cells was low and only noted when polymyxin was added to the medium.
Stem cells isolated from dental pulp possess the capacity for self-renewal and the potential for multi-lineage differentiation. However, dental pulp stem cells have different characteristics in terms of their culture conditions. The success of stem cells culture is governed by its micro-environmental niche. Therefore, we studied the effects of culture niche on long-term expansion of dental pulp stem cells in terms of cell morphology, growth kinetics, senescence pattern, cell surface marker expression differentiation capacity, and seeding plating density of dental pulp stem cells in four different, widely used media composition Among the various basal media tested, α-minimum essential media and knock out-minimum essential media supplemented with 10% fetal bovine serum were found to be the most optimal media composition in preserving the phenotypic characteristics and differentiation potential for prolonged periods as compared with DMEM-F12 and DMEM-LG. Plating density has been shown to affect overall yield. As a conclusion, the adoption of an appropriate culture system significantly improved cell yield, thus enabling the attainment of sufficient yields for therapeutic applications economizing in terms of cost of production and minimizing seeding cell density for maximum yield.
This paper introduces sludge palm oil (SPO) as a novel substrate for biosurfactant production by liquid state fermentation. Potential strains of microorganism were isolated from various hydrocarbon-based sources at palm oil mill and screened for biosurfactant production with the help of drop collapse method and surface tension activity. Out of 22 isolates of microorganism, the strain S02 showed the highest bacterial growth with a surface tension of 36.2 mN/m and was therefore, selected as a potential biosurfactant producing microorganism. Plackett-Burman experimental design was employed to determine the important nutritional requirement for biosurfactant production by the selected strain under controlled conditions. Six out of 11 factors of the production medium were found to significantly affect the biosurfactant production. K(2)HPO(4) had a direct proportional correlation with the biosurfactant production while sucrose, glucose, FeSO(4), MgSO(4), and NaNO(3) showed inversely proportional relationship with biosurfactant production in the selected experimental range.
GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
Leptospirosis is recognized as one of the important zoonotic diseases in the world including Malaysia. A total of 145 soil and water samples were collected from selected National Service Training Centres (NSTC) in Kelantan and Terengganu. The samples were inoculated into modified semisolid Ellinghausen McCullough Johnson Harris (EMJH) medium, incubated at room temperature for 1 month and examined under the dark-field microscope. Positive growth of the leptospiral isolates were then confirmed with 8-Azaguanine Test, Polymerase Chain Reaction (PCR) assay and Microscopic Agglutination Test (MAT). Fifteen cultures (10.34%) exhibited positive growths which were seen under dark field microscope whilst only 20% (3/15) were confirmed as pathogenic species. based on 8-Azaguanine Test and PCR. Serological identification of the isolates with MAT showed that hebdomadis was the dominant serovar in Terengganu. Pathogenic leptospires can be detected in Malaysian environment and this has the potential to cause an outbreak. Therefore, precautionary steps against leptospirosis should be taken by camp authorities to ensure the safety of trainees.
Introduction: The vast majority of in vitro research on microglia are based on cells isolated from
neonatal animals (3-5 days of age). Studying microglia of adults has been limited by the lack of a suitable culture system that supports their growth. In this study, we describe a protocol for growing microglia of adults based on modifications of the technique for culturing microglia isolated from neonatal rats. Methods: Mixed glia isolated from adult rats (age range of 1 month to 3 years old) were seeded in
culture flasks coated with poly-L-lysine. Cells were maintained in DMEM media supplemented with
insulin-transferrin-selenium (ITS) and recombinant human macrophage colony-stimulating factor
(M-CSF). Mild trypsinisation was carried out to isolate microglia from mixed glia culture. Results:
Microglia cells of adult rats were successfully grown in vitro. For the expansion of adult microglia,
it was observed that coating the cell culture flasks with poly-L-lysine was crucial to encourage cell
adherence. The substitution of insulin in culture media with ITS was found to improve cell yield and
reduced the number of days required for culture from 28 days to 14 days. Addition of M-CSF to cell
culture medium, along with the improvisations described above provided the best adult microglia cell
yield (2.91 ± 0.56 x 106 cells) compared to the technique of replating cells (0.91 ± 0.65 x 106 cells;
p
The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.
A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses.
The search for antimicrobial agents from plants has been a growing interest in the last few decades. However, results generated from many of these studies cannot be directly compared due to the absence of standardization in particular antimicrobial methods employed. The need for established methods with consistent results for the evaluation of antimicrobial activities from plant extracts has been proposed by many researchers. Nevertheless, there are still many studies reported in the literature describing different methodologies. The aim of this study was to find optimal methods to give consistent quantitative antimicrobial results for studying plant extracts. Three different agar-based assays (pour plate disc diffusion (PPDD), streak plate disc diffusion (SPDD) and well-in agar (WA)) and one broth-based (turbidometric (TB)) assay were used in this study. Extracts from two plant species (Duabanga grandiflora and Acalypha wilkesiana) were tested on two bacterial species, namely Escherichia coli and Staphylococcus aureus. Amongst the agar-based assays, PPDD produced the most reproducible results. TB was able to show the inhibitory effects of the test samples on the growth kinetic of the bacteria including plant extracts with low polarity. We propose that both agar- (i.e PPDD) and broth-based assays should be employed when assessing the antimicrobial activity of plant crude extracts.
Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.