Solid Culture Methods Research Articles

A liquid culture method assisted by ATP analysis for accelerating laboratory experiments on ultraviolet disinfection of airborne bacteria

The solid culture method for measuring the efficiency of ultraviolet (UV) disinfection of airborne bacteria is time-consuming, typically taking 12–48 h. To expedite such experiments, this study proposed a liquid culture method assisted by adenosine triphosphate (ATP) analysis, as a liquid culture is faster than a solid culture, and measurement of ATP does not require waiting for visible colonies to form. Escherichia coli (E. coli) was used as the experimental bacterium. This study first compared the log reduction of bacteria in liquid as measured by the proposed method and by the traditional solid culture method. The minimum liquid culture time was determined for different bacterial concentration ranges. Finally, the feasibility of the proposed method was validated by UV disinfection experiments on airborne bacteria. The results indicated that the proposed method measured a similar log reduction to that of the solid culture method in liquid experiments. The minimum liquid culture time for E. coli in 105-106 colony forming units (CFU)/mL was 2 h. The validation experiments demonstrated that the proposed method is capable of measuring the UV disinfection efficiency of airborne bacteria. The proposed method can accelerate laboratory experiments on UV disinfection of airborne bacteria, which in turn can support the effective design and utilization of UV disinfection in real life.

New endophytic fungal species of Chaetomiaceae (Ascomycota) in Iraq

Abstract. Al-Rifaie AA, Ameen MKM. 2023. New endophytic fungal species of Chaetomiaceae (Ascomycota) in Iraq. Biodiversitas 24: 5270-5277. The present study was conducted to isolate and identify some endophytic fungi from vegetable crops in Iraq. Samples of eight vegetable plants, namely Abelmoschus esculentus, Mentha piperita, Vicia faba, Petroselinum sativum, Ocimum basilicum, Lawsonia inermis, Beta vulgaris and Apium graveolens were collected from three regions in Basrah (Abu Al-Khaseeb, Karmat Ali and the Centre of Basrah). Isolation was done by solid culture method and moist culture method. The recovered endophytic fungi were purified and axenic cultures of each isolated species were then identified on the basis of their macro and micro-morphological features. Morphological identification was further confirmed by molecular analysis through DNA extraction and sequencing by PCR amplification of the ITS4 and ITS5 gene primers. Phylogenetic examination revealed that five novel endophytic fungal species related to the family Chaetomiaceae were isolated from vegetable plants, including Chaetomium cucumericola, C. madrasense, Amesia atrobrunnea, A. cymbiformis and Botryotichum verrucosum. Three species, including C. cucumericola, A. cymbiformis and B. verrucosum, were documented for the first time in the Iraqi mycobiota. To the best of our acknowledgment, this study is the first to investigating endophytic fungi from vegetables in Iraq.

Modern Approaches to In Vitro Clonal Banana Production: Next-Generation Tissue Culture Systems

In this study, the solid culture method, and Plantform™ and SETIS™ temporary immersion bioreactor systems were used comparatively to propagate, root, and acclimatize ‘Grande Naine’ and ‘Azman’ banana varieties for rapid, cheap, and mass production in in vitro conditions. Micropropagation rate, plant height, number of leaves, and fresh and dry weight parameters were investigated in the micropropagation stage across eight subcultures. Rooting rate, plant height, number of leaves, number of roots/plant, root length, fresh and dry weight parameters were investigated in the rooting stage. Photosynthetic pigment analyses and stoma examinations were performed throughout all stages. In the micropropagation stage, a 20% increase in the Plantform™ system, a 12% increase in the SETIS™ system in ‘Grande Naine’, an 82% increase in the Plantform™ system, and a 98% increase in SETIS™ system in ‘Azman’ were determined compared to the solid culture. At the rooting stage, higher data were obtained from bioreactor systems than solid culture. Plants from bioreactor systems acclimatized faster and developed healthier in the greenhouse stage. It was determined that stomata were more active, and pigment accumulation was higher in bioreactor systems. Genetic variations across subcultures are among the most critical issues in banana clonal propagation. Leaf samples were taken from each system, and plant variation was investigated using SSR (Simple Sequence Repeat) markers. No variation was observed from the initial stage to the greenhouse stage. As a result, it has been determined that bioreactor systems are an essential alternative for the mass production of bananas.

Effect of Bioreactor Cultures on the Proliferation and Biological Activity of Protocorm-like Bodies of Dendrobium loddigesii

Dendrobium loddigesii has long been used in traditional folk medicine. The purpose of this study was to optimize the culture conditions for its protocorm-like bodies (PLBs) and explore their biological activities. The use of an air-lift bioreactor demonstrated superior PLB proliferation compared to agitated and solid culture methods. The optimal inoculum quantity of 30 g/vessel, cultured for 28 days in the bioreactor, yielded the highest PLB biomass production. Analysis of PLB extracts revealed that the ethyl acetate (EtOAc) extract exhibited the highest levels of flavonoids and alkaloids, as well as potent antioxidant activity demonstrated by DPPH free radical scavenging assay and reducing power. Furthermore, the antiproliferative effects of the PLB extracts were assessed using MTT assays, and the EtOAc extract showed significant efficacy by reducing cell viability by over 60% in the human colon carcinoma cell line SW620 at the highest tested concentration (200 μg/mL). Mechanistic analysis revealed the downregulation of key regulatory apoptosis genes, including survivin, p53, caspase-3, and caspase-9. These results demonstrate the potential of the bioreactor culture method for the efficient production of D. loddigesii PLBs and the biological activities of the EtOAc extract, suggesting its therapeutic potential.

Mycelium as sustainable textile material – review on recent research and future prospective

PurposeMycelium is an upcoming bio-based alternative material that has various applications in different industries. Mycelium materials used as composites, leather, construction materials and some are even available for commercial purposes. However, there was not much research found when it came to the application of mycelium as a textile alternative. The purpose of this paper is to examine the potential of mycelium in the textile industry and its possible applications.Design/methodology/approachThis review consolidates literature that refers the two major methods used in fungal mycelium production namely; as a composite and as a pure self-grown mycelium sheet. The study compared the current research status in this respective field and reported the scope in the pure mycelium development.FindingsThe results of the review reported that several research works are performed in composite production with different feedstock. The production methods and product development steps were well established for several applications from home utilities to construction materials. Whereas, in the case of self-grown mycelium sheet production only limited research works were found. Though the possibilities of engineered composite sheets are developed with various properties, research on self-grown pure mycelium sheets are at infant stage. Sensitive production methods, lower tensile, tearing, poor handle properties with brittle structure and non-uniformity in thickness are noted as limitations. Sustainable nature, self-grown three-dimensional nano-fibril network with porous structure are found to be advantageous.Originality/valueThe solid culture method was identified as a potential method to develop a sheet-like self-grown mycelium with different dimensions. The review results clearly show the lack of research in the direct application of self-grown pure mycelium area concerning feedstock material, fungal species selection and characterization of the developed product. Addressing the existing limitations will yield a sustainable textile material for fashion and textile industry with great potential.

Cartridge-based nucleic acid amplification test in drug-resistant Mycobacterium tuberculosis as compared to solid cultures: Is it time to look beyond cartridge-based nucleic acid amplification test?

Background: There is a need to study the performance, validity, and accuracy of cartridge-based nucleic acid amplification test (CB-NAAT) for accessing drug resistance among pulmonary tuberculosis (TB) compared with the solid culture drug susceptibility test (DST).Methods: Patients with symptoms of cough for more than 2 weeks with anyone symptoms such as night sweats, fever, and unintentional weight loss were studied. Cases with previously diagnosed drug-resistant pulmonary TB by sputum CB-NAAT having constitutional symptoms but not on any ATT for a minimum of 2 months were also included in the study. The patient's information, including age, immune surveillance status, clinical features, and chest X-rays, were recorded. Each sputum sample was divided into three aliquots and tested for smear microscopy, liquid culture (LC), and genotypic DST. Results of all three diagnostic modalities were compared with CB-NAAT reports.Results: Of 236 patients with sputum-positive CB-NAAT (n = 236), 49.4% (n = 117) were rifampicin resistant, while 50. 4% (n = 119) were rifampicin sensitive. The genotypic DST assays carried out of all enrolled patients showed that 76.3% (n = 181) patients were resistant to one or more first-line antitubercular drugs (FL ATTs) or second-line (SL) ATTs, while 23.7% (n = 55) patients were sensitive to all ATTs. Among all the study participants, 56.4% (n = 133) of patients had sputum smear-positive by ZN stain, while 88.6% (n = 209) showed growth on LC (BACTEC) media. On concordant analysis of CB-NAAT with DST assays, we found that among 119 CB-NAAT rifampicin-sensitive patients, 66 patients were drug-resistant (DR) TB to any of the FL or SL ATTs. The sensitivity, specificity, positive predictive value, and negative predictive value of CB-NAAT for detecting rifampicin resistance on sputum for pulmonary TB when compared with the gold-standard DST assays were 97.67%, 76.67%, 70.59%, and 98.29%, respectively.Conclusions: This study found that the use of rapid molecular technique (CB-NAAT) in screening DRTB at the community level is suboptimal compared to the gold-standard solid culture method. Although CB-NAAT's sensitivity in detecting DR pulmonary TB is significantly higher, the specificity is lower in that population who have received ATT earlier.

Koji Starter and Koji World in Japan.

Koji is made by culturing koji mold on grains. Koji has wide-ranging applications, for example, in alcoholic beverages and seasonings. The word ‘mold’ generally has a bad image, but in Japan, koji mold is valued for its usefulness, and over the years, efforts have been made to make safe, stable, and delicious food products from it. Koji mold spores, essential when making koji, are called koji starter in the industry. From the many available strains, those suitable for the production of each fermented food are chosen based on indicators such as growth rate and enzyme production capacity. In manufacturing using microorganisms, purity and yield are prioritized. However, the production of fermented foods using koji is more complex, with focus not only on the degree of decomposition of raw materials but also on factors influencing overall product design, including palatability, color, smell, and texture. Production can be facilitated by the variety of koji brought about by the diversity of koji mold combined with the solid culture method which increases the amount of enzyme production. In this report, we introduce the history of koji starter in Japan, the characteristics of koji mold in practice, and various fermented foods made from it. In addition, the factors affecting the quality of koji in solid culture are described.

Genexpert rapid detection of rifampicin resistance among patients with pulmonary tuberculosis

The routine conventional phenotypic bacteriological tests available for pulmonary tuberculosis (PTB) diagnosis, such as microscopy by Ziehl Neelsen staining, and Lowenstein Jensen method of solid culture, requires a long time for revealing positivity, resulting in a delayed diagnosis. After Mycobacterium tuberculosis (MTB) genome was discovered, nucleic acid amplification techniques were developed with more rapid detection and identification of rifampicin resistance in respiratory and extra-pulmonary specimens. GeneXpert is a DNA-polymerase chain reaction technique based on NAA, which allows an accurate genotypic method for a quick diagnosis of PTB diagnosis. The aim of the study was to assess retrospectively the yield of genotypic assay GeneXpert in revealing multidrug resistant TB compare to phenotypic confirmation by solid culture. 512 patients with positive and negative smears of suspected PTB were investigated by conventional phenotypic assays. PTB diagnosis assessed by positive genotypic assay and confirmed by phenotypic conventional solid culture method was 78.12%. The yield of GeneXpert in revealing rifampicin resistance was high (n=79/512; 15.43%) with 12.5% additional resistance against isoniazid revealed by positive solid cultures and drug susceptibility test. GeneXpert assay is a very useful method for rapid detection of MTB and drug resistance, which facilitates timely diagnosis and appropriate management of pulmonary tuberculosis.

Antibiotic Efficacy Assessment of Certain Species of Penicillium fungi

This study aimed to evaluate the antimicrobial activity of Penicillium species on soil-isolated gram-positive coccus bacteria. The antimicrobial efficacy of Penicillium species filtrate extracts were determined via disc diffusion and broth dilution assay. A kill curve study was conducted with treated and untreated bacterial populations. There were P. oxalicum, and P. purpurogenum showed the maximum zone in solid surface culture methods, followed by other spices with fewer antibiotic properties. The kill curve study has shown an exposure-dependent inhibition for bacterial populations. With the increase in the exposure periods, microbial growth was significantly reduced. In the broth dilution assay methods, among all treated Penicillium species, only P. oxalicum and P. funiculosum species were shown potent antibiotic properties; other species had given the very least activity. The study interpreted that screening of the suitable strain of microbes for very essentially for industrially important bioactive compounds scale up at industrial set up.

An efficient and rapid Rhizobium rhizogenes root transformation protocol for Lemna minor

Duckweeds belong to the smallest aquatic flowering plant family, Lemnaceae, and have a rapid doubling time, making this group an excellent system to study reduced morphology and wide environmental adaptability at the molecular level. Despite the availability of genomic and transcriptomic data for duckweed member, Lemna minor, lack of an efficient genetic transformation system has limited its use in plant molecular biology research. The present study reports an efficient and rapid Rhizobium rhizogenes-mediated root transformation system for L. minor. Two different factorial experiments were designed to test the effect of explant type, age, culture media and inoculation methods on transformation efficiency. Leaf and root tip cut explants were inoculated with R. rhizogenes strain MSU 440 harboring pBIN-YFP vector using yellow fluorescent protein (YFP) as a reporter gene for identification of transgenic roots. In addition, two different culture media, full MS and 0.25X Hoagland, and four different infection methods, solid culture, centrifugation, liquid culture and sonication, were compared. After 8 weeks, about 17% of the root tip-cut explants infected via the solid culture method and maintained in 0.25X Hoagland medium had YFP-expressing roots. These transgenic L. minor roots were morphologically similar to normal roots and PCR analysis demonstrated that the YFP-expressing roots were positive for the integration-expected rol genes. The described optimized root transformation procedure is a valuable tool for pursuing high-throughput gene characterization studies in L. minor.

Growth and development of carnation ‘Dreambyul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods

The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation ‘Dreambyul’ plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H2O2 level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation ‘Dreambyul’ plantlets cultured in the TIS.

Development and validation of external quality assessment panels for mycobacterial culture testing to diagnose tuberculosis in China.

Mycobacterial culture remains the gold standard for detection of Mycobacterium tuberculosis (MTB) in clinical samples. However, no external quality assessment (EQA) tools exist to validate results obtained using this sophisticated method. Therefore, we developed EQA panels to assess the quality of mycobacterial culture results produced by designated TB hospitals in China. Artificial sputum containing methylcellulose was used to supplement quantified mycobacterial solutions to simulate culture-negative and culture-positive clinical sputum samples of low or high mycobacterial concentration, respectively. After storage of the quantified simulated EQA panels for 4weeks at 4°C, experimental bacterial quantification of the panels was again conducted, with no impact of artificial sputum on mycobacterial culture results observed. Next, 47 tuberculosis (TB) hospitals were recruited for evaluation of the EQA panels. Overall, 29 hospitals (61.7%) produced mycobacterial culture test results matching expected results for the EQA panels, while the remaining 18 (38.3%) hospitals did not. False-negative results for the low mycobacterial concentration panel sample accounted for 33 (73.3%) diagnostic errors. Compared with hospitals using solid culture methods as a control group, hospitals using the liquid culture method were less likely to produce uncertified results (aOR 0.064, 95% CI 0.005-0.770). In conclusion, we first developed then evaluated EQA panels for validation of mycobacterial culture testing in China. Our data demonstrate that approximately one-third of TB hospitals failed to produce results that met criteria for classification as certified mycobacterial culture testing providers, emphasizing the importance of quality control and quality assurance in TB diagnostics.

Declining tuberculosis prevalence in Saharia, a particularly vulnerable tribal community in Central India: evidences for action

BackgroundIn spite of an alarmingly high tuberculosis (TB) burden amongst the Saharia tribe of central India, there is hardly any study to investigate the impact of DOTS implementation on the magnitude of tuberculosis disease and the changes over time. This article present the findings of TB prevalence surveys conducted amongst this indigenous population in two different time periods to know the change in the prevalence of TB.MethodsA cross sectional survey was conducted among Saharia population in Shivpuri district, Madhya Pradesh during February 2013 to May 2013 and resurvey during March 2015 to July 2015. All individuals (≥15 years) were examined for chest symptoms suggestive of TB. Sputum samples were collected from all presumptive TB cases and were confirmed by laboratory examination by Ziehl-Neelsen smear microscopy and solid media culture methods. All detected cases were referred to health facility for anti-tuberculosis treatment as per RNTCP guidelines.ResultsThere was significant reduction (trend Chi square 19.97; OR = 1.521; p = 0.000) in the prevalence of TB at the endline (1995 per 100,000) as compared to baseline (3003 per 100,000). The reduction was significant among males as compared to females (OR 1.55; p = 0.000) and in the age group of 25–34 years (OR 2.0; p = 0.007) and 45–54 years (OR 4.39; p = 0.003). There was significant reduction in the prevalence in both smear (OR 1.29; p = 0.02) and culture positive (OR 1.57; p = 0.000) TB at the endline survey.ConclusionThe study findings highlight a reduction in the prevalence of TB among Saharia tribal population. Further studies are needed to identify the factors associated with reduction in prevalence among this population and also further surveys to monitor the prevalence trend over a period.

Evaluation of Genotype MTBDRplus Line Probe Assay in Detection of Rifampicin and Isoniazid Resistance in Comparison to Solid Culture Drug Susceptibility Testing in a Tertiary Care Centre of Western Uttar Pradesh

Background: Isoniazid (INH) and rifampicin (Rif) are the key first-line antituberculosis drugs, and resistance to these drugs i.e., multi-drug-resistant tuberculosis (MDR-TB), is likely to result in treatment failure and poor clinical outcomes. India has the highest burden of TB and MDR-TB in the world, disproportionately high even for India’s population. The GenoType® MTBDRplus molecular method allows rapid detection of Rif and INH resistance. Aim: The present study was done to compare the performance of line probe assay test (GenoType® MTBDRplus) with solid culture method for an early diagnosis of MDR-TB. Methods: Totally 1503 sputum samples of MDR-TB suspects were subjected to fluorescent microscopy. Decontamination was done by N-acetyl-L-cysteine and sodium hydroxide method. Fluorescent microscopy-positive samples were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay. Sixty-two random samples were compared with phenotypic drug susceptibility testing (DST) (1% proportion method) using solid culture method by Lowenstein–Jensen media. Results: The sensitivity, specificity, positive predictive value and negative predictive value for detection of resistance to Rif were 94.74%, 95.35%, 90% and 97.62% and to INH were 92.00%, 91.89%, 88.46% and 94.44%, respectively, in comparison with the phenotypic DST. Conclusion: GenoType® MTBDRplus has good sensitivity and specificity in detecting MDR-TB cases with a significantly lesser turnaround time as compared to conventional DST method and simultaneous detection of Rif and INH resistance. This technique saves several weeks of time required for culture and DST.

Comparative evaluation of loop-mediated isothermal amplification and conventional methods to diagnose extrapulmonary tuberculosis

Background: Extra-pulmonary tuberculosis is difficult to diagnose by conventional methods, because they are less sensitive and more time consuming. Loop-mediated Isothermal Amplification (LAMP) is a novel gene amplification method that has been developed to diagnose Mycobacterium tuberculosis complex in pulmonary and paucibacillary extrapulmonary specimens even in resource poor settings. Aims and Objectives: To evaluate the sensitivity and specificity of LAMP to detect Mycobacterium tuberculosis complex in paucibacillary extra-pulmonary specimens and to compare the results with other conventional methods in extrapulmonary specimens. Materials and Methods: 45 specimens from suspected extrapulmonary tuberculosis patients were collected and tested by LAMP method after DNA extraction. Simultaneously, these specimens were tested by smear microscopy, solid and liquid culture methods. Culture positivity, either in solid or liquid culture, was considered as a confirmed case of extrapulmonary tuberculosis. Results: Sensitivity of LAMP was 95.6% whereas for liquid culture, solid culture and smear microscopy were 69.6%, 65.2% and 17.4% respectively. Specificity for LAMP was 95.4% and for other 3 conventional methods were 100%. Positive predictive values, negative predictive values and likelihood ratios were also evaluated. Turn-around time (TAT) for LAMP was 8 hours only whereas for liquid culture was 2-4 weeks, and for solid culture it was 4-8 weeks. Conclusion: LAMP was a simple, rapid and cost-effective procedure with good sensitivity and specificity. It was found to be better than conventional methods to diagnose extrapulmonary tuberculosis.

Rapid Screening of MDR-TB in Cases of Extra Pulmonary Tuberculosis Using Geno Type MTBDRplus.

BackgroundDrug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes.MethodsA total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ) media. Phenotypic drug susceptibility testing (DST) was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method.ResultsOf 510 specimens cultured, the total culture positivity obtained was 11.8% (60) encompassing 54 (10.6%) Mycobacterium tuberculosis and 6 (1.2%) non-tubercular mycobacteria (NTM). DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB) in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours.ConclusionThe problem of MDR in extra pulmonary tuberculosis (EPTB) cannot be overlooked and due attention on patients should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens.

Performance of the BacT Alert 3D System Versus Solid Media for Recovery and Drug Susceptibility Testing of Mycobacterium tuberculosis in a Tertiary Hospital in Korea.

BackgroundTuberculosis (TB) is a major health problem, and accurate and rapid diagnosis of multidrug-resistant (MDR) and extended drug-resistant (XDR) TB is important for appropriate treatment. In this study, performances of solid and liquid culture methods were compared with respect to MDR- and XDR-TB isolate recovery and drug susceptibility testing.MethodsSputum specimens from 304 patients were stained with Ziehl-Neelsen method. Mycobacterium tuberculosis (Mtb) isolates were tested for recovery on Löwenstein-Jensen (LJ) medium and the BacT Alert 3D system. For drug susceptibility testing of Mtb, isolates were evaluated on M-KIT plates and the BacT Alert 3D system.ResultsThe recovery rates were 94.9% (206/217) and 98.2% (213/217) for LJ medium and the BacT Alert 3D system, respectively (kappa coefficient, 0.884). The rate of drug resistance was 13.4% for at least one or more drugs, 6.0% for MDR-TB and 2.3% for XDR-TB. M-KIT plate and BacT 3D Alert 3D system were comparable in drug susceptibility testing for isoniazid (97.7%; kappa coefficient, 0.905) and rifampin (98.6%; kappa coefficient, 0.907). Antibiotic resistance was observed using M-KIT plates for 24 of the total 29 Mtb isolates (82.8%).ConclusionThe liquid culture system showed greater reduction in the culture period, as compared with LJ medium; however, drug susceptibility testing using M-KIT plates was advantageous for simultaneous testing against multiple drug targets.

A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria.

Cyanobacteria can be utilized as a potential biocatalyst for the production of biofuels and biochemicals directly from CO2. Useful mutants of cyanobacteria, which can grow rapidly or are resistant to specific metabolic products, are essential to improve the productivity of biofuels. In this study, we developed a single cell culture system to effectively screen mutant cyanobacteria using magnetite nanoparticles and magnetic force. Lens culinaris Agglutinin (LCA) was selected as a lectin, which binds to the surface of Synechococcus elongatus PCC7942 cells and the LCA-conjugated magnetite cationic liposomes (MCLs) were developed for magnetic labeling of PCC7942 cells. The MCL-labeled PCC7942 cells were magnetically patterned at a single cell level by using 6,400 iron pillars of the pin-holder device. The device enabled 1,600 single cells to be arrayed in one square centimeter. We cultured the patterned cells in liquid medium and achieved higher colony-forming ratio (78.4%) than that obtained using conventional solid culture method (4.8%). Single cells with different properties could be distinguished in the single cell culture system depending on their growth. Furthermore, we could selectively pick up the target cells and subsequently perform efficient isolation culture. The ratio of successful isolation culture using the developed method was 13 times higher than that of the conventional methods. Thus, the developed system would serve as a powerful tool for screening mutant cyanobacteria.

THE ANTI-TB DRUG SENSITIVITY OF Mycobacterium tuberculosis FROM CEREBROSPINAL FLUID AND BONE TISSUE BIOPSY SPECIMENS OF PATIENTS SUSPECTED TUBERCULOUS MENINGITIS AND SPINAL TB IN Dr SOETOMO HOSPITAL INDONESIA

Tuberculous meningitis (TBM) is an infection of meningens which potentially life threatening with significant morbidity and mortality. Spinal TB has the same problem with TBM, infection in bone and joint, the delayed diagnosis worsens the prognosis. The rapid and accurate diagnosis plus promt adequate treatment is essential for the good outcome. The aim of this research is to study the first line drug sensitivity of Mycobacterium tuberculosis isolated from specimens of cerebrospinal fluid from suspected tuberculous meningitis patients and bone tissue biopsy from suspected spinal TB patients. The method of this research is TB Laboratory examination in Department of Clinical Microbiology – Dr. Soetomo General Hospital, Indonesia, using the gold standard liquid culture method MGIT 960 System (Becton Dickinson) and solid culture method with Lowenstein-Jensen medium. The specimens CSF from 50 TBM patients at January 2013 until May 2014. Positive isolate detection of Mycobacterium tuberculosis complex were 11 isolates (22%), which sensitivity 100% (11/11 isolates) to Rifampin (R), Pyrazinamide (Z), Ethambutol (E), and Streptomycin (S); one isolate resistant to Isoniazid, sensitivity to Isoniazid 90,90% (10/11); and received 21 specimens of bone tissue biopsy which positive 5 isolates (23%), all isolates sensitive 100% (5/5 isolates) to Rifampin and Pyrazinamide, and 1 isolates resistant to Isoniazid, Ethambutol, and Streptomycin, in which sensitivity 80% (4/5 isolates) to Isoniazid, Ethambutol, and Streptomycin. The conclusion of this research is positivity detection 22% of CSF specimens, and 23% of bone tissue biopsy were low. All isolates sensitive 100% to Rifampin and Pyrazinamide, and 80-90% sensitive to Isoniazid.

Situation of drug resistant tuberculosis in Saharia tribe of central India.

Background & objectives:The information on multidrug resistant tuberculosis (MDR-TB) situation amongst Saharia, one of the Particularly Vulnerable Tribal Groups (PVTGs) in Madhya Pradesh, India, is not available. Hence, this study was undertaken to find the situation of MDR-TB amongst Saharia PVTG in two districts of Madhya Pradesh.Methods:Community based cross-sectional TB prevalence surveys were conducted among Saharia PVTG in Gwalior and Shivpuri districts of Madhya Pradesh. Chest symptomatics were identified from the individual registered for the study. Two sputum samples were collected from each of the eligible individuals, transported to the laboratory, and were examined by Ziehl-Neelsen (Z-N) smear microscopy and solid medium culture methods. Drug susceptibility testing of the isolates was done by indirect proportion method on solid medium.Results:MDR rate was 2.2 per cent of new cases and 8.2 per cent among the previously treated cases of TB in Gwalior while it was two per cent among the previously treated cases in Shivpuri district.Interpretation & conclusions:Though the prevalence of tuberculosis in these districts was alarmingly high, the MDR rates were more or less similar to national average. However, the findings of this study highlight the need for active intervention so that the MDR-TB is kept under control.