Rapid Multiplication Technique for Production of High-Quality Seed Potato (Solanum tuberosum L.) Tubers

Potato farmers in India and other developing countries lack timely availability of healthy and good quality potato seed tubers. This is mainly due to conventional seed multiplication techniques which, has suffered inherently from low multiplication rates. The soil medium is generally used for seed tuber production. In order to overcome bottlenecks, a study was conducted to evaluate mini-tuber production potential for selected baskets of potato varieties in different soil-less solid media types compared with soil as a control. Soil-less media technique would also assist in planning for economical and rapid seed multiplication program, along with pathogen-free seed tubers, which will increase production of good quality reliable seed material in the country. This will finally ensure increased productivity of potato crop. With these objectives, an experiment was conducted during the years, 2015–2016 and 2016–2017. Three different soil-less solid propagation media (kalpeat plus, soilrite mix and soilrite mix TC) were tested against control (soil:sand 3:1); for five different varieties (1001, 1002, 1003, 1004 and 1005). Plantlets grown on soilrite mix performed better with higher mini-tuber yield of 548.58 gm/ container (1/2 m²), while for other propagation media, 283.39 gm/container (1/2 m²), 96.08 gm/container (1/2 m²), and 52.61 gm/container (1/2 m²) respectively, were observed for kalpeat plus, soilrite mix TC and control (soil:sand 3:1). Among the varieties tested, viz., 1005 and 1004 produced maximum mini-tubers, between 9 tubers/plant and 5 tubers/plant respectively. Soilrite mix increased the number and size of mini-tubers. Thus, this study concluded that soil-less solid media induced seed potato multiplication is better than traditional soil-based techniques

Filamentous fungi and media for cellulase production in solid state cultures

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.

Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity

The selection of high-producing mammalian cell lines is a crucial step in process development for the production of biopharmaceuticals. Previously, cloning by limiting dilution method was used to isolate monoclonal NS0 cells secreting high levels of humanized-C2 monoclonal antibodies. However limiting dilution method is time consuming, has low probability of monoclonality and is significantly limited by the number of clones that can be feasibly screened. In order to minimize the duration and to increase the probability of obtaining high-producing clones with high monoclonality, an automated colony picker, Clone Pix FL system was used to replace limiting dilution method. We were able to screen 1 x 10(5) clones secreting humanized monoclonal antibodies and high producer clones were selected in just 7 days. Briefly, semi-solid media was used to immobilize single cells separately and allow them to proliferate into discrete clones. The high viscosity nature of the semi-solid media retains the secreted products in the vicinity of the associated clones. Using Clone Pix FL system, all clones were screened and the producer clones with different exterior fluorescent intensities were automatically isolated. We were able to isolate rare high-producers (> 3000 FU) with frequency of as low as 0.003 percent of the population. A quantitative ELISA was also performed to evaluate the correlation between the fluorescence intensity of clones with its corresponding antibody productivity. Clones with fluorescence intensity of < 1000 FU showed relatively low antibody productivity compared with those greater than 1000 FU; however above this there was no correlation of production with the increase in fluorescence intensity. Hence, although the high-throughput, rapid and automated nature of Clone Pix FL system allows the screening of large number of cells in a short period of time with also an increased in the probability of obtaining rare and precious high-producing clones...

Evaluation of Automated Blood Culture System for Body Fluids Culture Other Than Blood / 대한임상미생물학회지

BACKGROUND: We investigated whether culture using an automated blood culture system enhances the recovery of bacteria and fungi from body fluids other than blood when compared to conventional solid media culture methods. METHODS: A total of 734 specimens [ascites (n=457), bile (n=5), CAPD (n=28), CSF (n=32), joint fluids (n=165), pericardial fluid (n=17), and pleural fluid (n=30)] were included in the study. Half of the volume of each specimen was inoculated directly into automated blood culture bottles (bioMeriux, Marcy-I'Etoile, France). The remaining volume was inoculated onto conventional solid media (sheep blood agar, chocolate agar, and phenylethyl alcohol agar) after centrifuging at 3,000 rpm for 10 min. RESULTS: Clinically significant microorganisms were isolated from 62 specimens (8.5%) by automated blood culture and 61 specimens (8.3%) by the conventional solid media culture (kappa index: 0.81, 95% confidence interval: 0.75~0.89). Contamination was observed in 11 (1.8%) of the automated blood culture specimens and 3 (0.4%) of the solid media culture specimens. The mean turnaround times of the automated blood cultures and the conventional solid media cultures were 3.7 and 2.8 days, respectively (P<0.0001). CONCLUSION: Compared with conventional culture methods, no improvement in the recovery of clinically significant microorganisms was noted with the use of the automated blood culture system for the culture of body fluids other than blood.

Evaluation of MGIT 960 System for Recovery of Mycobacteria from Body Fluids / 대한임상미생물학회지

BACKGROUND: In this study, we evaluated the BACTEC MGIT 960 system (Becton Dickinson Microbiology Systems, Sparks, Md, USA), which is fully automated, noninvasive and nonradiometric fluorescent indicator broth detection system, for the growth and detection of mycobacteria with body fluid specimens. METHODS: Total of 1,891 body fluid specimens were included (pleural fluid 752, ascitic fluid 629, cerebrospinal fluid 214, joint fluid 79, peritozol 54, others 163). Specimens were inoculated into MGIT and solid media (3% ogawa, Japan). Polymerase chain reaction was performed for the discrimination of Mycobacterium tuberculosis from Mycobacterium other than tuberculosis (MOTT). RESULTS: A total of 62 isolates of mycobacteria were recovered from all culture system. With MGIT system, 56 isolates were recovered, compared with solid system recovered 33 isolates. 29 isolates were recovered with MGIT only and 6 isolates recovered with solid media only. Among 62 isolates recovered, 11 isolates were positive in acid fast stain. 10 isolates were recovered with MGIT. One isolate was recovered with solid system. 51 isolates were negative in acid fast stain. Among this, 46 isolates were recovered with MGIT. The mean detection time was 14.2 days with MGIT system, and 38.2 days with solid media. Contamination rate for each system with body fluid specimens were 4.1% for MGIT and 1.7% for solid media. CONCLUSION: In body fluid, the MGIT system has the advantages of improved detection rate and rapid recovery than solid media to recover mycobacteria.

Evaluation of the BACTEC MGIT 960 system for the recovery of mycobacteria / 대한임상병리학회지

BACKGROUND: We evaluated the BACTEC MGIT 960 system(Becton Dickinson Microbiology Systems, Sparks, Md, USA), which is a fully automated, noninvasive and nonradiometric fluorescent indicator broth detection system for the growth and detection of mycobacteria with a capacity to incubate and continuously monitor 7 ml MGIT(Mycobacterium growth indicator tube) culture tube. METHODS: We studied 1,690 specimens(1,258 respiratory and 432 non-respiratory specimens). Processed specimens with 2% NaOH-NALC(final NaOH concentration: 1%) were inoculated into MGIT and solid media(3% Ogawa, Japan). Polymerase chain reaction was performed for the discrimination of Mycobacterium tuberculosis complex from Mycobacterium other than tuberculosis(MOTT). RESULTS: From all culture system, a total of 181 isolates of mycobacteria were recovered. The greatest number of isolates of mycobacteria was recovered with MGIT 960 system(174, 96.1%), followed by solid media(73, 40.3%). 108 isolates(59.7%) were recovered with MGIT only and 7(3.9%) recovered with solid media only( P value < 0.0001). From a total of 1,617 AFB smear negative specimens, 101(6.3%) were recovered with MGIT 960 system and 34(2.1%) recovered with solid media. The mean times to detection were average 11.1 days for MGIT 960 system and 31.6 days for solid media(P value < 0.0001). Contamination rates for each system were 14.9% for MGIT 960 and 2.6% for solid media. CONCLUSIONS: The newly introduced MGIT 960 system is easy to use and has advantages of high detection rate and rapid recovery than solid media, but there are some problems such as high contamination rate and high cost in the management of this liquid system.

Comparison of Three Egg-based Solid Media for Culture of Mycobacteria from Clinical Specimens / 대한임상병리학회지

BACKGROUND: Single egg-based Ogawa medium is used for mycobacterial culture in nearly all tertiary care or medical school-affiliated hospital in Korea. However, it is expected that some mycobacteria would grow so lately or never in only one media, even if mycobacteria are present sufficiently in the specimen. To estimate the efficiency of inoculation to two or more egg-based media for isolation of mycobacteria, positive culture rates and detection time were compared among three media. METHODS: 193 clinical specimens, which referred to laboratory of Pusan National University Hospital for 2 months, including 123 specimens of patients with mycobacteriosis, were processed and inoculated to Eiken, VITE and KT media. The isolates were identified and classified to Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) by AccuProbe method. The positive culture results were analyzed by McNemar test and t-test. RESULTS: M. tuberculosis and NTM were isolated in 65 (34.0%) and 14 (7.4%) specimens, resulting in positive culture rates of 41.4%. Of the 123 patients' specimens, 63 cases were positive in culture of VITE media, which were significantly higher than 50 cases of Eiken and 45 cases of KT media. In 43 positive-stained specimens, twelve to twenty samples (29.3-48.8%) failed to grow in at least one media. The colonies were visible at 24.9 to 30.7 days after inoculation, and VITE and KT media detected visible colonies 4.5 and 2.5 days more rapidly than Eiken media, respectively. CONCLUSIONS: Inoculation to two or more egg-based media is useful for increasing culture positivity and early detection of colonies for mycobacterial culture.