Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The analysis of internal transcribed spacer and its applications on the detection of orchid bacterial diseases

壹、 摘要 Abstract …………………………………………………03 貳、前言…………………………………………………………04 一、國內蘭花產業的發展及重要性………………………04 二、蝴蝶蘭花常見細菌性病害及介紹……………………05 三、ITS 在細菌病害檢測上之應用………………………07 參、材料與方法…………………………………………………12 一、供試菌株………………………………………………12 二、菌株培養………………………………………………12 三、染色體DNA的抽取……………………………………12 四、病原細菌ITS資料之收集與分析……………………13 五、病原細菌性病害引子之設計…………………………14 六、專一性引子之設計……………………………………18 七、PCR 增幅………………………………………………18 八、Multiplex PCR ………………………………………19 九、電泳分析………………………………………………19 十、引子對敏感度測試……………………………………20 肆、結果與討論…………………………………………………21 伍、圖表…………………………………………………………29 陸、參考文獻……………………………………………………52[[abstract]]造成台灣蝴蝶蘭細菌性病害的主要病原有三種，分別為 Pectobacterium chrysanthemi、 Acidovorax avenae subsp. cattleyae以及 Burkholderia gladioli。本研究利用自行建置之ITS資料庫，進行多序列比對分析。可對A.avenae subsp. cattleyae以及 B.gladioli分別設計出具專一性之AAC-ITS引子對及BG-ITS引子對，所有供試菌株經PCR電泳分析皆可產生專一性條帶。在P.chrysanthemi的鑑定係利用P.chrysanthemi所特有之藍色素相關基因之序列來設計專一性引子對，計有pecS、pecM及idgA等3組引子對，可專一性偵測到P.chrysanthemi。本研究中所設計5組引子對AAC-ITS、BG-ITS、pecS、pecM及idgA，測試之結果顯示100﹪之專一性。在敏感度分析上，idgA引子對偵測P.chrysanthemi之敏感度分析，敏感度為1.2x104 cells。BG-ITS引子對偵測B.gladioli之敏感度為1x102 cells。AAC-ITS引子對偵測A.avenae subsp. cattleyae之敏感度為1x102 cells。在多引子對之分析時以各引子對可精確地檢測出各病原菌的存在。 There are three species of bacterial can infect the local Phalaenopsis, three of them are Pectobacterium chrysanthemi, Acidovorax avenae subsp. cattleyae and Burkholderia gladioli. In this study, we use the in-house bacterial internal transcribed spacer database to analysis the sequence between the bacterial that can infect the Phalaenopsi. We design primers AAC-ITS and BG-ITS based on the results of the analysis of Acidovorax avenae subsp. cattleyae and Burkholderia gladioli ITS sequence, and the primers pecS、pecM and idgA designed by indigoidine related gene, to detect the Pectobacterium chrysanthemi. The primers we use in this study reveals perfect results to their on response bacterial. In the sensitivity test, primer idgA can detect the Pectobacterium chrysanthemi with 1.2x104 cells, primer AAC-ITS can detect Acidovorax avenae subsp. cattleyae with1x102 cells and primer BG-ITS can detect Burkholderia gladioli with 1.2x104 cells. The primers designed in this study can obviously and accurately detect the bacterial that can infect the local Phalaenopsis

Carbon Monoxide Inhibits Receptor Activator of NF-κB (RANKL)-Induced Osteoclastogenesis

Background: Low concentrations of carbon monoxide (CO) have anti-inflammatory effects and can reduce bone erosion in a murine collagen-induced arthritis model. The objective of this study was to assess the effects of CO on receptor activator of NF-γB ligand (RANKL), one of the key stimulators of osteoclastogenesis. Methods: The in vivo effects of CO on RANKL expression were assessed in a collagen antibody-induced arthritis model in mice. Cell proliferation and apoptosis were assessed in the RAW246.7 cell line stimulated with RANKL and exposed to either air or CO. The number of tartrate resistant acid phosphatase (TRAP)-positive RAW246.7 cells was also examined after treatment with RANKL and the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, Troglitazone. Results: CO reduced RANKL expression in the synovium of arthritic mice. Although CO slightly increased RAW246.7 cell proliferation, no differences in activated caspase 3 levels were detected. In addition, Troglitazone ameliorated the inhibitory effects of CO on RANKL-induced TRAP expression by RAW246.7 cells. Conclusions: CO suppresses osteoclast differentiation by inhibiting the RANKL-induced activation of PPAR-γ. Given the role of the PPAR-γ/cFos (AP-1) pathway in regulating the transcription factor, NFATc1, the master regulator of osteoclastogenesis, further studies are warranted to explore CO in treating inflammatory bone disorders