Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this meth...Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.展开更多
In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for su...In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.展开更多
Fermented chile pepper mash is a major food product in New Mexico. There are few reports on the fermentation process or on methods to monitor it, In the current study we examined a pour plate procedure with an overlay...Fermented chile pepper mash is a major food product in New Mexico. There are few reports on the fermentation process or on methods to monitor it, In the current study we examined a pour plate procedure with an overlay using plate count agar and 3 MTM PetrifilmTM Aerobic Count （AC） plates for determination of total aerobic bacterial counts during the fermentation of chile mash. Fifty chile mash samples were obtained directly from commercial fermentation vats and examined within 2 h of collection. Serial dilutions of the chile mash were prepared in Butterfield＇s Phosphate Buffer. 1 mL portions of the diluted samples were aliquoted in duplicate onto the AC plates and into empty Petri dishes. Plate count agar was poured and once the plates had solidified, they were overlaid with about 10 mL of PCA to minimize spreaders. Plates were incubated at 30 ℃ for 48 h and enumerated. Paired difference tests were conducted on log transformed data to compare the results of the two plating procedures. For commercial chile mash samples, we did not show any significant differences between the AC plate counts and the pour plate counts （α = 0.05）, 3 MTM PetrifilmTM AC plates are a good alternative to pour plates for the determination of the total aerobic counts in fermented chile mashes.展开更多
文摘Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.
基金supported by the National Natural Science Foundation of China(Nos.32070002 and 41876166)Science&Technology Fundamental Resources Investigation Program(Grant Nos.2022FY101100 and 2019FY100700)+1 种基金China Postdoctoral Science Foundation(2022M721923)Natural Science Foundation of Shandong Province,China(ZR2023QD187).
文摘In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.
文摘Fermented chile pepper mash is a major food product in New Mexico. There are few reports on the fermentation process or on methods to monitor it, In the current study we examined a pour plate procedure with an overlay using plate count agar and 3 MTM PetrifilmTM Aerobic Count （AC） plates for determination of total aerobic bacterial counts during the fermentation of chile mash. Fifty chile mash samples were obtained directly from commercial fermentation vats and examined within 2 h of collection. Serial dilutions of the chile mash were prepared in Butterfield＇s Phosphate Buffer. 1 mL portions of the diluted samples were aliquoted in duplicate onto the AC plates and into empty Petri dishes. Plate count agar was poured and once the plates had solidified, they were overlaid with about 10 mL of PCA to minimize spreaders. Plates were incubated at 30 ℃ for 48 h and enumerated. Paired difference tests were conducted on log transformed data to compare the results of the two plating procedures. For commercial chile mash samples, we did not show any significant differences between the AC plate counts and the pour plate counts （α = 0.05）, 3 MTM PetrifilmTM AC plates are a good alternative to pour plates for the determination of the total aerobic counts in fermented chile mashes.
