[Objectives]This study was conducted to investigate the spoilage microorganisms in the storage process of Chinese flowering cabbage.[Methods]Pathogenic bacteria were separated and purified from rotted Chinese flowerin...[Objectives]This study was conducted to investigate the spoilage microorganisms in the storage process of Chinese flowering cabbage.[Methods]Pathogenic bacteria were separated and purified from rotted Chinese flowering cabbage during storage.The gradient dilution culture method and streaking purification method were applied to selectively cultivate spoilage microorganisms for separation and observation.The isolated strains were identified through the ITS and sequence analysis of 16 S rDNA combined with the morphological characteristics and physiological and biochemical properties of the microbes.On the basis of morphology,combined with gene sequence analysis,the isolated pathogenic bacteria A1,A2,A3,and A4 were identified by PCR using the bacterial universal primer 16 S rDNA sequences,and B1 was amplified using the fungal universal primer ITS sequence.The gene sequences obtained by sequencing were subjected to homologous sequence alignment in the NCBI gene library to determine the biological classification of the spoilage bacteria.[Results]The results showed that the four bacteria numbered A1,A2,A3,and A4 were Klebsiella,Acinetobacter baylyi,Staphylococcus epidermidis,and Pseudomonas,respectively.The saprophytic fungus labeled B1 was Streptomyces albus.Re-contacting it to Chinese flowering cabbage caused the cabbage to rot,so it was the main saprophytic fungus that caused the cabbage to rot after picking.Therefore,the main spoilage microorganisms during storage of Chinese flowering cabbage were Klebsiella,A.baylyi,S.epidermidis,Pseudomonas,and S.albus.[Conclusions]This study provides a certain scientific basis and theoretical basis for the storage and preservation of Chinese flowering cabbage.展开更多
Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impe...Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.展开更多
基金Huanggang Normal University High-level Cultivation Project(04201711903)Youth Talent Project of Hubei Provincial Department of Education(03201718402)。
文摘[Objectives]This study was conducted to investigate the spoilage microorganisms in the storage process of Chinese flowering cabbage.[Methods]Pathogenic bacteria were separated and purified from rotted Chinese flowering cabbage during storage.The gradient dilution culture method and streaking purification method were applied to selectively cultivate spoilage microorganisms for separation and observation.The isolated strains were identified through the ITS and sequence analysis of 16 S rDNA combined with the morphological characteristics and physiological and biochemical properties of the microbes.On the basis of morphology,combined with gene sequence analysis,the isolated pathogenic bacteria A1,A2,A3,and A4 were identified by PCR using the bacterial universal primer 16 S rDNA sequences,and B1 was amplified using the fungal universal primer ITS sequence.The gene sequences obtained by sequencing were subjected to homologous sequence alignment in the NCBI gene library to determine the biological classification of the spoilage bacteria.[Results]The results showed that the four bacteria numbered A1,A2,A3,and A4 were Klebsiella,Acinetobacter baylyi,Staphylococcus epidermidis,and Pseudomonas,respectively.The saprophytic fungus labeled B1 was Streptomyces albus.Re-contacting it to Chinese flowering cabbage caused the cabbage to rot,so it was the main saprophytic fungus that caused the cabbage to rot after picking.Therefore,the main spoilage microorganisms during storage of Chinese flowering cabbage were Klebsiella,A.baylyi,S.epidermidis,Pseudomonas,and S.albus.[Conclusions]This study provides a certain scientific basis and theoretical basis for the storage and preservation of Chinese flowering cabbage.
基金We thank for the funding:National Key R&D Program of China(grant numbers 2018 YFC0310800,2018YFC0310803)COMRA Project DY135-B2-12+3 种基金the National Natural Science Foundation of China(grant numbers 41525011,91751205,11774225)the Recruitment Program of Global Experts(Program for Young Professionals),and the Natural Science Foundation of Shanghai(grant no.18ZR1419800)This is also a contribution to the Center for Ocean Mega-Science,Chinese Academy of Sciences,the Senior User Project of RV KEXUE(KEXUE2019GZ06)the International Center for Deep-Life Investigation(IC-DLI).We thank Gunter Wegener for providing the ANME enrichment sample.
文摘Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.
