To Grow or Not to Grow: Isolation and Cultivation Procedures in the Genomic Age

Zengler, Karsten

doi:10.1002/9781118409855.ch12

Cited by 3 publications

(3 citation statements)

References 108 publications

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Section: Discussionmentioning

confidence: 99%

“…Marine pelagic bacteria often have complex growth and nutrient requirements ( Stewart, 2012 ; Zengler, 2013 ). In addition, they are generally considered to be oligotrophs, since they inhabit a nutrient-poor environment and grow only very slowly, which might also compromise the cultivation process ( Amann et al, 1995 ; Keller and Zengler, 2004 ; Zengler, 2013 ). Hence, a large part of the marine environmental microbiome has been considered non-cultivable using standard cultivation techniques ( Amann et al, 1995 ; Morris et al, 2002 ; Giovannoni and Stingl, 2005 ).…”

Section: Discussionmentioning

confidence: 99%

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Exploring the Cultivable Ectocarpus Microbiome

et al. 2017

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Coastal areas form the major habitat of brown macroalgae, photosynthetic multicellular eukaryotes that have great ecological value and industrial potential. Macroalgal growth, development, and physiology are influenced by the microbial community they accommodate. Studying the algal microbiome should thus increase our fundamental understanding of algal biology and may help to improve culturing efforts. Currently, a freshwater strain of the brown macroalga Ectocarpus subulatus is being developed as a model organism for brown macroalgal physiology and algal microbiome studies. It can grow in high and low salinities depending on which microbes it hosts. However, the molecular mechanisms involved in this process are still unclear. Cultivation of Ectocarpus-associated bacteria is the first step toward the development of a model system for in vitro functional studies of brown macroalgal–bacterial interactions during abiotic stress. The main aim of the present study is thus to provide an extensive collection of cultivable E. subulatus-associated bacteria. To meet the variety of metabolic demands of Ectocarpus-associated bacteria, several isolation techniques were applied, i.e., direct plating and dilution-to-extinction cultivation techniques, each with chemically defined and undefined bacterial growth media. Algal tissue and algal growth media were directly used as inoculum, or they were pretreated with antibiotics, by filtration, or by digestion of algal cell walls. In total, 388 isolates were identified falling into 33 genera (46 distinct strains), of which Halomonas (Gammaproteobacteria), Bosea (Alphaproteobacteria), and Limnobacter (Betaproteobacteria) were the most abundant. Comparisons with 16S rRNA gene metabarcoding data showed that culturability in this study was remarkably high (∼50%), although several cultivable strains were not detected or only present in extremely low abundance in the libraries. These undetected bacteria could be considered as part of the rare biosphere and they may form the basis for the temporal changes in the Ectocarpus microbiome.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Exploring the Cultivable Ectocarpus Microbiome

et al. 2017

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“…Since the 19th century, culture-based identification methodologies have been a mainstay for the diagnosis of pathogenic bacteria in the specimens. However, culture methods have the following drawbacks: i) the culture period for some bacteria requires up to 1 week, ii) anaerobic bacteria are generally difficult to culture, and iii) poor colony formation is inevitably associated with blood from patients treated with antibiotics [ 1 ]. To circumvent these problems, we sought to detect and identify bacteria in patient specimens based on their DNA sequences using next generation DNA sequencing (NGS) technology.…”

Section: Introductionmentioning

confidence: 99%

Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis

et al. 2018

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Prompt identification of causative pathogenic bacteria is imperative for the treatment of patients suffering from infectious diseases, including sepsis and pneumonia. However, current culture-based methodologies have several drawbacks including their limitation of use to culturable bacterial species. To circumvent these problems, we attempted to detect bacterial DNA in blood using next-generation DNA sequencing (NGS) technology. We conducted metagenomic and 16S ribosomal RNA (rRNA) gene amplicon sequencing of DNA extracted from bacteria-spiked blood using an Ion Personal Genome Machine. NGS data was analyzed using our in-house pipeline Genome Search Toolkit and database GenomeSync. The metagenomic sequencing analysis successfully detected three gram-positive and three gram-negative bacteria spiked in the blood, which was associated with a significant portion of non-bacterial reads, even though human blood cells were separated by low-speed centrifugation prior to DNA extraction. Sequencing analysis of seven variable regions of the 16S rRNA gene amplicon also successfully detected all six bacteria spiked in the blood. The methodology using 16S rRNA gene amplicon analysis was verified using DNA from the blood of six patients with sepsis and four healthy volunteers with potential pathogenic bacteria in the blood being identified at the species level. These findings suggest that our system will be a potential platform for practical diagnosis in the future.

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Isolation and Cultivation of Anaerobes

Börner

2016

Advances in Biochemical Engineering/Biotechnology

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Anaerobic microorganisms play important roles in different biotechnological processes. Their complex metabolism and special cultivation requirements have led to less isolated representatives in comparison to their aerobic counterparts. In view of that, the isolation and cultivation of anaerobic microorganisms is still a promising venture, and conventional methodologies as well as considerations and modifications are presented here. An insight into new methodologies and devices as well as a discussion on future perspectives for the cultivation of anaerobes may open the prospects of the exploitation of these microorganisms as a source for biotechnology.

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To Grow or Not to Grow: Isolation and Cultivation Procedures in the Genomic Age

Cited by 3 publications

References 108 publications

Exploring the Cultivable Ectocarpus Microbiome

Exploring the Cultivable Ectocarpus Microbiome

Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis

Isolation and Cultivation of Anaerobes

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