Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia

Abstract

One of the fundamental methods for cultivating bacterial strains is conventional plating on solid media, but this method does not reveal the true diversity of the bacterial community. In this study, we develop a new technique and introduce a new device we term, I-tip. The I-tip was developed as an in situ cultivation device that allows microorganisms to enter and natural chemical compounds to diffuse, thereby permitting the microorganisms to grow utilizing chemical compounds in their natural environment. The new method was used to cultivate microorganisms from Baikalian sponges, and the results were compared with conventional plating as well as a pyrosequencing-based molecular survey. The I-tip method produced cultures of 34 species from five major phyla, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, 'missing' only two major phyla detected by pyrosequencing. Meanwhile, standard cultivation produced a smaller collection of 16 species from three major phyla, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, failing to detect over half of the major phyla registered by pyrosequencing. We conclude that the I-tip method can narrow the gap between cultivated and uncultivated species, at least for some of the more challenging microbial communities such as those associated with animal hosts.

Cultivation strategies for growth of uncultivated bacteria

“…Other inventive systems for the in-situ cultivation of fastidious bacteria in natural environments include the I-tip method [45] and the hollow-fiber membrane chamber (HFMC) device [46]. Schematic diagrams of these novel devices are included in the respective papers.…”

<i>In Situ</i> Cultivation Allows for Recovery of Bacterial Types Competitive in Their Natural Environment

“…A group of methods based on in situ cultivation is becoming prominent with the recent surge of new cultivation strategies (2, 3, 5, 7, 9–12). …”

Emerging Technologies for Gut Microbiome Research

“…While iChip was intended for drug discovery and identification of microbes in environmental samples, the technology has the potential for isolation of uncultivable gut microbes. A derivative of this technology (I-tip), which uses the same premise as iChip, trapping individual microbes within a gel that allows for passage of metabolites and nutrients, but at a smaller scale, has already been applied for in situ isolation of microbes from invertebrate organisms yielding isolates from 34 novel microbial species across 5 major phyla [61]. …”