Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria

Abstract

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.

FIG. 1.

Preparation of the microcultivation SSMS. A garden soil slurry is prepared in an inverted tissue culture insert containing a fixed anopore membrane, which supplies nutrients for soil bacteria growing on a PC membrane (PCM) while acting as a barrier to bacterial contamination. The PC membrane is inoculated with a soil filtrate and is placed on the inverted TCI, and growth is monitored.

FIG. 2.

Microcolony cultivation of bacteria grown on PC membranes using garden soil slurry as the culture medium. (A) Fluorescent staining (SYBR Green II) reveals several microcolony morphotypes growing in coculture after 10 days of incubation. (B) Presence of a common morphotype after 10 days of incubation.

FIG. 3.

Multicolor staining with FISH probes specific for TM7 bacteria (red) identifies two TM7 morphotypes that are stained by the eubacterial probe (green). (A) Faster-growing TM7 morphotype consists of small rods and several hundred cells per microcolony. (B) Larger TM7 microcolony consists of less than 50 long rods that are up to 15 μm long and is typical of filamentous TM7 lineage bacteria.