Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation offastidious soil bacteria as mixed microbial communities. Weisolated established microcolonies from these membranes by usingfluorescence viability staining and micromanipulation. Thisapproach facilitated the recovery of diverse, novel isolates,including the recalcitrant bacterium Leifsonia xyli, a plantpathogen that has never been isolated outside the host.

INTRODUCTION

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INTRODUCTION

The majority of bacterial species have never been recoveredin the laboratory (1, 14, 19, 24). In the last decade, novelcultivation approaches have successfully been used to recover"unculturables" from a diverse range of divisions (23, 25, 29).Most strategies have targeted marine environments (4, 23, 25,32), but soil offers the potential for the investigation ofvast numbers of undescribed species (20, 29). Rapid advanceshave been made toward culturing soil bacteria by reformulatingand diluting traditional media, extending incubation times,and using alternative gelling agents (8, 21, 29).

The soil substrate membrane system (SSMS) is a diffusion chamberapproach that uses extracts from the soil of interest as thegrowth substrate, thereby mimicking the environment under investigation(12). The SSMS enriches for slow-growing oligophiles, a proportionof which are subsequently capable of growing on complex media(23, 25, 27, 30, 32). However, the SSMS results in mixed microbialcommunities, with the consequent difficulty in isolation ofindividual microcolonies for further characterization (10).

Micromanipulation has been widely used for the isolation ofspecific cell morphotypes for downstream applications in moleculardiagnostics or proteomics (5, 15). This simple technology offersthe opportunity to select established microcolonies of a specificmorphotype from the SSMS when combined with fluorescence visualization(3, 11). Here, we have combined the SSMS, fluorescence viabilitystaining, and advanced micromanipulation for targeted isolationof viable, microcolony-forming soil bacteria.

Microcultivation using the SSMS.

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Microcultivation using the SSMS.

Soil (100 g) was collected 15 cm below the surface at MacquarieUniversity and passed through a 2-mm-mesh sieve. Three replicatesof the SSMS were set up as described previously (10, 12). TheSSMS consists of a tissue culture insert that supplies nutrientsin the form of soil extract to a polycarbonate membrane thatcontains the inoculum and also serves as a support for bacterialgrowth. After 7 days of incubation, mixed micro-CFUs (mCFUs)were visualized on a section of membrane by using SYBR green11 staining and epifluorescence microscopy.

Immunofluorescent viability staining of microcolonies.

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Immunofluorescent viability...

A LIVE/DEAD BacLight bacterial viability kit (L13152; Invitrogen,Mount Waverley, Victoria, Australia) was used as suggested bythe manufacturer. This kit supplies applicator strips with SYTO9and propidium iodide nucleic acid stains. For fluorescent staining,each membrane section was placed in an aliquot (100 µl)of a 1x concentration of BacLight staining solution for 10 minat room temperature. Membranes were washed twice by flotationon 200 µl 0.9% physiological saline for 5 min. Cells werevisualized using a BX61 fluorescence microscope (Olympus, NorthRyde, Australia) equipped with appropriate filters for SYTO9and propidium iodide visualization. To confirm that there wasno loss in cell viability after fluorescence staining, Escherichiacoli cells were used as a control. An aliquot (200 µl)of E. coli cells (1 x 10⁵/ml) was split, and one sample wasstained with a 1x concentration of BacLight staining solution.Following two washes, stained and unstained E. coli cells (1x 10⁵/ml) were plated onto nutrient agar and colony formationwas examined. No loss in E. coli viability was observed afterBacLight staining.

Advanced micromanipulation and subcultivation of microcolonies.

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Advanced micromanipulation and...

Individual microcolonies of 10 µm in diameter, containing50 to 1,000 cells, were targeted for isolation (Fig. 1). Forisolation using an LMD6000 laser microdissection system (Leica,Sydney, NSW, Australia), a BacLight-stained membrane sectionwas placed onto a glass slide. A section containing a live microcolonywas isolated using a UV pulse laser. Excised regions fell into0.5-ml Microfuge tubes containing 0.01x Ravan medium (12). Foradvanced micromanipulation, a BX51WIF epifluorescence microscope(Olympus, Sydney, Australia) equipped with a piezo-powered microdissector(Eppendorf, North Ryde, NSW, Australia) was used. A BacLight-stainedmembrane section was mounted onto 15 µl physiologicalsaline on a glass slide. Aliquots of physiological saline (5to 10 µl) were continually placed onto the edge of membranesto prevent them from drying out, with care being taken not todisturb the microcolonies. Live microcolonies were isolatedusing aspiration into sterile glass capillaries (TransferTips;Eppendorf) and transferred into a 96-well microtiter tray containing200 µl 0.01x Ravan medium. Cultures were incubated at22°C and subcultured every 7 days. After 3 subcultures,mCFUs were plated onto 0.01x Ravan agar. Pure colonies wereobtained and identified using 16S rRNA gene PCR and sequencing(10).

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FIG. 1. Fluorescence viability staining of microcolonies following microcultivation using the SSMS system. Live mCFUs (green) were easily differentiated from dead mCFUs (red) at a magnification of x400. Differentiation of both live and dead cells enables specific morphotypes of mCFUs to be targeted for micromanipulation in growth membranes for subsequent cultivation in oligophilic media.

Isolation of live microcolony-forming soil bacteria from growth membranes.

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Isolation of live microcolony...

After 7 days of incubation, live (green) and dead (red) mCFUscould be differentiated by epifluorescence microscopy (Fig.1). We attempted to isolate live microcolonies, but the lasermicrodissection caused damage to the membranes, resulting inno cell viability. By comparison, no detrimental effects onviability were observed following fluorescence staining andmicromanipulation in growth membranes. All isolated microculturessubsequently formed macroscopic colonies (Table 1).

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TABLE 1. Soil isolates recovered following fluorescence viability staining and advanced micromanipulation in the SSMS

Molecular analysis of pure cultures demonstrated that novelbacterial species were recovered, even within this pilot study(Table 1). 16S rRNA gene sequencing showed that four isolatesexhibited high levels of similarity to bacteria from the Actinobacteria(97 to 99% similarity). While Actinobacteria are well representedby pure cultures, there is a great diversity of undescribedspecies within this phylum in Australian arid soils, warrantingfurther investigation (18). Of particular interest was Leifsoniaxyli (mCFU65), a slow-growing, small coryneform and gram-positivephytopathogen thought to be widespread in the environment butwhich had, until these experiments, never been directly isolatedoutside the host cell (6, 7, 31). The remaining isolates weremost similar to bacteria within the Proteobacteria, includingmCFU17, a putative novel species most similar to Novosphingobiumsubterraneum (Table 1).

Fluorescence has been widely applied to the differentiationof active and dead bacterial cells (2, 16, 17, 22). When combinedwith epifluorescence microscopy or flow cytometry, analysisof single cells can be performed (9, 16, 28). The BacLight fluorescenceviability kit is a popular viability indicator, but the ongoingviability of bacterial cells following staining has not yetbeen reported. Here, the BacLight viability stain was used tosuccessfully differentiate active and dead mCFUs, with no detrimentaleffects on downstream cell viability. It is important to notethat the kit utilized here did not contain dimethyl sulfoxideas a cryoprotectant (13).

The SSMS is a powerful approach for the cultivation of recalcitrantsoil bacteria (10, 12, 26). However, the method has been limitedby the difficulty of isolating pure microcolonies (10). Whileflow cytometry offers the opportunity for high-throughout isolationof single cells, advanced microdissection combined with fluorescenceviability staining offers an alternative approach that facilitatesthe isolation of established intact microcolonies. Moreover,it offers the opportunity to select specific bacterial morphotypesof interest and is applicable to a wide range of microbial ecologystudies.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

Partial 16S rRNA gene sequences were deposited into the GenBankdatabase under the accession numbers FJ362389 (mCFU17), FJ362390(mCFU23), FJ362391 (mCFU54), FJ362392 (mCFU59), FJ362393 (mCFU61),FJ362394 (mCFU63), FJ362395 (mCFU65), and FJ362396 (mCFU68).

ACKNOWLEDGMENTS

This research was funded by a Macquarie University ResearchFellowship and a Macquarie University Research and DevelopmentGrant.

We thank Meredith Wallwork of the Adelaide Microscopy Unit atthe University of Adelaide for help with laser cutting microdissectionand Tristrom Winsley of UNSW for help with epifluorescence microscopy.

FOOTNOTES

* Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Randwick, NSW 2052, Australia. Phone: 612 9385 2032. Fax: 612 9385 1438. E-mail: b.ferrari{at}unsw.edu.au

Published ahead of print on 20 March 2009.

REFERENCES

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