In Situ Isolation and Culturing of Recalcitrant Soil Bacteria using an Isolation Chip (iChip)

Summary

This protocol describes the design and use of an isolation chip (iChip) for in situ cultivation of recalcitrant soil microbes in their natural environment. It also describes the in situ cultivation of antimicrobial-producing soil bacteria. The protocol can be adapted to recover bacteria from diverse niches with other desirable properties.

Abstract

Ecosystems harbor millions of environmental microbes, some of them capable of producing biotechnologically relevant products such as enzymes, vitamins, amino acids, organic acids, anti-parasitic agents, and antimicrobial agents. Most of these environmental bacteria are deemed "unculturable" as the laboratory culturing methods fail to meet their nutritional/environmental requirement. Moreover, as they coexist in nature, they may also be dependent on a nutrient/metabolite produced by another member of the microbial community. Bringing the "uncultured" microbial diversity into the culture will present an opportunity to explore the vast array of bioactive products they may encode. This article describes the implementation of the iChip technology, a multichannel diffusion chamber developed for a high-throughput, in situ culturing of the "unculturable" bacteria. Given the global menace of antimicrobial resistance, the article provides a detailed protocol for in situ cultivation and recovery of isolates from the microbial 'dark matter' capable of producing antimicrobial agents, as an example. This protocol describes the steps involved, right from the soil sample collection step to the recovery of isolates, and highlights the plausible factors that may influence the success of isolation. The use of this technology not only facilitates the isolation of otherwise "unculturable" bacteria for antibiotic discovery but also enables researchers to delve into the complex soil ecology, which can then be tapped into for a myriad of other applications.

Introduction

Rich repositories of previously unexplored microbes exist in natural environments such as soil, water bodies, and extreme environments. However, it is challenging to tap into this enormous biodiversity of microbes, as most of them cannot be easily cultured using traditional laboratory media and methods. The great plate count anomaly describes this phenomenon, highlighting the discordant numbers of microorganisms that can be visually observed versus those that are observed to grow in culture media¹. Bypassing this barrier and enabling access to the unculturable diversity of microbes and their products could possibly address various biotechnological needs.

For example, the development of antimicrobial resistance has become a threat to global public health. Major pharmaceutical corporations have been reluctant to pursue the discovery of new antimicrobials, causing the rate of discovery to dwindle in comparison to the rate of emergence of resistance to existing antibiotics^2,3. Thus, there is a critical need for the discovery of novel antimicrobials⁴. Since microbial communities have evolved together concomitantly or in competition⁵, they have developed the potential to produce novel chemical compounds that enhance their chances of survival. The enormous diversity of environmental microbes and their products has not been fully explored, as traditional laboratory culture-based methods often lead to the isolation of similar, known microbes. This is because conventional culture media and methods are biased toward the cultivation of human-relevant microbes and fail to mimic natural environments. Designing culture media to cultivate environmental microbes is challenging, as the growth of some species may require various nutrients, often produced by other microbes in the vicinity, and specific environmental conditions.

To address this issue and to cultivate environmental microbes in situ, diffusion chambers were first developed⁶. The diffusion chamber is a stainless-steel O-ring. The space within the ring is filled with agar seeded with an appropriate sample dilution. The top and bottom of the ring are then sealed with polycarbonate membranes, which allow the diffusion of soil-derived nutrients and metabolites while preventing the infiltration of other microbes into the chamber⁶. After seeding, the plate is placed in the environment from which the sample is collected to allow culture in situ. The diffusion chamber was highly efficient and resulted in the cultivation of numerous previously unculturable microbes. However, the O-ring design with agar became laborious and low-throughput, leaving open a problem space for further innovations⁶. Soon after, an isolation chip (iChip), which was capable of in situ culturing and simultaneous high-throughput recovery of pure cultures, was designed⁷. This design is conceptually the same as a diffusion chamber but consists of multiple channels where each channel acts as an independent diffusion chamber, allowing potential recovery of a single microcolony from each chamber⁸. The reimagination of the diffusion chamber design greatly enhanced the throughput of the technology and enabled the isolation and cultivation of a greater biodiversity of taxa from soil, marine water⁹, hot springs¹⁰, etc. Teixobactin, a recently described antibiotic, was discovered following the technology described above¹¹. It exhibited a novel mechanism of antimicrobial action and was found to be lethal against pathogens causing invasive infections, demonstrating that high-throughput isolation of microbes from microbial "dark matter" can be efficiently streamlined for the discovery of biotechnologically relevant natural products using this technology¹².

This article serves as a procedural guide for the effective implementation of this high-throughput technology for in situ cultivation and recovery of antimicrobial-producing recalcitrant bacteria from soil microbial "dark matter". The protocol outlines the design of an isolation chip, the collection of samples, sample preparation, seeding, incubation, and recovery. Though the presented protocol demonstrates in situ culture of microbes from soil, the same can be easily adapted for in situ cultivation of microbes from any environment. The high-throughput capabilities of this technology could increase the likelihood of discovering new microbes and utilizing their biosynthetic potential for various biotechnology applications.

Protocol

1. Design and 3D printing

1. Choose the design and size based on the type of sample, volume per diffusion chamber, dimensions of the membrane to be used, and the throughput required (number of diffusion chambers).
   NOTE: The thickness of the middle plate and the diameter of each channel determine the volume of each cylindrical diffusion chamber. The size depends on the number of individual diffusion chambers to be accommodated, and the dimensions of the membrane depend directly on the number of diffusion chambers covered (Figure 1).
2. Use the insert shapes option in any presentation software program to insert circles of the necessary dimensions to serve as the iChip. Insert smaller circles within to represent the channels. Arrange the smaller circles in the desired pattern to prepare a preliminary design for the top/bottom and the middle plate. Select the design and use the right-click menu to save the preliminary design as a picture.
3. Using the preliminary design as a template, begin creating a 2-dimensional (2D) design using computer-aided design (CAD) software.
   1. From the sketch menu, insert predefined shapes, predominantly circles, to create the basic design for the plates and the array of channels.
   2. Use the dimension option to set the actual dimensions of the plates and channels. Click on OK to finalize the design and then use the extrude function to convert the 2D design to a 3D model of the desired thickness.
   3. Finally, use the file menu Save to save a copy of the 3D model in the stereolithography (STL) file format, compatible for 3D printing.
4. Choose the material for printing based on the desired application, considering factors such as the preferred method of sterilization and the site of planting. For example, choose a temperature-resistant material if autoclaving will be used for sterilization or if they will be planted in a site where the temperature is high.
   NOTE: 3D printing for this experiment was performed using a ceramic-like material that can withstand high temperatures of up to 268 °C and was, therefore, autoclavable.
5. Choose an appropriate 3D printing method, depending on the dimensions (detail and resolution) and the material used.
   NOTE: 3D printing using stereolithography, an additive manufacturing method, can create intricate structures by curing a thermoset resin with an ultraviolet (UV) laser. Stereolithography can be used with several materials and permits excellent surface finishes and high-detail resolution.

2. Soil sample collection

1. Download and install a geocoding mobile application that allows identification of 3 m x 3 m sites around the globe by providing a unique 3-word address for the site from any mobile application store. Identify a site for soil sample collection. Then, open the geocoding mobile application to identify the site of interest and its unique three-word address.
2. Record the unique three-word address, date, time, depth at which the sample is to be collected, the weather conditions, plant and animal life nearby, exposure of the site, and the temperature of the site (surface and sub-surface at the desired depth).
   NOTE: Soil may contain pathogenic bacteria. Therefore, use appropriate personal protective equipment (gloves, lab coats, etc.) and follow protocols appropriate for biosafety level 2. Minimize contact to prevent sample contamination and exposure. Disinfect the work surface before and after handling soil samples.
3. Decontaminate the shovel by spraying it with 70% ethanol and wiping it clean. Decontaminate the polystyrene box, in which the soil is to be collected, by spraying it with 70% ethanol and wiping it clean. Allow any residual ethanol to dry out completely before proceeding further.
   NOTE: Although this protocol uses a polystyrene box to collect the sample and perform incubation, alternatives such as glass and metal containers may also be used if desired.
4. Remove any plant material, leaf litter, and/or stones from the surface to expose the topsoil. Decontaminate the shovel again as before, clear out the topsoil, and assess the soil at a depth of 3-6 inches subsurface. Use a ruler to measure the depth from the surface. Using the surface-sterilized shovel, collect 4-5 kg of soil from the desired depth into the polystyrene box.
5. Seal the box with a lid. If more than one sample is to be collected, decontaminate the shovel between two sampling sites to avoid cross-contamination. Once all samples have been collected, transport the samples back to the lab immediately.

3. Sample preparation for seeding

1. From the collected soil sample, weigh out 1 g of soil free from debris, rocks, and leaf litter into a sterile 50 mL centrifuge tube containing 10 mL of sterile saline.
   NOTE: Preserve some of the soil sample as glycerol stocks^13,14 at -80 °C to conserve the native microbial population for future requirements.
2. Mix the soil with the saline by vortexing for about 10 s to create a soil slurry. Leave the centrifuge tube undisturbed for 5 min to allow the large soil particles in the tube to settle down. Then, carefully transfer the supernatant soil suspension to a fresh test tube labelled as "S".
3. Using the soil suspension as the undiluted sample, perform serial dilution by transferring 1 mL of the sample into 9 mL of sterile saline in a fresh tube. Prepare dilutions from 10^-1 to 10^-5.
   NOTE: Plate count of the diluted soil suspensions on non-selective media can provide some insights into the density of bacteria in the sample, which can be used to choose the appropriate dilution for seeding. However, note that this is only an estimate of the culturable bacteria present in the sample.

4. Seeding and assembly

1. Inside a biosafety cabinet, surface decontaminate the top plate, bottom plate, and middle plate by soaking in 70% ethanol for 10 min. Rinse off the excess ethanol by washing the plates with sterile deionized water and allow them to air dry in a sterile Petri dish.
2. Place the middle plate in a sterile Petri dish, ready for seeding. Then, melt the SMS agar medium in a microwave, allow it to cool down, and transfer 22.5 mL of SMS agar at 45 °C into a 50 mL centrifuge tube.
3. To this, add 2.5 mL of an appropriate dilution of the soil sample and mix well by gentle inversion, while avoiding the introduction of air bubbles.
   NOTE: Choose an appropriate dilution for seeding. To achieve a final seeding density as in the 10^-4 dilution, choose the 10^-3 suspension as the sample will be further diluted 10-fold in the medium used for seeding.
   1. Choose the seeding dilution to immobilize 1 cell per chamber, which can then develop into a colony in situ.
      NOTE: Distilled water may also be used in place of SMS medium to mimic the soil environment and to permit the growth of microorganisms that grow only under such conditions. Optionally, antifungal agents like cycloheximide may also be added to the seeding medium to avoid fungal growth.
4. Slowly pour 3-4 mL of the seeded SMS agar medium over the middle plate, covering the entire surface. Then allow the SMS agar medium to set for about 5-10 min. Check if the SMS agar medium is set. Then, with a sterile razor blade, scrape off the excess SMS agar from the surface of the plate, leaving behind the agar plugs in the individual diffusion chambers.
5. After removing the protective sheet from one of the 0.03 µm polycarbonate membrane filter, place it carefully on the inside of the top plate using sterile forceps. Then, place another 0.03 µm polycarbonate membrane filter on the exposed side of the seeded middle plate using a sterile forceps.
6. Place the top and bottom plates, sandwiching the middle plate and the polycarbonate membranes, taking care to align the screw holes in all the plates, to complete the assembly (Figure 1B). Tighten the nuts and bolts to secure the assembly.
   NOTE: It is good practice to assemble a control iChip with only sterile SMS agar, without seeding, in parallel and plant it in the soil along with the seeded one to verify the seal. This would rule out the possible entry of microbes from the environment and act as a negative control.

5. In situ cultivation and recovery

1. Create a depression larger than the size of the iChip in the soil collected in the polystyrene box using a sterile shovel. Plant the assembly vertically within the depression and pack with soil firmly, ensuring that all surfaces are completely covered. Then allow for in situ culture for the desired duration from 2-4 weeks. During incubation, use sterile distilled water to moisten the soil periodically to prevent the seeded SMS agar plugs in the diffusion chambers from drying out.
   NOTE: Placing the assembly in a vertical position prevents continuous water seepage through the diffusion chambers during regular soil moistening, as may occur if it is planted horizontally.
2. After the desired incubation, gently dislodge the soil around the planted iChip to recover it. Then, wash it with sterile saline to remove adhering soil particles and transfer it to a biosafety cabinet for further processing.
3. Unfasten the nuts and bolts from the assembly. Then, carefully disassemble it. Using sterile forceps, remove the polycarbonate membranes and transfer the middle plate into the prepared Petri dish.
4. Use sterile wooden applicators to recover the individual agar plugs within each diffusion chamber. Inoculate the agar plugs into appropriate media like SMS agar or 0.1x tryptic soy broth agar to attempt laboratory cultivation.
   NOTE: Cultivated isolates can then be screened for antimicrobial production by routine methods such as agar overlay¹⁵.
5. Alternatively, stain and image the recovered agar plugs containing in situ cultivated, recalcitrant soil bacteria to confirm the growth of microcolonies under in situ conditions.
   NOTE: To enhance the sensitivity of detection, the crushed agar plug can also be stained using fluorescent dyes that can stain bacteria, followed by fluorescence microscopy.

Results

The iChip used in this protocol was designed, 3D-printed, and assembled as shown in Figure 1. The overview of the protocol described is shown in Figure 2. The soil sample used for the experiment was collected from IIT Jodhpur campus in Rajasthan, India. The unique three-word address of the location was 'communicate.supervising.recurrence.' The chosen location was a semi-arid zone with a temperature of 40 °C on the day of sampling. The soil sample was collected i...

Discussion

The numerous unique habitats on Earth host distinct microbial communities that represent enormous diversity. This remains unexplored due to limitations in the provision of appropriate growth conditions required for their cultivation. Some bacteria depend on the metabolites released by others for their survival and hence are unable to survive independently in laboratory conditions. Additionally, bacteria also play a crucial role in orchestrating biogeochemical cycles, which cannot be emulated in the laboratory environment...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D Printer	Protolabs, USA	1631-833	3D printing service provider
Agarose	Himedia, India	MB002	Media component
Casamino Acids	Sisco, India	68806	Media component
Casein Digest	ASES, India	C5890	Media component
Polycarbonate membrane	Sterlitech, USA	PCT00347100	Consumable
Potato Starch	Himedia, India	GM403	Media component
PowerPoint	Microsoft, USA	N/A	Used for creation of iChip components preliminary design
PTC Creo	PTC, USA	N/A	Used for 3D computer aided modeling of iChip components
Tryptic Soy Broth	Himedia, India	M011	Laboratory medium
What3Words	What3Words, UK	N/A	Used for recording coordinates of the sampling location