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

Rich repositories of microbes exist in diverse natural environments, such as soil, water bodies, and extreme environments. However, tapping into this enormous biodiversity is largely limited by laboratory methods and media. By passing this culture barrier and tapping into this biodiversity has the potential to address various biotechnological needs, as well as solve global challenges.

For example, the development of anti-microbial resistance has become set to global public health. Soil bacteria that are difficult to cultivate in laboratory may produce novel bioactive compounds with potent anti-microbial activity that could be valuable in addressing global challenge of anti-microbial systems. The enormous variety of environmental microbes and their product have not been explored as traditional laboratory culture-based technologies often leads to the isolation of similar known microbe. The goal of this experiment is to demonstrate high throughput culture of recalcitrant soil bacteria in-situ using IChip technology. In this protocol, we'll demonstrate the construction and use of this IChip technology that can be used for high throughput in-situ cultivation of difficult to cultivate bacteria and recovery of anti-microbial producing bacteria as an application of this technology.

[Presenter] To begin, create a preliminary design of the top, bottom, and middle plates of the isolation chip in a presentation software. Use the insert shape option in the presentation software to insert circles of necessity dimensions to serve as the IChip. Insert smaller circles within to represent IChip channels. Arrange the smaller circles in the desired pattern to prepare a preliminary design for the top, bottom, and the middle plate of the IChip. Select the design and use the right click menu to save the preliminary design as a picture. Using the preliminary design as a template, begin creating a two-dimensional design using a computer-aided design software. From the sketch menu, insert predefined shapes, predominantly circles to create the basic design for the IChip plates and the area of channels. Use the dimension option to set the actual dimensions of the plates and channels. Click on okay to finalize the design and then use the extrude function to convert the 2D design to a 3D model of desired thickness. Finally, use the file menu to save a copy of the 3D model in the still lithography file format compatible for 3D printing.

[Presenter] Collect all essential items that will be required for soil sample collection and reach the site from where the sample is to be collected. Open the geocoding mobile application and find the unique three-word address of the sampling site and record it. Next, record relevant sample metadata, such as the temperature, date, time, depth of sampling, weather, and site exposure to the elements. Spray a shovel and the polystyrene box in which the IChip is to be planted with 70% ethanol. Wipe them clean with tissue paper and allow the ethanol to air dry completely. Then use the decontaminated shovel to clear out any surface debris, such as the leaf litter or rocks to expose the top soil. Decontaminate the shovel again as before. Using the shovel, access the soil at a depth of three to six ingest subsurface, and transfer around four to five kg of soil from a uniform depth into the polystyrene box. The box should be approximately 75% full. Transport the collected soil back to the laboratory immediately. From the collected soil sample, weight out one gram of soil free from debris, rock, and leaf litter, into a sterile 50-mil centrifuge tube containing 10 ml of sterile saline. Mix the soil with the saline while vortexing for about 10 seconds to create a soil slurry. Leave the centrifuge tube undisturbed for five minute to allow the large soil particles in the tube to settle down. Then carefully transfer the supernatant soil suspension to a fresh test tube labeled as S. Using the soil suspension as the undiluted sample, perform zero dilution by transferring one ml of the sample into nine ml of sterile saline in a fresh tube. Prepare dilution from 10 is to power minus one to 10 is to power minus five. Inside a biosafety cabinet, surface decontaminate the top plate, bottom plate, and middle plate, of the IChip by soaking in 70% ethanol for 10 minutes. Rinse off the excess ethanol by washing the IChip components with sterile the deionized water, and allow them to air dry a sterile Petri dish. Place the middle plate of the IChip in a sterile Petri dish ready for seeding. Melt the SMS agar medium in a microwave. Allow it to cool down, and then transfer 22.5 ml of SMS agar at 45 degrees into a sterile 50 ml centrifuge tube. To this, add 2.5 ml of an appropriate dilution of soil sample and mix well by gentle inversion while avoiding introduction of air bubbles. Slowly pour around three to four ml of the seeded SMS agar medium over the middle plate of the IChip covering the entire surface. Then allow the SMS agar medium to set for about five to 10 minutes. Check if the SMS agar medium is set. Then with the sterile razor blade, scrape off the excess SMS agar from the surface of the IChip metal plate leaving behind the agar plugs in the individual diffusion chambers. After removing the protective sheet from one of the 0.03 micrometer polycarbonate membrane filter, place it carefully on the inside of the top blade of the IChip using a sterile forceps. Then place another 0.03 micrometer polycarbonate membrane filter on the exposed side of the seeded metal plate using a sterile forceps. Place the top and bottom plates of the IChip, sandwiching the middle plate, and the polycarbonate membranes, taking care to align these screw holes in all the plates to complete the IChip assembly. Tightly fasten the nuts and bolts to secure the IChip assembly. Create a depression larger than the size of the IChip in the soil collector in the polystyrene box using a sterile shovel. Plant the IChip assembly vertically within the depression and pack with soil firmly ensuring that all surfaces of the IChip are completely covered. Then allow for in-situ in culture for the desired duration from two to four weeks. During incubation, use sterile distilled water to moisten the soil surroundings, the planted IChip, periodically to prevent the seeded SMS agar plugs in the diffusion chamber from drying out. After desired incubation, gently dislodge the soil around the planted IChip to recover it. Then wash the IChip with sterile saline to remove adhering soil particles and transfer it within a biosafety cabinet for further processing. Unfasten the nuts and bolt from the IChip assembly and carefully disassemble the IChip. Using a sterile forcep, remove the polycarbonate membranes and transfer the middle plate of the IChip into the prepared Petri dish. Use sterile wooden applicator 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. Cultivated isolates can then be screened for anti-microbial production by routine methods, such as agar overlay using tester strains. Here we show representative screening results for 12 cultures, IC-1 through 12 obtained from IChip agar plugs against Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii B8342. Note that cultures IC-6 and IC-8 show activity against gram-positive and gram-negative tester strains respectively. Alternatively, stain and image the recovered agar plugs containing in-situ cultivated recalcitrant soil bacteria to confirm growth of micro colonies under in-situ conditions. Using this approach, it is possible to recover recalcitrant soil bacteria in-situ in a high throughput manner. The 3D-printed IChip can accommodate 156 agar plugs. However, it is necessary to choose the right dilution while seeding to ensure one bacterial cell is seeded into each channel of the IChip. The required bacteria can then be screened for desired bioactivities, such as metabolite production, enzyme production, biopolymer production, et cetera, if they can be cultured in the lab conditions. However, it is also likely that some bacteria may not grow in lab conditions, and in such cases, we may have to adopt to metagenomic approaches to realize the microbial presence and to access their biosynthetic potential. The described method is very easy and can be easily used for bioprospecting microbes in diverse environmental conditions with minor modifications.