Construction of the oak seedling microcosms

rm(list = ls())
graph_par <- c(las=1, bty="l", col.axis="gray30")

Objective

This is a step by step guide to assemble 100 microcosms as described in Abdelfattah et al. (2021). The patent for the device can be find on Google Patents.

Material for 100 microcosms

• 200 x Magenta Vessels GA-7 (Merck, ref. V8505-100EA)
• 100 x conical Falcon tubes 50 mL (Greiner bio-one, ref. 227261)
• 100 x Membrane Filter, Mixed Cellulose Ester, 47 mm dia., 0.22 µm (Kinesis, SLS ref. FIL1023)
• a power drill
• 1 x 25 mm whole saw for power drills (facultative)
• 1 x 29 mm whole saw for power drills
• 1 small saw
• 1 small round file
• 240 grit sand paper
• a scalper
• some cardboard to make a template
• a sharpie
• DIY gloves (mendatory!)
• a dust mask
• All purpose silicone sealant
• Spatula to apply silicone (ideally curved at the end)

Note

I initially tried some plastic superglue but it did not survive well the autoclave. Silicone is a bit better although not perfect, and has the advantage of being easily removed if we need to replace the filter

Figure 1: Overview of the tools needed for constructing the microcosms (note that I did not use this glue in the end)

Microcosm construction

Figure 2: Overview of the microcosm construction. 1. magenta boxes 2. Magenta boxes lids 3. Hole of 28 mm dia. 4. Hole of 25 mm dia. 5. 50 mL tube cut at the 25 mL mark 6. Cut of the bottom of the tube at 2 mm from the edge (Adapted from pattent SE543621)

1. Using some cardbord, make a template to place the center of the hole in the top magenta box (Figure 3).

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Figure 3: Creation of a template to position the center of the filter hole. 1. Create a corner with some cardbord and tape, place a Magenta box in the corner and trace the contour of the box on the cardbord. Find the position of the center of the filter hole and make a small hole in the cardbord to be able to get the tip of a sharpie through it. 2. Position back the Magenta box in the template. 3. Flip the box and template around and mark the center of the filter hole using a sharipe

2. Drill a 25 mm (or 29 mm depending of filter size) hole in the Magenta box as shown in Figure 4 (only 100 out of the 200 boxes should be drilled). I found that high speed but medium pressure on the drill works the best. Make sure to use gloves as the cut plastic is sharp

3. Smooth the edges of the whole with the round file

Figure 4: Whole drilling and filing

5. Using the whole saw once again, drill a 28 mm dia. whole in the 200 Magenta box lids. The center of the lid is already obvious and can be used as a guide. Use a scalpel to smooth the edges of the whole.

6. Cut the 100 tubes at the bottom and the middle following the red lines in Figure 5

Figure 5: Tubes should be cut on the 25 mL mark and around 2 mm away from the tip

Note

I originally cut the tubes on the 25 mL line, but shorter can be done if the seedlings are not expected to grow a lot

4. Glue the 0.22 µm filter on the whole in the upper box using silicon sealant. Follow the manufacturer’s instructions for the silicon, but usually it takes 24h to completely cure.

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Figure 6: Steps for glueing the filter to the top Magenta box. 1. Overview of the tools needed. 2. Place a ring of silicone around the filter hole. 2. Place the filter on the silicone, press and let set for 24 h

Microcosms sterilisation and assembly

All parts of the microcosms can now be autoclaved. Microcosms then need to be be assembled under the laminar flow hood so they remain steriles.

1. The two lids of the magenta box are assembled with lid top facing each other and alining the wholes. They are secured together with a piece of parafilm (50 x 100 mm or two squares folded in half along the long edge, Figure 7 1-3)
2. A piece of parafilm (50 x 100 mm or two squares) can be folded twice and wrapped around the top of the tube to make it fit into the wholes of the magenta lids (Figure 7 4-6). The tube should stay in place and not drop through the wholes

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Figure 7: Legend

3. Assemble the bottom Magenta box (without filter), middle section (Magenta lids and tube) and tom Magenta box (with filter) as shown in Figure 8. The microcosm is now ready to be filled with sterile type II water or other substrate.

Figure 8: Fully assembled microcosm

References

Abdelfattah, Ahmed, Michael Wisniewski, Leonardo Schena, and Ayco J. M. Tack. 2021. “Experimental Evidence of Microbial Inheritance in Plants and Transmission Routes from Seed to Phyllosphere and Root.” Environmental Microbiology 23 (4): 2199–2214. https://doi.org/10.1111/1462-2920.15392.