This research was funded by Plymouth University&#39;s School of Humanities and Performing Arts. The authors would like to acknowledge Functionalize for supplying samples of their conductive PLA.

1. Fabrication of a 3D Printed Receptacle
<ol>
	<li>Chambers, lids, and base
	<ol>
		<li>Load a 3D printer with High-Impact Polystyrene (HIPS) by using the printer interface to set the print bed temperature to 85 &#176;C and the extruder to 230 &#176;C. When the temperatures are reached, loosen the idler arm, insert the filament, and push down until it starts to extrude out of the hot end. Then, retighten the filament idler arm and remove the extruded material.</li>
		<li>Import the 3D receptacle STL model file into a 3D printing slicing software, which can normally be achieved by navigating to the file tab and selecting the import/open options (Figure 2).</li>
		<li>If slicing software offers high and low quality print settings, select high quality while also ensuring that the correct material profile is selected. 
		NOTE: If printing several receptacles in one run, make sure that the software is set to print each object one at a time. If this step is skipped, the print quality may be reduced, which will likely cause tolerance issues when fitting the parts together.</li>
		<li>Once the printing is complete, wait until the print bed temperature is below 50 &#176;C to remove the parts.</li>
		<li>Using a thin wire brush, gently clear the electrode socket of any imperfections that may cause obstructions when fitting the chamber with an electrode.</li>
	</ol>
	</li>
	<li>Electrodes
	<ol>
		<li>Replace the HIPS filament for a cleaning filament and run plenty of the material through the print head.</li>
		<li>Load the printer with an electrically conductive polylactic acid (PLA) filament that has a volume resistivity of 0.75 &#937;-cm or less.</li>
		<li>Set the print bed temperature to 60 &#176;C and the extruder to 230 &#176;C (see step 1.1.1 for guidance).</li>
		<li>When the temperatures are reached, extrude several centimeters of the filament through the print head. This process will help ensure that all particulates from previous sessions are removed.</li>
		<li>Using a 3D printing slicing software, load the electrode STL file (Figure 3).</li>
		<li>In the print settings, specify the following: Layer height = 0.16 mm, Shell thickness = 1.7 mm, Bottom/Top thickness = 0.74 mm, Fill density = 100% (Figure 4).</li>
		<li>If printing several electrodes in one run, set the printer to print one at a time.</li>
		<li>Once printed, leave the electrodes on the print bed until they have cooled to room temperature. This ensures the part does not become warped and misshaped.</li>
	</ol>
	</li>
	<li>Receptacle assembly
	<ol>
		<li>Slot an electrode into each of the two chambers. If step 1.1.5 has been completed correctly, the electrodes should go into the chambers without much force.</li>
		<li>Using a sharp scalpel, cut a 10 mm piece of Polyvinyl chloride (PVC) tubing (4 mm inner diameter and 6 mm outer diameter) taking care to ensure that each end is cut straight and cleanly.</li>
		<li>Gently ease each end of the 10 mm PVC tubing over the rim of the two electrodes.</li>
		<li>Once connected, the two chambers clip into the base.</li>
	</ol>
	</li>
</ol>
2. Receptacle Preparation and P. polycephalum Inoculation
<ol>
	<li>2% agar medium preparation
	<ol>
		<li>Put 2 g of non-nutrient microbiological agar powder into a 250 mL glass bottle.</li>
		<li>Add 100 mL of deionized water and mix well.</li>
		<li>Autoclave the bottle for 12 - 15 min at 121 &#176;C or place in a boiling water bath for 15 - 20 min.</li>
	</ol>
	</li>
	<li>Setting the agar substrate into the receptacle&#39;s chambers
	<ol>
		<li>Melt the agar using a water bath or microwave.</li>
		<li>Fill a 2 mL pipette with molten agar.</li>
		<li>Fill each of the receptacle&#39;s chambers by hovering the nib of the pipette approximately 5 mm above the internal base and slowly filling the wells up to the bottom of the connecting tube hole.</li>
		<li>Immediately after filling the wells, place a lid on each of the chambers and set the receptacle aside until the agar has set and reached room temperature.</li>
	</ol>
	</li>
	<li>P. polycephalum inoculation
	<ol>
		<li>Place an oat flake in each of the two chambers.</li>
		<li>Remove one 2 mL blob of pseudopods from a starved (approximately 12 h) culture of plasmodium and place it in one of the two chambers. To promote speedy growth, try to take the protoplasm from the most active anterior of the organism.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Chua, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memristor-The+Missing+Circuit+Element.">Memristor-The Missing Circuit Element.</a> IEEE Transactions on Circuit Theory. 18 (5), 507-519 (1971).</li><li>Trefzer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memristor+in+a+Nutshell.">Memristor in a Nutshell.</a> Guide to Unconventional Computing for Music. , 159-180 (2017).</li><li>Strukov, D. B., Snider, G. S., Stewart, D. R., Williams, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+missing+memristor+found.">The missing memristor found.</a> Nature. 453 (7191), 80-83 (2008).</li><li>Howard, G., Gale, E., Bull, L., De Lacy Costello, B., Adamatzky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+plastic+learning+in+spiking+networks+via+memristive+connections.">Evolution of plastic learning in spiking networks via memristive connections.</a> IEEE Transactions on Evolutionary Computation. 16 (5), 711-729 (2012).</li><li>Sah, M. P., Kim, H., Chua, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brains+are+made+of+memristors.">Brains are made of memristors.</a> IEEE Circuits and Systems Magazine. 14 (1), 12-36 (2014).</li><li>Miranda, E. R., Kirke, A., Braund, E., Antoine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+Unconventional+Computing+for+Sound+and+Music.">On Unconventional Computing for Sound and Music.</a> Guide to Unconventional Computing for Music. , 23-62 (2017).</li><li>Volkov, A. G., Tucket, C., Reedus, J., Volkova, M. I., Markin, V. S., Chua, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memristors+in+plants.">Memristors in plants.</a> Plant Signal Behav. 9 (2), 37-41 (2014).</li><li>Grimnes, S., L&#252;tken, C. A., Martinsen, &. #. 2. 1. 6. ;. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memristive+properties+of+electro-osmosis+in+human+sweat+ducts.">Memristive properties of electro-osmosis in human sweat ducts.</a> World Congress on Medical Physics and Biomedical Engineering, September 7-12, 2009, Munich, Germany. , 696-698 (2009).</li><li>Gale, E., Adamatzky, A., de Lacy Costello, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slime+Mould+Memristors.">Slime Mould Memristors.</a> BioNanoScience. 5 (1), (2014).</li><li>Braund, E., Sparrow, R., Miranda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physarum-based+memristors+for+computer+music.">Physarum-based memristors for computer music.</a> Advances in Physarum Machines. , 755-775 (2016).</li><li>Daniel, J. W., Rusch, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pure+culture+of+Physarum+polycephalum+on+a+partially+defined+soluble+medium.">The pure culture of Physarum polycephalum on a partially defined soluble medium.</a> Journal of General Microbiology. 25 (1901), 47-59 (1961).</li><li>Adamatzky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physarum+wires:+Self-growing+self-repairing+smart+wires+made+from+slime+mould.">Physarum wires: Self-growing self-repairing smart wires made from slime mould.</a> Biomedical Engineering Letters. 3 (4), 232-241 (2013).</li><li>Tarabella, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+living/organic+electrochemical+transistor+based+on+the+Physarum+polycephalum+cell+endowed+with+both+sensing+and+memristive+properties.">A hybrid living/organic electrochemical transistor based on the Physarum polycephalum cell endowed with both sensing and memristive properties.</a> Chemical Science. 6 (5), 2859-2868 (2015).</li><li>Braund, E., Miranda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+Building+Practical+Biocomputers+for+Real-world+Applications:+Receptacles+for+Culturing+Slime+Mould+Memristors+and+Component+Standardisation.">On Building Practical Biocomputers for Real-world Applications: Receptacles for Culturing Slime Mould Memristors and Component Standardisation.</a> Journal of Bionic Engineering. 14 (1), 151-162 (2017).</li><li>Walter, X. A., Horsfield, I., Mayne, R., Ieropoulos, I. A., Adamatzky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+hybrid+circuits+exploiting+thermistive+properties+of+slime+mould.">On hybrid circuits exploiting thermistive properties of slime mould.</a> Scientific reports. 6, (2016).</li><li>Romeo, A., Dimonte, A., Tarabella, G., D'Angelo, P., Erokhin, V., Iannotta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bio-inspired+memory+device+based+on+interfacing+Physarum+polycephalum+with+an+organic+semiconductor.">A bio-inspired memory device based on interfacing Physarum polycephalum with an organic semiconductor.</a> APL materials. 3 (1), (2015).</li><li>Whiting, J. G., de Lacy Costello, B., Adamatzky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+function+of+protoplasmic+tubes+of+Physarum+polycephalum.">Transfer function of protoplasmic tubes of Physarum polycephalum.</a> Biosystems. 128, 48-51 (2015).</li></ol>

The authors have nothing to disclose.

This paper presented a method for growing memristors out of the myxomycete P. polycephalum. The organism is grown inside 3D printed receptacles that were designed to overcome some of the constraints that are associated with implementing the bio-memristors. Such limitations include setup time, sample growth time, and lack of standardization for sample-to-sample growth conditions and electrical observations.
Our receptacle was first revealed in 2015 in the printed publicity material for the Peninsula Arts Contemporary Music Festival 2016 (PACMF) and respective website15. Here, our technology was used to develop a hybrid hardware-bioware interactive music system that was capable of generating musical accompaniments to a live musician. In reference14, we reported on the extensive testing of our receptacle and compared the results against previous approaches9,10. Following these developments, another group of researchers subsequently explored creating growth environments to study the organism&#39;s thermistive properties16, but these are not the same as memristive properties. There have, however, only been two other attempts at developing a controlled approach to implementing P. polycephalum memristors13,17. In these experiments, wells were made from a gel-like biocompatible elastomer material called polydimethylsiloxane (PDMS), and electrodes were created using various metals or poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS). Although these materials are routinely used in electronics, microfluidics, and bionic engineering, they are expensive and require some expertise to use. For example, PEDOT:PSS needs spin-coating and doping to improve its conductivity. Therefore, the techniques are out of reach for people who do not have access to specialist resources. The receptacles presented in this article use methods and materials that are easily accessed and inexpensive. Furthermore, the design provides a hospitable environment for the plasmodium to habitat, which is in contrast to the other P. polycephalum memristor prototypes where no attempt was made to keep the cell alive for any duration of time.
Until now, it has been difficult to obtain consistent I-V measurements using previous methods for culturing the organism on Petri dishes (Figure 5, left). Our methods improved this scenario significantly (Figure 6). The results of our receptacle&#39;s testing have demonstrated that the design has decreased growth time, increased lifespan, standardized component responses, and created a protected microenvironment to encapsulate the organism. Furthermore, the device provides feasible means of integrating the organism as a component of an electrical scheme.
The presented method alleviates a number of issues related to harnessing P. polycephalum memristors within electrical systems. There are, however, limitations that require further research and development. Firstly, condensation can gather on the connecting tube&#39;s inner surface if the receptacles are subjected to a quick change in temperature or if a high voltage is applied for long durations. The latter is due to the organism&#39;s high resistance causing electrical energy to be transferred into heat. If significant, the condensation can create a low resistant pathway between the two electrodes at either end of the connecting tube. This limitation can be managed effectively by ensuring that the memristors are not overloaded. Secondly, the overall resistance of memristors produced using the presented method can vary from component-to-component. Such a phenomenon may be a result of the approach not restricting the outer diameter of the protoplasmic tube. Consequently, users may need to incorporate a calibration process into their application of the memristors.
Thanks to this methodology, we can now begin to study the biological processes that are causing memristive observations in P. polycephalum. It is likely that such processes have dynamic parameters that we might be able to exploit to augment the element&#39;s usage. We have begun running some preliminary experiments where extracellular ion concentrations are altered to review if voltage-gated ion channels play a role in memristance.
The presented receptacles were designed solely for implementing P. polycephalum memristors. These devices are likely, however, to have uses beyond that of implementing a single component. For example, in references12,18, the protoplasmic tube was studied as a self-assembling and self-repairing biological wire. In both these investigations, the researchers expressed that further work was needed to establish methods of growing the protoplasmic tube according to a scheme. The receptacles put forward in this paper provide a method of delineating the production of the tube between two, or potentially more, points. Figure 7 shows two photographs illustrating that the receptacles can be used to grow healthy tubes at lengths more than 100 mm. In reference18, the transfer function of the protoplasmic tube was investigated. Results from this investigation indicated that the agar required to grow the tubes may cause an issue if the organism was to be integrated into an electrical system. This is due to the substrate&#39;s capacitance. The receptacles presented here still require agar to keep humidity high. However, with small changes to the receptacle&#39;s design, it is possible to create a detachable tube. This set up may allow for the tube to be disconnected from the chambers once growth is complete and clipped into an electrical system. Furthermore, once the tube&#39;s health begins to deteriorate, it could be reconnected to new chambers for food and respite until it has repaired itself and can be used again. Figure 8 shows a photo of long tubes that have been disconnected from the chambers. Future research is needed to investigate the protoplasmic tube&#39;s electrical properties without the agar and when grown at lengths using the presented method.

Today&#8217;s computers are built using the three two-terminal fundamental passive circuit elements: the capacitor, the resistor, and the inductor. Passive elements are only capable of dissipating or storing energy, not generating it. These elements were established in the 18th and 19th century and are linked through Maxwell&#8217;s equations. We define each of these three circuit components in terms of their relationship between two of the four circuit variables namely, current (I), voltage (V), charge (Q), and flux-linkage (&#966;). The charge is the time integral of the current and Faraday&#8217;s law defines the voltage as the time integral of the flux. Thus, a capacitor is defined by a relationship between voltage and charge, a resistor is defined by a relationship between voltage and current, and the inductor is defined by a relationship between flux and current. For well over a century, these elements were a cornerstone of electronics. However, they only represent three of the possible four relationship pairs between the circuit variables, leaving flux-linkage and charge unlinked. In 1971, Leon Chua published a paper1 where he postulated that there was a missing fourth element that linked the remaining two variables, which he called the memristor. The memristor can be described as a resistor that remembers its history, hence the contraction &#39;memory resistor.&#39;&#160;This element functions by altering its resistance according to the magnitude of the previously applied voltage and its duration. Moreover, the memristor retains its last resistance state once the voltage is no longer applied. Unlike the capacitor, resistor, and inductor, the memristor&#39;s behaviour is nonlinear, which is evident in its I-V profile where a pinched hysteresis loop is formed under an AC voltage. This loop takes the form of a Lissajous figure containing two perpendicular oscillations of high and low resistant states. Before Chua&#8217;s formalized memristance theory, other researchers had reported on memory resistance effects at certain frequencies when experimenting with materials such as polymers and metal oxides, along with developing electrical devices at the micrometer scale2. However, in many cases, these effects were considered undesirable. It took almost forty years for Chua&#8217;s formalization to be connected to a physical device and for researchers to begin developing methods of exploiting memristive effects. A team at the HP Laboratories succeeded in fabricating a memristive device in 20083 that ignited huge interest in the element.&#160;
Computer scientists have a keen interest in the memristor due to it being credited as the first element to combine processing and memory abilities in a single unit. It also displays behaviors that are analogous to certain neurological processes such as Spike-Timing-Dependent Plasticity (STDP)4, to name but one. Such behaviors are giving rise to perspectives of building brain-like computing technologies that relinquish the distinction between memory and central processing unit (or CPU)5. In contrast to the popular approaches to developing memristors (using TiO2, for example), our ambition is to develop an organic bio-memristor. Furthermore, we are interested in how this component may provide means of exploring paradigms beyond conventional approaches to engineering computing devices; e.g., creative applications in the field of Computer Music6.
Memristance is an effect that researchers have recently found throughout a range of biological systems. For example, memristive properties have been observed in aloe vera plants7 and human skin8, to cite but two. These discoveries indicate that it may be possible to implement processing and memory devices on biological substrates. Harnessing organic systems within technology may allow us to explore exciting concepts such as self-assembly, self-repair, low environmental impact, and self-powering. Before we can investigate these opportunities however, several challenges need to be addressed. Many of the biological systems that have memristive properties have significant constraints that limit their viability as an actual electronic component. For example, an aloe vera leaf7 needs light, has a limited lifespan, and would be difficult to integrate into a circuit. Furthermore, several other in vivo memristive phenomena, such as human sweat ducts8, are not currently feasible options for developing systems for use outside of the laboratory and in everyday electronic systems. However, of all the memristive phenomena, there is one potential candidate: P. polycephalum.
The plasmodium of P. polycephalum is an amorphous unicellular system that has been discovered to act as a memristive component9,10. This organism is an ideal candidate for research in hybrid hardware-wetware electronics for a number of reasons. Firstly, the organism is non-pathogenic, macroscopic, and requires no specialist equipment use, which renders the plasmodium accessible to engineers and non-biologists. Secondly, the cell is amorphous, forms networks of wire-like veins, and will grow on most substrates (Figure 1). These properties allow the cell&#39;s morphology to be easily delineated to conform to a conventional electrical scheme. There is also research demonstrating that the plasmodium can live for over four years11, and that its veins can act as self-repairing conductive pathways12. Several laboratory studies have confirmed the organism&#39;s memristive abilities9,10,13 and now the time is ripe to explore its potential.
The idea of using P. polycephalum memristors is relatively new. As a result, there are no established standards for measuring and observing its electrical properties. Such a lack of uniformity in experimental procedures within the same research group and between groups may be the reason there are inconsistencies amongst published results9,10. It is likely that such variation is most prominent in sample growth conditions and handling. Thus, we need to establish methods for producing and testing P. polycephalum memristors where factors that might cause errors are better controlled and monitored.&#160; Furthermore, we need to create methods of implementing P. polycephalum memristors that allow for stable and easy integration into electrical systems.
The method presented in this paper provides a platform for exploration of practical applications of P. polycephalum memristors by providing means of incorporating the organism as a component into an electrical schematic. It is likely that these techniques will appeal to engineers looking to explore real-world uses of hybrid hardware-wetware systems. Furthermore, it is accessible to non-experts (e.g., open-source electronic prototyping enthusiasts) who may be interested in experimenting with aspects of unconventional computing but have found it difficult to find prototypes to adapt to their needs. Some potential applications may include implementing probabilistic models harnessing the memristors spiking behavior, developing approaches to performing stateful logic operations, and modelling neurological processes for information storage and processing.

<table><tbody><tr><td>Conductive PLA filament 2.85mm</td><td>Functionalize</td><td>FE_1LB_2.85MM</td><td>Conductive 3D Printing Filament</td></tr><tr><td>HIPS Filament 3mm 1KG (black)</td><td>NuNus</td><td>104856</td><td>3D printing filament</td></tr><tr><td>Cleaning Filament, 3mm, 0.1 kg, Natural</td><td>3D Prima</td><td>3DPCLEAN300</td><td>3D cleaning filament</td></tr><tr><td>Lulzbot Taz 5</td><td>Lulzbot</td><td>TAZ 5</td><td>3D printer</td></tr><tr><td>Agar powder</td><td>Sigma-Aldrich</td><td>0504</td><td>Non-nutrient microbiological Agar powder</td></tr><tr><td>4mm ID x 6mm OD Clear PVC Tubing Pipe Hose 5 Metres</td><td>Amazon</td><td>B008NC4JUO</td><td>Roll of PVC tubing</td></tr><tr><td>Physarum polycephalum Plasmodium, Living, Plate</td><td>Carolina Biological Supply Company</td><td>156193</td><td>Plasmodium culture.</td></tr><tr><td>Oat Flakes</td><td>Carolina Biological Supply Company</td><td></td><td>Oak flakes to feed the Plasmoidum</td></tr><tr><td>Cura</td><td>Lulzbot</td><td>Cura LulzBot Edition</td><td>https://www.lulzbot.com/cura</td></tr><tr><td>230 Programmable Voltage Source</td><td>Keithley Instruments</td><td></td><td>Voltage source instrument.</td></tr><tr><td>617 Programmable Electrometer</td><td>Keithley Instruments</td><td></td><td>Electrometer to measure low currents.</td></tr></tbody></table>

a method for growing bio-memristors from slime mold

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and computing architectures based on biology. This specific paper focuses on harnessing the unicellular slime mold Physarum polycephalum to develop bio-memristors (or biological memristors) and bio-computing devices. The memristor is a resistor that possesses memory. It is the 4th fundamental passive&#160;circuit element (the other three are the resistor, the capacitor, and the inductor), which is paving the way for the design of new kinds of computing systems; e.g., computers that might relinquish the distinction between storage and a central processing unit. When applied with an AC voltage, the current vs. voltage characteristic of a memristor is a pinched hysteresis loop. It has been shown that P. polycephalum produces pinched hysteresis loops under AC voltages and displays adaptive behavior that is comparable with the functioning of a memristor. This paper presents the method that we developed for implementing bio-memristors with P. polycephalum and introduces the development of a receptacle to culture the organism, which facilitates its deployment as an electronic circuit component. Our method has proven to decrease growth time, increase component lifespan, and standardize electrical observations.

To produce representative results, we set up 5 samples using the exact method described above. For a control, 5 samples were also arranged using the method described in the early P. polycephalum memristor investigations9,10. Here, we positioned two electrodes spaced at a distance of ~10 mm within 60 mm Petri dishes. Each electrode consisted of a circle (~20 mm in diameter) of tinned copper wire (16 stands at 0.2 mm) filled with a 2% non-nutrient deionized agar (~2 mL). All samples were monitored via time-lapse imagery to review growth time. Here, the 5 receptacle samples connected the two electrodes within 10 h of inoculation. The fastest of these grew in under 2 h, and the longest was 10 h, with the mean average growth time across all 5 samples of 7 h 24 min. Four&#160;of the control samples produced a linking protoplasmic tube and one&#160;propagated off the inoculation electrode but dried out before it made the required connection. The quickest of the control samples made its connection within 19 h while the slowest took 36 h, with an average growth time across control samples of 26 h 15 min. These data show a significant decrease in growth time for memristors grown using the presented method.
The I-V profile of a memristor is its most defining feature. As such, we performed I-V measurements on the samples to produce representative results for this paper. Here, instantaneous current measurements were made at each point of a 160-step voltage sine wave. Each voltage step had a static dwell time of 2 s. Electrical measurements were made using a 230 Programmable Voltage Source and a 617 Programmable Electrometer. These devices were selected as they are capable of sourcing voltage and taking measurements at high resolutions. Experiments were conducted at room temperature in an unlit room.
Figure 6 shows typical I-V curves produced from tests on P. polycephalum memristors. Figure 6c and 6d show plots with the representative measurements from components implemented in the Petri dishes. Results using this method show that, although curves measured on the same sample are morphologically similar, hysteresis varies heavily from sample-to-sample. Such variation includes the location of pinch points, the magnitude of both positive and negative lobes, and the symmetry between measurements in the negative and positive voltage domains. Thus, I-V curves measured on memristors using the Petri dish method are not the footprint of an &#8216;ideal&#8217; memristor because pinch points are not at zero voltage and current. Figure 6a and 6b show graphs with representative measurements from memristors grown in the receptacles. The pinch point locations and lobe sizes of these hysteresis loops are relatively consistent both in discrete sample curves tested under different voltage ranges and time-steps, and sample-to-sample curves. Therefore, receptacle I-V curves were more reminiscent of an &#8216;ideal&#8217; memristor&#39;s footprint, where pinch points were always singular and almost consistently at zero voltage and current. However, although hysteresis morphologies were similar sample-to-sample, there was variation in overall resistance between samples. .
After the initial I-V measurements were completed, tests were made on each sample once a day until they presented no memristive curves. Of the 4 control samples, 2 dried up within 2 days of initial testing, while the remaining 2 continued to record pinched curves for a further 2 days. The receptacle samples maintained their memristance for at least 7 days, with 3 samples exceeding that. Over time, each of the receptacle sample&#39;s protoplasmic tubes became thicker, and there was a decrease in overall resistance, with some samples measuring in the A x 10-04 range for 10 V runs against A x 10-05 in their earlier tests.
The reader is referred to the article by Braund14 for results on the extensive testing of the presented receptacle.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56076/56076fig1.jpg" /> 
Figure 1: A photograph of a 2 day-old culture of plasmodium of P. polycephalum. <a href="https://cloudflare.jove.com/files/ftp_upload/56076/56076fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56076/56076fig2.jpg" /> 
Figure 2: A screenshot of the receptacle STL file after it is loaded into the slicing software. <a href="https://cloudflare.jove.com/files/ftp_upload/56076/56076fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56076/56076fig3.jpg" /> 
Figure 3: A screenshot of the electrode STL file after it is loaded into the slicing software. <a href="https://cloudflare.jove.com/files/ftp_upload/56076/56076fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56076/56076fig4.jpg" /> 
Figure 4: A screenshot of the settings configuration for printing the electrode STL model.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56076/56076fig5.jpg" /> 
Figure 5: Two photographs depicting P. polycephalum memristors implemented in a Petri dish (left) and using the method presented in this paper (right). <a href="https://cloudflare.jove.com/files/ftp_upload/56076/56076fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56076/56076fig6.jpg" /> 
Figure 6: Four I-V graphs that were produced from two memristors grown in the receptacles (a, b) and two implemented in Petri dishes (c, d). <a href="https://cloudflare.jove.com/files/ftp_upload/56076/56076fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56076/56076fig7.jpg" /> 
Figure 7: Photographs showing the receptacle being used to grow tubes at various lengths.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56076/56076fig8.jpg" /> 
Figure 8: A photograph showing protoplasmic tubes that have been disconnected from the chambers.

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A Method for Growing Bio-memristors from Slime Mold

This paper introduces an improved method for growing bio-memristors out of the plasmodium of Physarum polycephalum. Such a method has proven to decrease growth time, increase component lifespan, standardize electrical observations, and create a protected environment that can be integrated into conventional circuitry.

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and ...

a-method-for-growing-bio-memristors-from-slime-mold

Research

JoVE Journal

Bioengineering

8.9K Views.  Plymouth University. Deze paper introduceert een verbeterde methode voor het kweken van bio-memristors uit de plasmodium van Physarum polycephalum. Deze methode heeft bewezen groei tijd inkorten, verhogen van levensduur van de component, standaardiseren elektrische opmerkingen en maken een beschermde omgeving dat conventionele circuits geïntegreerd kan worden.

Video: A Method for Growing Bio-memristors from Slime Mold

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Genetics

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Biology

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Neuroscience

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

A Method for Growing Bio-memristors from Slime Mold

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