This research was supported by the Swiss Agency for Development Cooperation and the REACH programme funded by UK Aid from the UK Department for International Development (DFID) for the benefit of developing countries (Aries Code 201880). The views expressed and information contained in it are not necessarily those of or endorsed by these agencies, which can accept no responsibility for such views or information or for any reliance placed on them. The authors also thank Arnt Diener for his contributions to early iterations of the polystyrene foam incubator prototype.

1. Heating Unit
<ol>
	<li>Gather the following components (Figure 1): 
	<img alt="Equation" src="/files/ftp_upload/58443/01.jpg" /> Support plate (280 x 250 mm) with required anchorage holes 
	<img alt="Equation" src="/files/ftp_upload/58443/02.jpg" /> Axial fan (60 x 60 x 25 mm); 2x 
	<img alt="Equation" src="/files/ftp_upload/58443/03.jpg" /> Spacer (length 20 mm, internal diameter 4.25 mm (M4)); 4x 
	<img alt="Equation" src="/files/ftp_upload/58443/04.jpg" /> Luster terminal with three pins 
	<img alt="Equation" src="/files/ftp_upload/58443/05.jpg" /> Screw nut (M4); 4x and (M3); 1x 
	<img alt="Equation" src="/files/ftp_upload/58443/06.jpg" /> Washer (M4); 8x and (M3); 1x 
	<img alt="Equation" src="/files/ftp_upload/58443/07.jpg" /> Screw (M4); 4x and (M3); 1x</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58443/58443fig1.jpg" /> 
Figure 1: Individual components of the heating unit.&#160;<img alt="Equation" src="/files/ftp_upload/58443/01.jpg" /> Support plate, <img alt="Equation" src="/files/ftp_upload/58443/02.jpg" /> axial fans, <img alt="Equation" src="/files/ftp_upload/58443/03.jpg" /> spacers, <img alt="Equation" src="/files/ftp_upload/58443/04.jpg" /> luster terminal, <img alt="Equation" src="/files/ftp_upload/58443/05.jpg" /> screw nuts, <img alt="Equation" src="/files/ftp_upload/58443/06.jpg" /> washers and <img alt="Equation" src="/files/ftp_upload/58443/07.jpg" /> screws. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Drill the required holes (Figure 2) into the support plate <img alt="Equation" src="/files/ftp_upload/58443/01.jpg" /> to secure the axial fans <img alt="Equation" src="/files/ftp_upload/58443/02.jpg" /> as well as the luster terminal <img alt="Equation" src="/files/ftp_upload/58443/04.jpg" /> (Figure 1).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58443/58443fig2.jpg" /> 
Figure 2: Schematic diagram of support plate. Indications to drill the anchorage holes into the support plate to fix the axial fans as well as the luster terminal. Distances are given in millimeters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Anchor the axial fans <img alt="Equation" src="/files/ftp_upload/58443/02.jpg" /> in the center on the support plate <img alt="Equation" src="/files/ftp_upload/58443/01.jpg" /> as shown in Figure 3 with two M4 screws, screw nuts, and washers <img alt="Equation" src="/files/ftp_upload/58443/05.jpg" />, <img alt="Equation" src="/files/ftp_upload/58443/06.jpg" />, <img alt="Equation" src="/files/ftp_upload/58443/07.jpg" /> per fan. Use the spacers <img alt="Equation" src="/files/ftp_upload/58443/03.jpg" /> to leave a distance between the fans and the support plate (Figure 3).</li>
	<li>Anchor the luster terminal <img alt="Equation" src="/files/ftp_upload/58443/04.jpg" /> to the support plate <img alt="Equation" src="/files/ftp_upload/58443/01.jpg" /> using M3 screw, screw nut, and washer. Secure the cable fans.&#160;(Figure 3).</li>
	<li>Connect the fan cables with the luster terminal. Connect the positive cables of each fan together and the negative cables of each fan together (Figure 3). The speed sensor is not required.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58443/58443fig3.jpg" /> 
Figure 3: Axial fans fixed on support plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: The cable colors mentioned correspond to the ones used in the figures. The cable colors might change depending on the material used.
2. Control Unit (Power Supply)
<ol>
	<li>Gather the following components: 
	<img alt="Equation" src="/files/ftp_upload/58443/08.jpg" /> Universal enclosure (here 200 x 120 x 60 mm, but dimensions will depend on the size of the DC/DC converter and the PID temperature controller) 
	<img alt="Equation" src="/files/ftp_upload/58443/09.jpg" /> On/off-Switch 
	<img alt="Equation" src="/files/ftp_upload/58443/10.jpg" /> DC/DC converter, input voltage range 9 - 36V, output voltage 12V 
	<img alt="Equation" src="/files/ftp_upload/58443/11.jpg" /> PID temperature controller, 12 - 35 V/DC operating voltage 
	<img alt="Equation" src="/files/ftp_upload/58443/12.jpg" /> Cable gland, M12 x 15 mm, clamping range 2 - 7.5 mm (or according to the cable used) 
	<img alt="Equation" src="/files/ftp_upload/58443/13.jpg" /> Temperature sensor Pt100 
	<img alt="Equation" src="/files/ftp_upload/58443/14.jpg" /> AC power supply 
	NOTE: The incubator can be connected to the mains power supply or to a battery. In the case of the mains operation, the AC power supply is required and if the unit is exclusively connected to mains, the DC/DC converter is not mandatory. In the case of battery operation, the DC/DC converter is highly recommended, and a two-wire cable is required instead of the AC power supply. This protocol presents the version with the DC/DC converter and the AC power supply. An electrical diagram of the incubator electrical core is detailed in the supplementary material (Figure S1).</li>
	<li>Mill the openings for the PID temperature controller, on/off switch, and cable glands into the enclosure with a drill and jigsaw, or an equivalent tool (Figure 4).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58443/58443fig4.jpg" /> 
Figure 4: Schematic diagram of the universal enclosure. (a) Indications to place the temperature controller <img alt="Equation" src="/files/ftp_upload/58443/21.jpg" />, on/off switch <img alt="Equation" src="/files/ftp_upload/58443/22.jpg" /> and the cable glands <img alt="Equation" src="/files/ftp_upload/58443/23.jpg" /> into the universal enclosure; distances are given in millimeters. (b) 3D view of the universal enclosure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Connect the DC/DC Converter to the on/off switch: connect the positive cable of the AC power adapter to the on/off switch and the negative cable of the AC power adapter to the &#34;- Vin&#34; of the DC/DC converter (Figure 5).</li>
	<li>Use a cable to connect the on/off switch to the &#34;+ Vin&#34; of the DC/DC converter (Figure 5).</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58443/58443fig5v2.jpg" /> 
Figure 5: Mounted control unit. Universal enclosure <img alt="Equation" src="/files/ftp_upload/58443/08.jpg" /> with DC/DC converter <img alt="Equation" src="/files/ftp_upload/58443/10.jpg" /> connected to PID temperature controller <img alt="Equation" src="/files/ftp_upload/58443/11.jpg" /> and on/off switch <img alt="Equation" src="/files/ftp_upload/58443/09.jpg" />. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Connect the cables from the heating unit to the PID temperature controller as follows (Figure 6):
	<ol>
		<li>Connect the terminal &#34;1&#34; of the PID temperature controller to the &#8220;DC &#8211;&#8220; wire from the heating unit connection and to the &#34;- Vout&#34; terminal of the DC/DC converter.</li>
		<li>Connect the &#8220;DC +&#8221; wire going to the heating unit to the terminal &#34;4&#34; of the PID temperature controller as well as to the terminal &#34;2&#34; of the PID temperature controller (see point 3.2).</li>
		<li>Connect the terminal &#34;2&#34; of the PID temperature controller to the &#34;+ Vout&#34; terminal of the DC/DC converter.</li>
		<li>Connect the terminal &#8220;5&#8221; of the PID temperature controller to the &#8220;command&#8221; wire going to the heating unit. (see point 3.2).</li>
		<li>Connect the temperature sensor to the terminals &#34;10&#34;, &#34;11&#34; and &#34;12&#34;. 
		NOTE: The red cable of the temperature sensor must be connected to terminal &#8220;11&#8221; of the PID temperature controller.</li>
	</ol>
	</li>
	<li>Anchor the DC/DC converter with Velcro tape at the bottom of the enclosure, and close the universal enclosure.</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58443/58443fig6v2.jpg" /> 
Figure 6: Cable connection of DC/DC converter with PID temperature controller. DC/DC converter <img alt="Equation" src="/files/ftp_upload/58443/10.jpg" />, PID temperature controller <img alt="Equation" src="/files/ftp_upload/58443/11.jpg" />, connection to Incubator (cable A) and connection to temperature sensor (cable B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: The functions of the PID temperature controller terminals used are given in Table 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PID temperature controller terminal</td>
			<td>Function</td>
		</tr>
		<tr>
			<td>Terminal &#34;1&#34;</td>
			<td>Supply input +</td>
		</tr>
		<tr>
			<td>Terminal &#34;2&#34;</td>
			<td>Supply input -</td>
		</tr>
		<tr>
			<td>Terminal &#34;4&#34;</td>
			<td>Control Output Common Contact</td>
		</tr>
		<tr>
			<td>Terminal &#34;5&#34;</td>
			<td>Control Output Normally Open Contact</td>
		</tr>
	</tbody>
</table>
Table 2: Functions corresponding to the PID temperature controller terminals.
3. Assembly of the Incubator Electrical Core
<ol>
	<li>Gather the following components: 
	Heating unit from section 1 
	Control unit from section 2 
	<img alt="Equation" src="/files/ftp_upload/58443/15.jpg" /> Heating foils, self-adhesive, 100 x 200 mm, 12 V/20 W, 2x</li>
	<li>Link the connection cables from the control unit to the heating unit as follows (Figure 7):
	<ol>
		<li>Connect the &#8220;DC-&#8221; wire from the control unit with one conductor of each of the heating foils and the negative wire of each fan.</li>
		<li>Connect the &#8220;DC+&#8221; wire coming from the control unit with the positive cable of each fan.</li>
		<li>Connect the &#8220;command&#8221; wire from the control unit to the remaining two conductors of the heating foils.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58443/58443fig7v2.jpg" /> 
Figure 7: Cable connection of heating foils <img alt="Equation" src="/files/ftp_upload/58443/15.jpg" /> with PID temperature controller. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: The completed field incubator electrical core of the incubator is shown in Figure 8.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58443/58443fig8v2.jpg" /> 
Figure 8: Complete field incubator electrical core. Heating unit <img alt="Equation" src="/files/ftp_upload/58443/21.jpg" />, control unit <img alt="Equation" src="/files/ftp_upload/58443/22.jpg" /> and temperature probe <img alt="Equation" src="/files/ftp_upload/58443/23.jpg" />. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Assembly of the Incubator
<ol>
	<li>Gather the following components: 
	Incubator electrical core 
	Incubator shell (here a polystyrene foam box, but can be any type of box made of insulating material) 
	Support rack (here a metal rack, but can be another material)</li>
	<li>Place the incubator components together as follows (Figure 9):
	<ol>
		<li>Place the incubator shell on its side, so that the opening of the incubator (door) is located on a side.</li>
		<li>Place the support plate with the heating unit at the bottom of the incubator shell.</li>
		<li>Place the support rack on top of the heating unit, leaving a space of a minimum of 10 cm between the heating unit and the support rack.</li>
		<li>Place the temperature probe on the support rack and secure it in the incubator.</li>
	</ol>
	</li>
	<li>Drill holes in the door of the incubator to allow for the entry of the cables (Figure 9).</li>
	<li>Connect the incubator to the power source.</li>
	<li>Turn the incubator on and adjust the settings of the PID temperature controller (see Table S1 in the supplementary material for detailed settings).</li>
</ol>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58443/58443fig9.jpg" /> 
Figure 9: Complete field incubator.&#160;Open (left) and closed (right). Heating unit <img alt="Equation" src="/files/ftp_upload/58443/21.jpg" />, support rack <img alt="Equation" src="/files/ftp_upload/58443/22.jpg" />, temperature probe <img alt="Equation" src="/files/ftp_upload/58443/23.jpg" />, control unit <img alt="Equation" src="/files/ftp_upload/58443/24.jpg" />, incubator shell <img alt="Equation" src="/files/ftp_upload/58443/25.jpg" /> and holes for cables in the incubator shell (circled area). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: The shell of the incubator can be a box of any type of material. It is recommended to use an insulating material, and that the box closes tightly to avoid dissipation of the heat. The support rack should contain large holes to avoid the accumulation of heat in the rack, and the material can be metal or other (e.g. plastic).

<ol>
	<li>Bain, R., 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+summary+catalogue+of+microbial+drinking+water+tests+for+low+and+medium+resource+settings.">A summary catalogue of microbial drinking water tests for low and medium resource settings.</a> International Journal of Environmental Research and Public Health. 9 (1609-1625), (2012).</li><li>K&#246;ster, W., 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=Analytical+methods+for+microbiological+water+quality+testing.">Analytical methods for microbiological water quality testing.</a> Assessing Microbial Safety of Drinking Water. , 237-277 (2003).</li><li>World Health Organization (WHO). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for Drinking Water Quality. , (2011).</li><li>World Health Organization (WHO). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safely Managed Drinking Water - Thematic Report on Drinking Water. , (2017).</li><li>Peletz, R., Kumpel, E., Bonham, M., Rahman, Z., Khush, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+what+extent+is+drinking+water+tested+in+sub-Saharan+Africa?+A+comparative+analysis+of+regulated+water+quality+monitoring.">To what extent is drinking water tested in sub-Saharan Africa? A comparative analysis of regulated water quality monitoring.</a> International Journal of Environmental Research and Public Health. 13 (3), 275 (2016).</li><li>Diener, A., 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=Adaptable+drinking-water+laboratory+unit+for+decentralised+testing+in+remote+and+alpine+regions.">Adaptable drinking-water laboratory unit for decentralised testing in remote and alpine regions.</a> 40th WEDC International Conference. , 1-6 (2017).</li><li>Malkin, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+health+care+technologies+for+the+developing+world.">Design of health care technologies for the developing world.</a> Annual Review of Biomedical Engineering. 9 (1), 567-587 (2007).</li><li>Rahman, Z., Khush, R., Gundry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatest:+Expanding+Microbial+Water+Quality+Testing+for+Drinking+Water+Management.">Aquatest: Expanding Microbial Water Quality Testing for Drinking Water Management.</a> Drinking Water Safety International. 1 (4), 15-17 (2010).</li><li>DelAgua Water Testing Ltd. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DelAgua Portable Water Testing Kit: User Manual Version 5.0. , (2015).</li><li>Aquagenx LLC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Portable Incubator Fabrication Instructions. , (2015).</li><li>Nair, J., Mathew, K., Ho, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiences+with+implementing+the+H2S+method+for+testing+bacterial+quality+of+drinking+water+in+remote+aboriginal+communities+in+Australia.">Experiences with implementing the H2S method for testing bacterial quality of drinking water in remote aboriginal communities in Australia.</a> Water for all life: A decentralized infrastructure for a sustainable future. , (2007).</li><li>Kandel, P., Kunwar, R., Lamichhane, P., Karki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+of+fecal+contamination+of+household+drinking+water+in+Nepal:+Further+analysis+of+Nepal+Multiple+Indicator+Cluster+Survey+2014.">Extent of fecal contamination of household drinking water in Nepal: Further analysis of Nepal Multiple Indicator Cluster Survey 2014.</a> American Journal of Tropical Medicine and Hygiene. 96 (2), 446-448 (2017).</li><li>Edberg, S. C., Rice, E. W., Karlin, R. J., Allen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli:+the+best+biological+drinking+water+indicator+for+public+health+protection.">Escherichia coli: the best biological drinking water indicator for public health protection.</a> Journal of Applied Microbiology. 88 (51), 1065-1165 (2000).</li><li>Taylor, D. D. J., Khush, R., Peletz, R., Kumpel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+microbial+sampling+recommendations+and+practices+in+sub-Saharan+Africa.">Efficacy of microbial sampling recommendations and practices in sub-Saharan Africa.</a> Water Research. 134, 115-125 (2018).</li></ol>

The authors have nothing to disclose.

Under Sustainable Development Goal 6.1, the demand for water quality sampling is increasing, especially in remote rural areas where monitoring practices are less established14. A major barrier to implementing regular water quality testing in these settings is poor access to laboratories capable of supporting microbial methods6. This paper presents a method for a reliable incubator constructed from materials that are relatively cheap and widely available. The electrical components are relatively easy to source and assemble, requiring only limited expertise. Moreover, the incubator shell design is flexible and therefore can be constructed from locally available materials. This is especially desirable for those traveling to remote locations, since luggage space is not needed for a heavy and bulky shell. Depending on the shell used, the volume of the incubator is also adaptable and can be sized to accommodate a specific sample size. The presented set-up can be used on- and off-grid, which makes it robust to power cuts or absence of reliable electrical supply. While certain design limitations were observed, this set-up up generally proved to be effective under a range of ambient temperature conditions (3.5 &#176;C to 39 &#176;C).
There are several steps in the protocol that are critical for achieving an incubator design suitable to one&#39;s needs. The first is the selection of the electrical components of the incubator. Alternative components can be chosen based on the price or the local availability. Depending on the material selected and their technical specifications, the incubator may have altered performances as compared to the results presented. Another critical step in the protocol is the choice of shell material, which should be made based on the expected range of ambient temperatures, local power supply, and availability of materials. At lower ambient temperatures (&#60; 25 &#176;C), a shell constructed of polystyrene foam or a hard cooler box is recommended to achieve a set temperature of 37 &#176;C to 44.5 &#176;C. Based on the experimental data presented, these set ups can be expected to reach the set temperature in 45 - 96 minutes and consume 0.78 - 1.05 kWh/24h in cold environments (3.5 to 7.5 &#176;C). The cardboard box with survival blanket is not recommended for use at lower ambient temperatures since this set up never reached a stable set temperature during the experimental observation period. At moderate ambient temperature (27 &#176;C) any of the shell types tested are acceptable, with similar to slightly greater power consumption observed for the cardboard box set up. At higher ambient temperatures (39 &#176;C), the incubator designs presented here were prone to overheating if unless the set temperature was even higher (i.e., 44.5 &#176;C). Therefore, such conditions would require a cooling device or use in a climate controlled space.
The cost of constructing the incubator presented here was about 300 USD when materials were sourced in Switzerland. However, these costs may be considerably lower in different locations, especially if shipping fees for the electronic core components can be kept to a minimum. Modification of the various components described in the protocol can further reduce costs. The protocol presented here is limited in that it compares only three shell material types at two set temperatures, as well as verification of microbial growth for E. coli only. Future research should test the suitability of this incubator design under a greater range of temperature parameters and using additional microbial indicator species (e.g., Enterococcus) and pathogens (e.g., salmonella, Vibrio cholerae). Future research should also focus on development of effective cooling techniques within the incubator, which would allow for its use in extremely warm environments (&#62; 40 &#176;C).
To our knowledge, there is no other known field incubator that offers adaptable volume capacity and is easily dismountable, while remaining transportable and low-cost. This innovative alternative to commercially available incubators fulfills a need for governments and organizations with water quality and other culture-based testing objectives where few laboratory facilities are available. When paired with simple water quality testing equipment, this incubator can help practitioners with limited capacities to establish permanent or seasonal laboratories at a reasonable cost. By increasing the number of laboratories in remote areas, efforts to conduct regular water quality surveillance or achieve punctual monitoring of system operations will become increasingly feasible.

Culture-based methods for the detection of microbial contaminants are the state-of-the-art for water quality analysis in both industrialized and developing countries1,2. Microorganisms exist in many environments and require different temperature conditions for optimal growth. Therefore, creating a temperature-stable incubation environment is a precondition for the reliable detection of microbial contaminants of concern in drinking water. According to the World Health Organization, Escherichia coli (E. coli) (or alternatively, thermotolerant coliforms (TTC)) are the most suitable indicators of fecal contamination in drinking water3. Detection of these organisms consists of, for example, filtering a 100 mL water sample through a membrane followed by incubation of the membrane on selective media at 35 - 37 &#176;C (E. coli) or 44 - 45 &#176;C (TTC)3.
Field-based applications of culture-based methods have become increasingly relevant in recent years. Under Sustainable Development Goal 6, Target 6.1, governments have committed to regularly report bacteriological quality of drinking water at the national level4. In addition to such public health surveillance efforts, operational monitoring of water infrastructure is regularly undertaken at the local or regional level5. These surveillance and monitoring campaigns are often in remote locations where the required laboratory infrastructure is inadequate or unavailable.6 Similarly, culture-based methods are widely used in medical diagnosis and microbiological research where local clinics and research institutions may be challenged by limited resources and insecure power supplies7.
In the above contexts, conventional incubators are often inadequate or unavailable. As an alternative, field incubators have been specifically developed for use outside the laboratory, e.g., the Aquatest project8, University of Bristol, United Kingdom; DelAgua9, Marlborough, United Kingdom; or Aquagenx10, University of North Carolina, United States. However, these devices are relatively small in volume, thus limiting the number of samples that can be simultaneously processed. Field incubators on the market are also not designed to operate under very low (&#60; 20 &#176;C) or very high (&#62; 40 &#176;C) ambient temperature conditions, making their use in desert or alpine environments difficult. Further alternative solutions include yogurt-making appliances11, body belts, and phase-change incubators12. However, such unconventional incubators may function unreliably or be burdensome to operate11.
There is thus a need for an incubator that offers the advantages of laboratory-based models (ease of use, larger volume, and temperature precision) while remaining suitable for field applications (low-cost, easily transported and maintained, robustness to a range of ambient temperatures, energy efficient, and resilient to intermittent power supplies) (Table 1). The purpose of this protocol is to detail the fabrication process of a low-cost incubator designed to optimize the advantages of both conventional and field-based models using widely available material.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Characteristic</td>
			<td>Laboratory-based</td>
			<td>Field</td>
			<td>Optimized</td>
		</tr>
		<tr>
			<td>User friendly design</td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Large capacity</td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Robust to wide range of ambient temperatures</td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Maintains constant temperature</td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Low cost</td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Easily transported</td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Energy efficient</td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
		<tr>
			<td>Resilient to intermittent power supply</td>
			<td><img alt="X" src="/files/ftp_upload/58443/58443eq2.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
			<td><img alt="Check" src="/files/ftp_upload/58443/58443eq1.jpg" /></td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of commercially available incubators (laboratory-based and field) and the optimized approach.
The following assembly protocol specifies the required materials and steps for building the incubator. It is structured in four steps: first, assembly of the heating unit; second, assembly of the control unit; third, assembly of the incubator electrical core; and fourth, assembly of the incubator. This protocol explains the construction of the electronic core of the incubator, that can work with a variety of incubator shells. See the Table of Materials for a full list of all components used in the Protocol and their technical specifications. The protocol below presents a functional example of the field incubator, but flexible use of different components is possible as long as they fulfill the electrical requirements. Using different components might influence the performances of the incubator. It is advised that the construction and wiring of electrical components be done by a person skilled in the electrical field.

<table>
	<tbody>
		<tr>
			<td>Heating foil</td>
			<td>Thermo</td>
			<td>2115337</td>
			<td>Self-adhesive 10x20 cm; Operating voltage 12V; Power 20W</td>
		</tr>
		<tr>
			<td>Axial fan</td>
			<td>Yen Sun Technology Corp.</td>
			<td>FD126025MB</td>
			<td>6x6x2.5 cm; Operating voltage 12VDC; Power 1.44W; Max. current consumption 60mA</td>
		</tr>
		<tr>
			<td>PID Temperature Controller</td>
			<td>Wachendorff Automation GmbH &#38; Co. KG</td>
			<td>UR3274S</td>
			<td>PID controller 32x74 mm; Universal input for process signals, thermocouples, Pt100; Operating voltage 24 VDC; Outputs (thermostats) 10 A relay, 5 A relay, SSR, RS 485</td>
		</tr>
		<tr>
			<td>Temperature sensor Pt100</td>
			<td>Conrad</td>
			<td>198466</td>
			<td>Temperature range -100&#176;C to 200&#176;C; Sensor Pt100, Type FS-400P</td>
		</tr>
		<tr>
			<td>Universal enclosure</td>
			<td>OKW Geh&#228;use System</td>
			<td>C2012201</td>
			<td>Dimensions 200 x 120 x 60 mm</td>
		</tr>
		<tr>
			<td>ON/OFF Switch</td>
			<td>SHIN CHIN INDUSTRIAL CO.</td>
			<td>R13-70A-01</td>
			<td>Connection Type C CEE 7/16 plug 6.3 mm; Contact resistance Max 50 m&#8486;; Switching voltage 24 VDC; Switching current (mx.) 10A; Insulation resistance Min 100 M&#8486;/500 Vdc</td>
		</tr>
		<tr>
			<td>DC/DC converter</td>
			<td>Traco Power</td>
			<td>TMDC 60-2412</td>
			<td>Nominal voltage 24 VDC; Input voltage 9-36 VDC; Output voltage 12 VDC; Max. output current 5 A; Power 60W</td>
		</tr>
		<tr>
			<td>AC power adapter</td>
			<td>Bicker Elektronik</td>
			<td>BET-0612</td>
			<td>Output voltage 12 VDC; Max. output current 5 A; Input voltage 115-230 VAC</td>
		</tr>
		<tr>
			<td>Spacer</td>
			<td>Sch&#228;fer Elektromechanik</td>
			<td>20/4</td>
			<td>Without thread; Thread size M4; Polystyrene; Distance 20 mm</td>
		</tr>
		<tr>
			<td>Cable gland</td>
			<td>WISKA</td>
			<td>10066410</td>
			<td>M12 x 1.5 cm; clamping range 3 &#8211; 7 mm</td>
		</tr>
		<tr>
			<td>Luster terminal</td>
			<td>Adels Contact</td>
			<td>125312</td>
			<td>Nominal current 25 A; Nominal Voltage 500V</td>
		</tr>
		<tr>
			<td>Screw M4 x 50</td>
			<td>Bossard</td>
			<td>1579010</td>
			<td>M4 x 50 mm</td>
		</tr>
		<tr>
			<td>Screw nut M4</td>
			<td>Bossard</td>
			<td>1241478</td>
			<td>M4</td>
		</tr>
		<tr>
			<td>Washer M4</td>
			<td>Bossard</td>
			<td>1887505</td>
			<td>M4</td>
		</tr>
		<tr>
			<td>Screw M3 x 25</td>
			<td>Bossard</td>
			<td>1211099</td>
			<td>M3 x 25 mm</td>
		</tr>
		<tr>
			<td>Screw nut M3</td>
			<td>Bossard</td>
			<td>1241443</td>
			<td>M3</td>
		</tr>
		<tr>
			<td>Washer M3</td>
			<td>Bossard</td>
			<td>1887483</td>
			<td>M3</td>
		</tr>
		<tr>
			<td>Support plate</td>
			<td>&#160;-&#160;</td>
			<td>&#160;-&#160;</td>
			<td>Insulating material (plastic or other); 28 x 25 cm</td>
		</tr>
	</tbody>
</table>

construction of a low-cost mobile incubator for field and laboratory use

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water quality. However, commercially available incubators are often costly, difficult to transport, not flexible in terms of volume, and/or poorly adapted to local field conditions where access to electricity is unreliable. The purpose of this study was to develop an adaptable, low-cost and transportable incubator that can be constructed using readily available components. The electronic core of the incubator was first developed. These components were then tested under a range of ambient temperature conditions (3.5 &#176;C - 39 &#176;C) using three types of incubator shells (polystyrene foam box, hard cooler box, and cardboard box covered with a survival blanket). The electronic core showed comparable performance to a standard laboratory incubator in terms of the time required to reach the set temperature, inner temperature stability and spatial dispersion, power consumption, and microbial growth. The incubator set-ups were also effective at moderate and low ambient temperatures (between 3.5 &#176;C and 27 &#176;C), and at high temperatures (39 &#176;C) when the incubator set temperature was higher. This incubator prototype is low-cost (&lt; 300 USD) and adaptable to a variety of materials and volumes. Its demountable structure makes it easy to transport. It can be used in both established laboratories with grid power or in remote settings powered by solar energy or a car battery. It is particularly useful as an equipment option for field laboratories in areas with limited access to resources for water quality monitoring.

The reliability of a robust field incubator lies in its ability to reach and maintain a set temperature under various conditions. To monitor the performance of the various incubator set-ups, the following measurements were made: time needed to reach the set temperature, effect of opening the door for one minute, power consumption over 24 hours of operation, inner temperature stability over 24 hours of operation, and observation of E. coli growth. The temperature inside the incubator was measured every minute with 4 temperature logging devices placed in different positions in the structure (support rack, wall, top, inside a growth plate). The set temperature was considered to be attained when all measurements were within plus or minus 2 &#176;C, which is the acceptable range for the incubation of E. coli.13
The electronic core was tested with three types of shells, using materials that are typically found in many countries: a polystyrene foam box (78 liters), a hard plastic cooler box (30 liters), and a cardboard box covered with a survival blanket (46 liters) (Figure 10). To cover a range of ambient conditions that can be experienced in the field, these incubator set-ups were tested at three ambient temperatures: ambient (about 27 &#176;C), cold (about 3.5 &#176;C and 7.5 &#176;C) and hot (about 39 &#176;C). Performance measures were tested setting the inner temperature at 37 &#176;C and 44.5 &#176;C.
The time to reach the set temperature in the incubators was influenced by the ambient temperature and the material of the incubator shell. At an ambient temperature of about 27 &#176;C, the three incubators set-ups reached the set temperatures (37 &#176;C and 44.5 &#176;C) in a similar time (Figure 11a and Figure 12a) and comparable with the performance of a standard incubator (Table 3). In cold environments (3.5 &#176;C and 7.5 &#176;C), the incubators with thicker shells, i.e., the polystyrene foam and cooler box, reached the target set temperatures (37 &#176;C and 44.5 &#176;C) in a similar time; about four times longer than under an ambient temperature of 27 &#176;C. With its lower insulation, the cardboard box with survival blanket never fully reached the set temperatures under cold ambient temperature conditions (Figure 11b and Figure 12b). In a warm environment (39 &#176;C), the three incubator set-ups reached the target temperature of 44.5 &#176;C in under 10 minutes (Figure 12c). However, when the set temperature was of 37 &#176;C, i.e., lower than the ambient temperature, none of the incubators could lower the temperature, resulting in overheating for all three incubator set ups (Figure 11c).
The ambient temperature and the type of incubator shell influenced the impact of opening the door of the incubator for one minute. The heat loss was greater in the cold environment, and the time to recover the inner set temperatures was longer, with the exception of the cardboard box incubator where the set temperatures were never reached (Figure 13b and Figure 14b). In the warmer environments, the heat loss was limited and the set temperatures were recovered in under 10 minutes (Figure 13a,c and Figure 14a,c). In an ambient temperature of 39 &#176;C and set temperature of 37 &#176;C, opening the door did not cause nor reduce overheating of the incubators (Figure 13c).
The power consumption increased with cold environments and with an increase in the set temperature. Better insulating incubator shells (polystyrene foam and cooler box) showed a reduced power consumption as compared to the cardboard box incubator. In similar environments (ambient temperature of about 27 &#176;C), the three incubator set-ups consumed 0.22 to 0.52 kWh/24h less energy than the standard incubators tested (Table 3).
The temperature in the incubator remained stable over 24 hours with all type of incubator shells and ambient temperature tested (Figure 13 and Figure 14). Slight variations of the measured temperature compared to the set temperature were observed according to the position of the temperature logging device in the incubator. With the exception of the tests with the ambient temperature (39 &#176;C) warmer than the set temperature (37 &#176;C) (Figure 13c), the variations in temperature were all within the 2 &#176;C acceptable range for E. coli incubation.
All tests were performed in the presence of E. coli and total coliform measurement materials (membrane filter placed on growth plate). Replicates of a sample were placed in each incubator set-up and in a standard incubator for comparison. In all set-ups and conditions, the growth of E. coli and total coliform was successful and comparable to the growth observed in the standard incubator. A summary of the incubator configurations and ambient temperature conditions tested with results are shown in Table 3.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Test 1: 
			Time to set temperature</td>
			<td>Test 2: 
			Side door opening one minute</td>
			<td>Test 3: 
			Power consumption over 24-hour period</td>
			<td>Test 4: 
			Temperature variation over 24-hour period</td>
			<td>Test 5: 
			E. coli growth observed</td>
		</tr>
		<tr>
			<td></td>
			<td>Ambient temperature</td>
			<td>Set temperature</td>
			<td>(min)</td>
			<td>Maximal loss of temperature (&#176;C); time to recover set temperature (min)</td>
			<td>(kWh/24 h)</td>
			<td>Absolute maximal temperature (&#176;C); absolute minimal temperature (&#176;C) *</td>
			<td>(Yes / No)</td>
		</tr>
		<tr>
			<td rowspan="6">Polystyrene foam box</td>
			<td>3.5 &#176;C</td>
			<td>37 &#176;C</td>
			<td>45</td>
			<td>10 &#176;C; 17 min</td>
			<td>0.78</td>
			<td>37; 35.5</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>7.5 &#176;C</td>
			<td>44.5 &#176;C</td>
			<td>74</td>
			<td>16.5 &#176;C; 31 min</td>
			<td>0.89</td>
			<td>44.5; 42.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">27 &#176;C</td>
			<td>37 &#176;C</td>
			<td>12</td>
			<td>2.5 &#176;C; 3 min</td>
			<td>0.28</td>
			<td>37.5; 36.5</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>20</td>
			<td>4.5 &#176;C; 7 min</td>
			<td>0.43</td>
			<td>44.5; 43.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">39 &#176;C</td>
			<td>37 &#176;C</td>
			<td>0 (overheat)</td>
			<td>2 &#176;C; 0 min (overheat)</td>
			<td>0.11</td>
			<td>42.5; 42</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>7</td>
			<td>3.5 &#176;C; 5 min</td>
			<td>0.17</td>
			<td>45; 43.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="6">Hard cooler box</td>
			<td>3.5 &#176;C</td>
			<td>37 &#176;C</td>
			<td>54</td>
			<td>8 &#176;C; 10 min</td>
			<td>0.86</td>
			<td>37.5; 36</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>7.5 &#176;C</td>
			<td>44.5 &#176;C</td>
			<td>96</td>
			<td>12 &#176;C; 30 min</td>
			<td>1.05</td>
			<td>45; 43</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">27 &#176;C</td>
			<td>37 &#176;C</td>
			<td>13</td>
			<td>1.5 &#176;C; 0 min</td>
			<td>0.27</td>
			<td>37.5; 36.5</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>25</td>
			<td>2 &#176;C; 4 min</td>
			<td>0.50</td>
			<td>45; 43.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">39 &#176;C</td>
			<td>37 &#176;C</td>
			<td>0 (overheat)</td>
			<td>1 &#176;C; 0 min (overheat)</td>
			<td>0.11</td>
			<td>43; 42.5</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>9</td>
			<td>4 &#176;C; 3 min</td>
			<td>0.19</td>
			<td>45.5; 44.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="6">Cardboard box with survival blanket</td>
			<td>3.5 &#176;C</td>
			<td>37 &#176;C</td>
			<td>Never reached (stable temperature after 109 min)</td>
			<td>6.5 &#176;C; stable temperature after 30 min</td>
			<td>1.24</td>
			<td>33.5; 30.5</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>7.5 &#176;C</td>
			<td>44.5 &#176;C</td>
			<td>Never reached (stable temperature after 120 min)</td>
			<td>8 &#176;C; stable temperature after 20 min</td>
			<td>1.28</td>
			<td>36.5; 32</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">27 &#176;C</td>
			<td>37 &#176;C</td>
			<td>15</td>
			<td>2.5 &#176;C; 6 min</td>
			<td>0.42</td>
			<td>36.5; 35.5</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>24</td>
			<td>3 &#176;C; 8 min</td>
			<td>0.70</td>
			<td>44.5; 42.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">39 &#176;C</td>
			<td>37 &#176;C</td>
			<td>0 (overheat)</td>
			<td>1.5 &#176;C; 0 min (overheat)</td>
			<td>0.11</td>
			<td>41.5; 40</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>9</td>
			<td>2 &#176;C; 0 min</td>
			<td>0.20</td>
			<td>45; 43.5</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td rowspan="2">Standard incubator</td>
			<td rowspan="2">27 &#176;C</td>
			<td>37 &#176;C</td>
			<td>18</td>
			<td>1 &#176;C; 0 min (overheat)</td>
			<td>0.64</td>
			<td>38.5; 36</td>
			<td>ND&#8224;</td>
		</tr>
		<tr>
			<td>44.5 &#176;C</td>
			<td>23 (overheat)</td>
			<td>2.5 &#176;C; 0 min</td>
			<td>0.95</td>
			<td>47.5; 43.5</td>
			<td>ND&#8224;</td>
		</tr>
	</tbody>
</table>
Table 3: Results summary for the incubator configurations and ambient temperature conditions tested. *Test 4: Absolute maximal and minimal temperatures recorded during the stable periods, i.e., from 10 minutes after the end of a disruptive event (time to reach set temperature, opening the door). &#8224;ND: No data, test not run.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/58443/58443fig10.jpg" /> 
Figure 10: Incubator shells tested. Open (upper row) and closed (bottom row).&#160;Polystyrene foam box (left), thickness of 3.5 cm, outer dimensions 39 x 56 x 36 cm; hard plastic cooler box (middle), thickness of 2.5 cm, outer dimensions 32 x 41 x 47 cm; cardboard box (right) covered with a standard survival blanket of a thickness of 12 &#181;m folded twice, outer dimensions 30 x 42 x 37 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/58443/58443fig11.jpg" /> 
Figure 11: Time to reach set temperature (37 &#176;C) of the incubator set-ups under different ambient temperature conditions. Performances of incubators with a shell made of a polystyrene foam box, a hard cooler box, and a cardboard box covered with a survival blanket. At room ambient temperature (a), cold ambient temperature (b), and warm ambient temperature (c). Temperatures recorded on the support rack of the incubators. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/58443/58443fig12v4.jpg" /> 
Figure 12: Time to reach set temperature (44.5 &#176;C) of the incubator set-ups under different ambient temperature conditions. Performances of incubators with a shell made of a polystyrene foam box, a hard cooler box, and a cardboard box covered with a survival blanket. At room ambient temperature (a), cold ambient temperature (b), and warm ambient temperature (c). Temperatures recorded on the support rack of the incubators.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig12v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/58443/58443fig13.jpg" /> 
Figure 13: Temperature variations over 24-hour period and effect of door opening under different ambient temperature conditions. Set temperature of 37 &#176;C. Performances of incubators with a shell made of a polystyrene foam box, a hard cooler box, and a cardboard box covered with a survival blanket. At room ambient temperature (a), cold ambient temperature (b), and warm ambient temperature (c). Circled areas show the temperature variations due to the door opening for one minute. Temperatures recorded on the support rack of the incubators. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/58443/58443fig14.jpg" /> 
Figure 14: Temperature variations over 24-hour period and effect of door opening under different ambient temperature conditions. Set temperature of 44.5 &#176;C. Performances of incubators with a shell made of a polystyrene foam box, a hard cooler box, and a cardboard box covered with a survival blanket. At room ambient temperature (a), cold ambient temperature (b), and warm ambient temperature (c). Circled areas show the temperature variations due to the door opening for one minute. Temperatures recorded on the support rack of the incubators. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" src="/files/ftp_upload/58443/58443fig15.jpg" /> 
Figure S1: Electrical diagram of incubator electrical core wiring. Alternatives for mains operation and battery operation are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58443/58443fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Parameter</td>
			<td colspan="2">Selected value</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="4">Type of control output</td>
			<td colspan="2">Control Q1 / Alarm Q2</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="4">Type of connected sensor</td>
			<td colspan="2">Pt100 (-200 to 140&#176;C)</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="4">Lower limit selectable for setpoint value</td>
			<td colspan="2">0</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="4">Upper limit selectable for setpoint value</td>
			<td colspan="2">50</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="4">Type of control</td>
			<td colspan="2">Heating</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="4">ON/OFF hysteresis or dead band for P.I.D. control</td>
			<td colspan="2">0</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="4">Proportional bandwidth of the process expressed as units (&#176;C if temperature)</td>
			<td colspan="2">1</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="4">Integral time. Intertia of the process expressed as seconds</td>
			<td colspan="2">80.0</td>
		</tr>
		<tr>
			<td>9</td>
			<td colspan="4">Derivative time for P.I.D.&#160;</td>
			<td colspan="2">20.0</td>
		</tr>
		<tr>
			<td>10</td>
			<td colspan="4">Cycle time for time-proportioning output</td>
			<td colspan="2">10</td>
		</tr>
		<tr>
			<td>11</td>
			<td colspan="4">Allow/deny modification of setpoint values by frontal keyboard</td>
			<td colspan="2">Allow modification of all setpoints</td>
		</tr>
		<tr>
			<td>12</td>
			<td colspan="4">Software filter. Number of readings to calculate the comparison value PV-SPV</td>
			<td colspan="2">10</td>
		</tr>
		<tr>
			<td>13</td>
			<td colspan="4">Type of degree</td>
			<td colspan="2">&#176;C</td>
		</tr>
		<tr>
			<td>14</td>
			<td colspan="4">Type of cooling liquid</td>
			<td colspan="2">Air</td>
		</tr>
	</tbody>
</table>
Table S1: PID temperature controller settings. Display of set values; other parameters not necessary to run the incubator were left to default values.

Watch this Scientific Journal Video about Construction of a Low-cost Mobile Incubator for Field and Laboratory Use at JoVE.com

Construction of a Low-cost Mobile Incubator for Field and Laboratory Use

This paper describes a method for building an adaptable, low-cost and transportable incubator for microbial testing of drinking water. Our design is based on widely available materials and can operate under a range of field conditions, while still offering the advantages of higher-end laboratory-based models.

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water ...

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Nanyang Technological University

Research

JoVE Journal

Environment

13.9K Views.  Swiss Federal Institute of Aquatic Science and Technology (Eawag). This paper describes a method for building an adaptable, low-cost and transportable incubator for microbial testing of drinking water. Our design is based on widely available materials and can operate under a range of field conditions, while still offering the advantages of higher-end laboratory-based models.

Video: Construction of a Low-cost Mobile Incubator for Field and Laboratory Use

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

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A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

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Laboratory and Field Protocol for Estimating Sheet Erosion Rates from Dendrogeomorphology

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to severe environmental impacts. The understanding of sheet erosion is, consequently, a worldwide issue with implications for both environment and economies. However, the knowledge on how erosion evolves in space and time is still limited, as well as its effects on the environment. Below, we explain a new dendrogeomorphological protocol for deriving eroded soil thickness (Ex) by acquiring accurate microtopographic data using both terrestrial laser scanning (TLS) and microtopographic profile gauges. Additionally, standard dendrogeomorphic procedures, dependent on anatomical variations in root rings, are utilized to establish the timing of exposure. Both TLS and microtopographic profile gauges are used to obtain ground surface pro&#64257;les, from which Ex is estimated after the threshold distance (TD) is determined, i.e., the distance between the root and the sediment knickpoint, which allows de&#64257;ning the lowering of the ground surface caused by sheet erosion. For each profile, we measured the height between the topside of the root and a virtual plane tangential to the ground surface. In this way, we intended to avoid small-scale impacts of soil deformation, which may be due to pressures exerted by the root system, or by the arrangement of exposed roots. This may provoke small amounts of soil sedimentation or erosion depending on how they physically affect the surface runoff. We demonstrate that an adequate microtopographic characterization of exposed roots and their associated ground surface is very valuable to obtain accurate erosion rates. This finding could be utilized to develop the best management practices designed to eventually halt or perhaps, at least, lessen soil erosion, so that more sustainable management policies can be put into practice.

Characterizing erosion from dendrogeomorphology has usually focused on accurately finding the starting time of root exposure, by examining macroscopic or cell level changes caused by exposure. Here, we offer a detailed description of different novel techniques to obtain more precise erosion rates from highly accurate microtopographic data.

Sheet erosion is among the crucial drivers of soil degradation. Erosion is controlled by environmental factors and human activities, which often lead to ...

Construction of a Compact Low-Cost Radiation Shield for Air-Temperature Sensors in Ecological Field Studies

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although cost-effective, if not deployed with proper solar radiation shielding, the observations recorded from these sensors will be biased and inaccurate. Manufactured radiation shields are effective at minimizing this bias, but are expensive compared to the cost of these sensors. Here, we provide a detailed methodology for constructing a compact version of a previously described custom fabricated radiation shield, which is more accurate than other published shielding methods that attempt to minimize shield size or construction costs. The method requires very little material: corrugated plastic sheets, aluminum foil duct tape, and cable ties. One 15 cm and two 10 cm squares of corrugated plastic are used for each shield. After cutting, scoring, taping and stapling of the sheets, the 10 cm squares form the bottom two layers of the solar radiation shield, while the 15 cm square forms the top layer. The three sheets are held together with cable ties. This compact solar radiation shield can be suspended, or placed against any flat surface. Care must be taken to ensure that the shield is completely parallel to the ground to prevent direct solar radiation from reaching the sensor, possibly causing increased warm biases in sun-exposed sites in the morning and afternoon relative to the original, larger design. Even so, differences in recorded temperatures between the smaller, compact shield design and the original design were small (mean daytime bias = 0.06 &#176;C). Construction costs are less than half of the original shield design, and the new design results in a less conspicuous instrument that may be advantageous in many field ecology settings.

With the advent of small, low-cost environmental sensors, it is now possible to deploy high-density networks of sensors to measure hyper localized temperature variation. Here, we provide a detailed methodology for constructing a compact version of a previously described custom-fabricated radiation shield for use with inexpensive thermochrons.

Low cost temperature sensors are increasingly used by ecologists to assess climatic variation and change on ecologically relevant scales. Although ...

BtM, a Low-cost Open-source Datalogger to Estimate the Water Content of Nonvascular Cryptogams

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the carbon and nitrogen cycles in many ecosystems. Being poikilohydric organisms, they do not actively control their internal water content and need a humid environment to activate their metabolism. Therefore, studying water relationships of nonvascular cryptogams is crucial to understand both their diversity patterns and their functions in the ecosystems. We present the BtM datalogger, a low-cost open-source platform for the study of the water content of nonvascular cryptogams. The datalogger is designed to measure ambient temperature, humidity, and conductance from up to eight samples simultaneously. We provide a design for a printed circuit board (PCB), a detailed protocol to assemble the components, and the required source code. All this makes the assembly of the BtM datalogger accessible to any research group, even to those without previous specialized knowledge. Therefore, the design presented here has the potential to help popularize the use of this type of device among ecologists and field biologists.

We present a simple and cost-effective method to build an open-source datalogger that measures the conductance of nonvascular cryptogams together with the environmental temperature and humidity. We describe the hardware design of the datalogger and provide step-by-step assembly instructions, the list of required open-source logging software, the code to run the datalogger, and a calibration protocol.

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the ...