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/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/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/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/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/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/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/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/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/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 &amp;amp; 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&amp;deg;C to 200&amp;deg;C; Sensor Pt100, Type FS-400P</td></tr><tr><td>Universal enclosure</td><td>OKW Geh&amp;auml;use System</td><td>C2012201</td><td>Dimensions 200 x 120 x 60 mm</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&amp;auml;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 &amp;ndash; 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>&amp;nbsp;-&amp;nbsp;</td><td>&amp;nbsp;-&amp;nbsp;</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/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/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/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/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/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/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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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

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Biology

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Neuroscience

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 ...

Simulating Temperature in a Soil Incubation Experiment

The study of warming impact on soils requires a realistic and accurate representation of temperature. In laboratory incubation studies, a widely adopted method has been to render constant temperatures in multiple chambers, and via comparisons of soil responses between low- and high-temperature chambers, to derive the warming impact on soil changes. However, this commonly used method failed to imitate both the magnitude and amplitude of actual temperatures as observed in field conditions, thus potentially undermining the validity of such studies. With sophisticated environmental chambers becoming increasingly available, it is imperative to examine alternative methods of temperature control for soil incubation research. This protocol will introduce a state-of-the-art environmental chamber and demonstrate both conventional and new methods of temperature control to improve the experimental design of soil incubation. The protocol mainly comprises four steps: temperature monitoring and programming, soil collection, laboratory incubation, and warming effect comparison. One example will be presented to demonstrate different methods of temperature control and the resultant contrasting warming scenarios; that is, a constant temperature design referred to as stepwise warming (SW) and simulated in situ temperature design as gradual warming (GW), as well as their effects on soil respiration, microbial biomass, and extracellular enzyme activities. In addition, we present a strategy to diversify temperature change scenarios to meet specific climate change research needs (e.g., extreme heat). The temperature control protocol and the recommended well-tailored and diversified temperature change scenarios will assist researchers in establishing reliable and realistic soil incubation experiments in the laboratory.

Laboratory soil warming experiments usually employ two or more constant temperatures in multiple chambers. By presenting a sophisticated environmental chamber, we provide an accurate temperature control method to imitate the magnitude and amplitude of in situ soil temperature and improve the experimental design of soil incubation studies.

The study of warming impact on soils requires a realistic and accurate representation of temperature. In laboratory incubation studies, a widely adopted ...

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

Bioengineering

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.&#160;The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.&#160;Eggs were quickly collected after laying at a broiler breeder farm.&#160;They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.&#160;The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

Immunology and Infection

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Medicine

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...