Portable Petrifilm™ Incubator for Inexpensive In-Field Analysis of Water Quality

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Construction of the incubator should only take about 2 hours

Materials (For online sources and prices see attached BillofMaterials.pdf)

• Insulated Container (recommended: Stanley Adventure Series 7 Quart Cooler)
• 2 Resistive Heating Pads
• Count Up Timer (optional)
• 12V Thermostat
• 12V Lithium Ion Battery 10Ah [7 X 18650 cells]
• Closed Cell Foam Insulation
• Inner Chamber (we used a 4"x4"x4" junction box)
• 3 Circuit Terminal Block Speaker Wire
• Thermometer
• Add-a-Fuse Crimp kit
• Printed PDF Templates (Note: Each of the six template should be printed on a single sheet of paper at 100% size.)

The total cost is estimated at $150 to $200.

Tools:

• Wire strippers
• Drill
• 5/32" and 3/8" drill bit
• Computer and Printer (to print 1:1 patterns)
• Box cutter
• Hot glue gun
• Screwdriver
• Multimeter

Attachments

• Bill_of_Materials.pdf
  Download
• IncubatorTemplates.pdf
  Download

In this step, use the templates printed in Step 2 to cut the foam insulation into the appropriate patterns.

1. Cut out one bottom foam layer pattern using the template labeled “BOTTOM”
2. Cut out one top foam layer pattern using the template labeled “TOP”
3. Cut out nine middle foam layer patterns using the template labeled “MIDDLE”

Together, these foam patterns should fully insulate your cooler. If they do not, adjust the number of middle foam layers until the cooler is full. Note: For best performance, we recommend use of closed cell foam with a high R-value (capacity of a material to resist heat flow).

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In this step, we will cut all of the cardboard components of the incubator using the templates provided.

1. Use the Petrifilm Holder template to cut an appropriate sized rectangle of cardboard
2. Use a sharp edge to crease the rectangle at the designated location.
3. This shelf can hold up to 20 Petrifilm plates. Please utilize a second shelf on top of the first if you wish to incubate more than 20 plates.

Next, we will be creating the heating pad spacer. This will be utilized to suspend the heating pads in the bottom of the chamber.

1. Again, cut out a cardboard rectangle using the template provided.
2. Cut four slits into the rectangle as shown, and fold the spacer in half
3. Finally, cut out a small divot in the base of the spacer. The thermocouple will be mounted here in a later step.

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In this step, a port is drilled for the system's thermometer.

1. Use a 5/32” drill bit to create a centered hole located 0.57” above the incubator's base. If you are using our recommended cooler, feel free to use the provided template as shown and simplify the aligning process.

Note: For this incubator, our foam bottom layer is 0.57" thick. If your foam has a different thickness, adjust the height of the thermometer port to account for this discrepancy. (With thicker foams, move the port up)

Next, we will continue this port into the inner chamber

1. Place the bottom layer of foam into the cooler
2. Place the inner chamber in the center of the cooler.
3. Utilize a middle layer of foam to ensure that the inner chamber is centered, then remove the layer
4. Continue the port into the inner chamber, utilizing the previously drilled hole as shown. This hole should be approximately ¼ inch from the bottom of the inner chamber.
5. Remove the inner chamber, and increase the diameter of the inner chamber's port to 3/8".
6. Ensure the thermometer can reach the inner chamber from the outside

In this step, the electrical heating system of the incubator is assembled.
The required components are:

• Thermostat
• 12 ohms of heating elements or 2 heating pads
• 3 position terminal block
• Add-a-fuse 5A Fuse
• 12V Battery
• 16 -18 gauge speaker wire

Wiring the Incubator:

1. Begin by setting the thermostat to 38°C. This is accomplished by setting position 2 of the red DIP switch on the thermostat to the ON (up) position. All other positions should be set to OFF (down).
2. Strip one end of 5.52mm male cable to expose positive and negative leads
3. Connect each of these ends to fork connectors, and wire these leads to terminals 1 and 2 of the block.
4. Wire the switch to positions 1 and 3 of the terminal block
5. Wire the thermostat to the terminal block as shown in picture 1.
6. Feed two foot long wires from the thermostat into the inner chamber. These will be connected to the heating pads
7. Connect the heating pad in series to these wires so they can rest in the inner chamber
8. Feed the thermocouple from the thermostat into the inner chamber as well

The heating pad and thermocouple will be mounted in the heating assembly in the next step.

The heating pad spacer is used to suspend the pads in the inner chamber while preventing direct contact with the Petrifilms.

1. Slide each heating pad into the cardboard, as shown in the pictures.
2. Use hot glue to firmly secure the pads in the spacer
3. Glue the thermocouple to the back of the heating assembly.
4. Slide the completed heating assembly into the inner chamber.

In this step, we will be mounting the thermometer in our incubator.

IMPORTANT: After this step, the inner chamber will be permanently mounted in the incubator. Ensure the heating pads and thermocouple are properly inserted in the system.

1. Ensure the inner chamber is placed on top of the bottom later of foam, and is centered in the incubator.
2. Slide the thermometer from the outside of the incubator into the inner chamber.
3. Firmly mount the thermometer by filling the incubator port with hot glue. Any air leaks will decrease the efficiency of the system.
4. Apply hot glue around the probe's circumference, then fully insert it, ensuring it is oriented correctly, holding it in place until the glue sets.
5. Apply hot glue around thermometer on the inside of the cooler. You may have to move the inner chamber back to access it.

A good seal is important for optimal insulation, and to ensure the incubator operates effectively.

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In this step we will trim one of the foam middle layers to accommodate the electrical system.

1. Use the modified middle FOAM template to trim one of the middle insulation pieces as shown
2. Place the fuse, thermostat, and terminal block with the connections facing down on top of the bottom foam layer in the corner. Excess wire should be laid flat on the bottom layer of foam.
3. Add the trimmed middle layer of foam, so the removed section accommodates the electrical components.
4. Ensure the electrical components do not protrude above the trimmed middle layer, and add a standard middle layer of foam
5. Continue adding layers of foam until the incubator is full. It is absolutely critical that the lid of the cooler still closes completely. Trim or remove foam until this is the case.

Now that you have constructed the Petrifilm™ Incubator, you should test it in a controlled environment before relying upon it in the field.

We recommend that you obtain the E. Coli Petrifilms™ from 3M and and establish a control using sterile water. Additionally, test water from an actual creek likely to be contaminated with fecal matter. Any slow-running creek where people walk their dogs without cleaning up is a likely target.

Follow 3M's instructions for inoculation. We hope to make a separate instructable about this step and the use of the Petrifilm™ in general, but this is a summary.

1. Lay the films on a flat surface, with the flaps opening towards you.
2. Fill a 1mL pipette with the water to be tested.
3. Lift the flap on the first film, and use a pipette to gently spread the solution on the film. Ensure that the pipette does not scratch the film surface, and that the water does not overflow the film's circular area.
4. Gently replace the clear sheet on the surface of the film. Make sure there are not significant air bubbles between the clear sheet and the film's surface.
5. Use a permanent marker to write the sample name on each film.
6. Repeat steps 2 - 5 for as many films as needed.

This instructional video by 3M may clarify the above steps.

1. Place a cardboard divider on top of the heating pad holder and place your petrifilms on top of the cardboard.
2. If you are using more than 20 films, use the cardboard divider to separate the first 20 films from the additional films inside the incubator.
3. Close the inner chamber by placing the lid on top and screwing it in. Place any more remaining layers of foam in the incubator.
4. Make sure the incubator's battery is fully charged.
5. When all the pieces are in place, place the top layer of foam in the incubator and close the lid. It is extremely important to ensure the lid is completely sealed.
6. Set a timer to incubate the films for 48 hours. Ensure the incubator's temperature does not fall below 37±1°C.

Check the thermometer a few times during the incubation period to see that it is very close to 37°C or 98°F. After 48 hours, remove the Petrifilms™. If you have colonies in the sterile water or if you have no colonies at all in the dirty creek water, you must investigate further until you understand why the incubator did not give both a negative and positive result. If however you understand the results and think the Petrifilm­™ Incubator is ready for use, then congratulations! You now have a portable, battery powered incubator that can give you useful field information without readily available electrical power. It is important to ensure you have a full charge in the battery. When not in use, disconnect it from the charger and ensure it is in the off position. Despite these precautions, batteries lose energy continuously and it is important that you charge it before embarking on your journey and, if possible, before heading into the field. A fully charged battery left for a month will likely be discharged! If the ambient temperature is very low, keep the incubator in a warm place to ensure battery life lasts through an entire incubation period.

For more details on Petrifilm™ incubation, see the 3M Petrifilm™ data sheet.

This project was developed and is maintained by the Engineers Without Borders USA, Greater Austin Chapter, in the 2015-2016 school year. This is a combined professional and student chapter. Many people contributed to this project, but Evan Bartilson was a key contributor.

We are actively seeking users to test this Incubator and report your experience back to us. Please contact us via the emails listed on our contact page or by sending email to Robert L. Read [email protected].