Protocol

Feces retrieval

1. Take small beakers (one for each sample you plan to run in parallel), tape, marker, and scale to the freezer room.
2. Set out the beakers on the counter near the freezer. Affix pieces of tape to the counter in front of each beaker.
3. Retrieve the box of samples from the -80C freezer. When out of the freezer, the box/samples should remain on ice at all times.
4. Remove as many sample baggies as is necessary, put them on ice, and return the stock box to the freezer.
5. Each bag will have (1) a paper label and (2) another baggie with the fecal sample:

6. On the tape, write the UID (e.g., R1125) and the recap ID (e.g., r1581).
7. Tare the scale with the beaker on it and measure out 1 g of feces.
1. If there is >1 g available, measure out only what is necessary and return the rest to the baggie.
2. If there is <1 g available, transfer it all to the beaker.
8. Return the baggies (even if all the sample was used) to the box in -80C labelled “Analyzed squirrel samples”.
9. Put the labelled tape on the relevant beaker.

Egg floatation

1. Bring the materials to P313.
2. Mix the 1 g of fecal matter with about 5-10 mL of flotation solution in the beaker
3. Once mixed, fully strain the mixture through a tea strainer or cheesecloth into a separate beaker
4. Pour the mixture into a 15 mL conical tube and fill the tube as full as possible with flotation solution.
5. lace a cover slip on top of the meniscus, so that the coverslip is touching the top edge of the tube and the floatation solution.

6. Spin in a centrifuge for 5 minutes on 2500 rpm
1. Note: the centrifuge should be automatically set (Program 1) to the correct settings. These settings include a slow acceleration and no breaking, so the entire spin cycle takes about 7.5 minutes.
7. Lift the cover slip slowly and carefully and put it on a slide labeled with tape.

Slide scanning

1. Take the slides to the Revolve microscope in P370.
2. Create a new album for the day using the current date and your initials (e.g., 20230315_MKW) as the album name:

3. Set the image name using the UID and recap ID (e.g., R1691_1360). Each image you snap will have a suffix attached as a counter (e.g., R1691_1360_0001, R1691_1360_0002).
4. Ensure the image type is set to tiff (not jpg).
5. Begin scanning the slide at 10X at the bottom-left corner of the slide.
6. Use the digital zoom on the iPad (pinch in/out) to quickly zoom in on objects of interest.
7. If you have found an egg or worm, switch to 20X, zoom to 170%, and snap a photo.

Image annotation

1. Click the Browse button in the top-left corner.
2. Scroll through the images, ignoring those that you deem not to contain a parasite.
3. Open images of parasites and click Create annotation in the bottom right corner.
4. Set the Objective setting to be 20X - this will allow you to measure each parasite in µm.
5. Use the digital zoom to get a clear, high-definition view of the parasite.
6. Click the Blue box and then set the measurement to be length or area:

7. Measure the length, width, and area of each egg and the width and area of each worm. Move the length measurement bars so that they don’t occlude the parasite. Record the area in the data sheet (WheelerLab/Data/project-squirrel_parasitome/fecal_data/fecal_data.xlsx), and then remove the drawn area shape. Save the annotation (a new image will be created with an automatically generated name).

Note: Only annotate/record the area measurements for whole worms, not partial carcasses.

8. Add a scale bar to the bottom-right corner.
9. Save the annotation.
10. When finished with a slide, dispose of it in the cardboard box (labelled “sharps”) above the microscope.

Annotating on Personal iPad

1. Go to browse option in upper left corner
2. select photos/ album
3. share (box with arrow)
4. Send to Offline iPad
5. airdrop to nearby devices
6. download Revolve Pro app on personal ipad
7. annotate like as seen above (with pencil if available)
8. (send back to microscope ipad?)

Recipes

33% Zinc Sulfate Solution

1. Mix water and 330 g of zinc sulfate together so that the volume of the solution is 1000 mL
2. Check the SPG with a hydrometer
3. Additional water may be added to get desired SPG

Sheather’s Sugar Solution

1. Mix 355 mL (12 fl oz) of water and 454 g (1 lb) of granulated sugar (sucrose).
2. Dissolve the sugar in the water over low heat.
1. If there are chunks grind it up with a motor or soak it and mix with water to soften it up
3. Once fully dissolve allow the mixture to cool to room temperature
4. Add 6 mL of formaldehyde
5. Check the SPG with a hydrometer