Preparation of Phytophthora zoospore suspensions

• Before carrying out tedious tasks, inspect the container of the P. capsici from ATCC
• Ensure that the packaging is intact, and free of damage before beginning process

• Gather the following materials
• Do not filter or alter the V8 juice

• Pour 800mL of DI water into a 2 L glass Erlenmeyer flask
• Add a stir bar to the flask and place on the stirring plate
• Stir at medium speed (approximately 200-300 rpm, depending on the stir bar size)
• Measure 3.0 g of CaCO3 on the analytical balance
• Add the CaCO3 powder into the flask and stir for at least 2 min at medium speed

Figure 1. Prepare V8 medium in a 2L Erlenmeyer flask

• Measure 200 mL of V8 tomato juice in a glass beaker
• Add the V8 juice to the 2L Erlenmeyer flask while continuously stirring
• Mix for at least 2 min at medium speed
• Measure 15.0 g of agar in a weigh boat, and then add the powder to the 2L beaker
• Mix for at least 2 min at medium speed, observing that the agar is not adhering to the wall of the flask

• Insert a calibrated pH probe into the V8 medium

Figure 2. Adjust the pH of the V8 medium to 7.2 ± 0.2.

• Using a transfer pipette, adjust the pH to 7.2 ± 0.2. See note for suggestions on pH adjustment

• Retrieve the stir bar using a magnetic stir bar retriever

• Place a 12-14 inch long piece of aluminum foil on the counter
• Fold the aluminum foil in half, and then fold in half one additional time
• Place the stir bar from step 1.3 into the center of the foil
• Create an aluminum foil "butterfly" around the stir bar and pinch into place
• Without dropping the stir bar into the flask, carefully wrap the aluminum foil around the mouth
• Place a small piece of autoclave tape on the foil to secure

• Autoclave the V8 medium at 121 C for 15 minutes.

• Place the 2L flask on the disinfected stir plate
• Pull the ends of the foil "butterfly lid" to release the stir bar into the flask but be careful to NOT remove the aluminum foil lid entirely
• Mix for at least 2 min at medium speed on the stir plate as the agar cools to approximately 45C

• Wait approximately 1 hour for agar to solidify
• Stack plates upside down to avoid condensation on the agar, being careful not to open the lids

Figure 3. Prepared V8 agar plates

• Store stacked plates (upside down) in a refrigerator or cold room.

• Retrieve the cryovial containing P. capsici ATCC 52771 from −80 °C storage.
• Allow the vial to thaw at room temperature for 2–3 minutes or until the contents are partially thawed.
• Gently mix the contents by flicking the vial. DO NOT VORTEX

Transfer to agar plate

• Flame sterilize a loop or use a sterile pipette tip.
• Transfer a small amount (5–20 µL) of the thawed culture onto the center of a V8 agar plate.
• Using the sterile loop, gently streak the culture onto the agar surface.
• Close the plate immediately.

Incubate culture

• Incubate plates under the following conditions:
• Temperature: 22–25 °C
• Light: dark or low light
• Duration: 48–96 hours

Expand culture for experiments

• Once a colony reaches 2–3 cm diameter, use a sterile cork borer or scalpel.
• Remove a 5–7 mm plug from the actively growing edge.
• Transfer to a fresh V8 agar plate.
• Incubate 2–3 days to produce an actively growing culture plate.
• This plate will be used for sporangia induction and zoospore production.

• Grow culture on V8 agar or corn meal agar until an actively growing colony margin is visible.
• Remove V8 agar plates from refrigerated storage and allow them to warm to room temperature.
• Using sterile technique, transfer a 5–7 mm plug of actively growing mycelium from a stock culture to the center of a fresh V8 agar plate.
• Incubate plates under the following conditions:
• Temperature: 22–25 °C
• Light: dark or low light
• Duration: 2–4 days

• Add 5–10 mL of room temperature sterile DI water to each plate.
• Allow plates to sit undisturbed at room temperature for 30–60 minutes to stimulate sporangia formation.

• Turn on microscope
• If using an older microscope, allow 10 minutes for the bulb to warm (for LED optics no waiting is necessary)
• Prepare a standard glass microscope slide

• Withdraw 50 µL of liquid from the plate surface using a sterile pipette.
• Place the droplet on a glass microscope slide.
• Carefully place a coverslip over the droplet.

• Observe the sample using a compound microscope
• Start at 100× magnification to locate zoospores
• Increase to 400× to evaluate motility

• Carefully remove liquid from each plate and discard into a waste vial
• Replace with 5-10 mL of cold sterile water (4–10 °C)
• Allow 5-10 minutes to stimulate zoospore release

• Using a sterile pipette, collect the liquid from each plate surface and transfer to a sterile tube
• Avoid scraping agar surface
• Label the sterile tube as P capsici zoospores and include standard labeling according to the lab SOP

• Turn on microscope
• If using an older microscope, allow 10 minutes for the bulb to warm (for LED optics no waiting is necessary)

• Prepare a standard glass microscope slide.
• Using a micropipette, place a 10 µL droplet of zoospore suspension on the slide.
• Carefully place a coverslip over the droplet.
• Observe the sample using a compound microscope from 100X to 400X as in step 5

• Prepare a solution with the following ingredients.
• Adjust pH to approximately 7.0

• Clean up used glassware
• Dispose of used weigh boats or weighing paper
• Ensure analytical balance is clean (no spilled powders)
• Ensure magnetic stir plate is clean (no liquid of powder spills)
• Dispose of all biological material in biohazard bags according to the local SOP
• Surface sanitize all counters and used materials according to the local SOP