Rcperez journal
03/29/2019
Check out this new protocol and video supporting reliable mycelium-based distributed bioproduction!
Video: [1]
Protocol: [2]
————Materials————
Kit contents: Pringles strains (4x) cheese cloth tissue coring tool Materials needed but not included: 1L glass beaker (2x) 1L glass bottles (2x) 250 mL glass beaker 1 L graduated cylinder vacuum filter flask funnel parafilm 9 cm petri dishes (24x) agar diH2O 70% ethanol spray bottle sticker labels Sharpie marker large sponge magic bullet, blender, or other grinding tool camera or camera phone analytical balance microwave incubator autoclave lab bench vacuum supply line bunsen burner and lab bench gas supply line
————Protocol————
Overview: Please use the contents of your MycoKit and a can of Pringles Original flavor that you have purchased from your local grocery store to measure growth rate of each strain on the two different Pringles substrates.
-Procedure (3-4 hours for setup, 1 hour per day for 7 days, 1 hour imaging on final day, total hours: 12 hours): Unpack your kit and take inventory.
-Take inventory of the “required but not included” materials and equipment.
-Prepare the substrate for aqueous extraction (steps 3 & 4, ~20 minutes): Weigh 14 grams of the substrate and grind the substrate for 2.5 minutes in the magic bullet, or using a blender or mortar and pestle until the substrate is pulverized.
-Setup aqueous extraction (~5 minutes): In a 1L glass beaker add pulverized substrate and 700 mL of diH2O and autoclave at 121°C for 45 minutes using standard safety precautions and standard autoclave tape
-Build your filtration system (steps 5-8, 15 minutes): While the aqueous extraction process it taking place in the autoclave, prepare the filtration system by first cutting the provided cheesecloth into 4 equal sized pieces.
-Set up your vacuum filter funnel by lining the outer surface of the funnel neck with parafilm.
-Using one piece of the cheese cloth, fold the cloth such that it will fit into the funnel without being pushed all the way through the funnel neck. Position the funnel into the flask, forming a tight seal.
-Connect the vacuum filter neck of the flask to the lab bench vacuum line.
-Collect aqueous extraction (~30 mins total): Pour the autoclaved substrate into the funnel, allowing time for the liquid aqueous extract portion to flow through the cheese cloth in the funnel and into the flask. Dispose of the solid materials that are captured by the cheesecloth in the funnel.
-Note: If the cheesecloth becomes clogged, replace the cheesecloth with a new piece.
-Prepare substrate mix (steps 10-11, ~15 minutes): Add 20 grams of agar to a 1 L glass bottle.
-Using the 1 L graduated cylinder, measure 500 mL of the aqueous extract and pour into the 1 L glass bottle containing the 20 grams of agar. Autoclave the bottle with aqueous extract and agar mix at 121°C for 30 minutes using standard safety precautions and standard autoclave tape.
-Pour plates (~1 hour): Under bench-top sterile conditions, using your bunsen burner, pour 20 mL/plate of the aqueous extract + agar mixture into ~25 Petri dish plates. Allow the plates to solidify under sterile conditions.
-Repeat steps 3-12 for each substrate.
-Plug your plates (~45 minutes total): Under sterile conditions, using sterile technique to maintain sterility of your tissue coring tool when working with each plug and taking care to prevent cross contamination of strains, you will inoculate 3 plates for each substrate for each strain in the following manner:
-Note: To sterilize your tissue coring tool and forceps simply spray the tips with 70% ethanol solution and flame very briefly.
-Take care not to melt your coring tool. If you do melt it a little, no biggie just press on with your tool trying not to melt it completely.
-Using your 5 mm tissue coring tool cut 6 plugs of tissue into the leading edge of the growing starter colony, such that 75% of the tissue plug surface is covered in mycelium tissue.
-Using your forceps, pick one tissue plug and place the plug, mycelium tissue facing down, onto the agar surface at one edge of the plate — keeping the agar surface facing upright.
-Mark the location of the tissue plug onto the bottom surface of the plate.
-Repeat 4 times, once for each strain: 3 plates per substrate/per strain, for 4 strains, 12 plates per substrate, for a total of 24 plates.
-Sterilize your tissue coring tool and forceps before working with the next strain.
-Incubate all plates, agar surface facing upright, at 30°C and ~80% humidity. To control humidity fill a 250 mL beaker with ~50 mL of water and soak the large sponge with the water. Place the beaker with the residual water and the damp sponge inside it into the incubator, as close as possible to the 24 plates.
-Colony edge tracing (~1 hour): Every 24 hours use your Sharpie to trace the leading edge profile of each growing colony. Repeat for 5 days.
-Note: To help visualize the colony edge against the substrate hold the plate up the the ceiling lights and trace the edge profile onto the bottom surface of the plate.
-Image your plates (~30 minutes): Image the bottom surface of each plate using your camera such that each image contains the full profile of the plate and is taken equidistant from its plate. We recommend you setup a camera support, perhaps an old 1000 uL pipette tip box if using a camera phone, so you can make sure each image is taken at an equal distance from it’s plate.
-Send the images to rcperez@stanford.edu.We crunch the numbers and share the results!
