Griffitts:Conetainers

Turface preparation

Note: For Best results do turface one week prior to desired planting day so that it has time to dry

• Get two large white tubs and fill them ½ full with Pro League turface
• Rinse the turface repeatedly until the water comes off clear
  • Your goal is to remove the smaller particles as well as leaves, roots, and salt buildup left over from prior experiments
• Drain off all of the water and autoclave
  • For small loads, use the autoclave in 758 WIDB
    • 30 minutes sterilize time, 30 minutes dry time
  • For large loads, use the autoclaves in 740 WIDB
    • 60 minutes sterilize time, 99 minutes dry time
    • If possible leave in the autoclave overnight for additional drying
• Remove from autoclave and immediately cover with foil to prevent contamination
  • It will be hot, so bring orange autoclave gloves

Cone preparation

• Fill the sink up with water and place a rack with 200 cones into it upside down
  • Make sure that all the roots from previous experiments have been removed—this may require scrubbing each one with the brush
• When they are all clean add bleach until you can smell it easily (about 1.5 L)
• Allow them to soak for several hours
  • We used to do this overnight, but the bleach was corroding the sinks
• After they are done soaking, take them out, and place them on a cart covered with benchcoat and leave them in the autoclave room for an additional day to dry
• Wipe down the rack well with 70% ethanol
• Wrap white tape around the top of the rack so you can label each row
• Wipe down the cover with 70% ethanol
• Proceed to batting square preparation

Batting square preparation

NOTE: This should be done the same day as the cone preparation

• Cut the white batting into 200 squares (~1 square inch each) for each conetainer being prepared
  • They sometimes stick together, so it doesn't hurt to cut a few extra
• Soak them overnight in a small tub with 4 L dH₂O and 130 mL bleach
  • Cover with aluminum foil to prevent contamination
• While wearing gloves, remove the squares from the bleach and squeeze them dry
• Place them on a cart covered with benchcoat and leave them in the autoclave room for an additional day to dry
  • This can be the same cart that the conetainer rack is drying on
• When the squares are dry, stuff them into the bottom of the cones using sterile technique (a sterile inoculating stick is good for this)

Rack preparation

• Wipe down a funnel and a scoop with 70% ethanol
• Using the sterile funnel and scoop, fill each cone to the top with turface
• Compact the turface by lifting it and pounding it against the counter
• Fill each cone again until it is about 1 cm from the top of the cone
  • It won’t compact much the second time, so be sure not to overfill
• Put on gloves and spray them with 70% ethanol
• Wipe off any extra turface not in the cones
• Cover rack with Saran wrap until you are ready to plant and place dome on it

Planting

• Add 3 mL sterile dH₂O to a row of ten cones
• Using flamed tweezers, dig a hole in the center of each of the cones
• Remove parafilm from the germinated plants and add enough dH₂O to the plates to allow all the plants to be kept moist
• Using the flamed tweezers, insert the appropriate plant into the desired cone
  • It may sometimes be desirable to plant two plants per cone
  • Be sure not to force the plant into the turface, so that you don’t damage the root
• Using the flamed tweezers, compact the turface around the plant
  • You want the top of the cotyledon showing and that is all
• After planting all 10 cones, add another 3 mL sterile dH₂O to each cone
• Proceed to the next row of cones
• After you have planted the entire rack, add another 3 mL sterile dH₂O to each cone
• Cover with Saran wrap, top with the dome, and place on the light bank
• On the second day of watering, remove Saran wrap and only use the dome from then on to cover them

Watering

Note: On average we set an experiment for 25–28 days

• Use a black marker to mark ten 3-mL intervals on a 25-mL pipette
• Being careful not to touch the rack, turface, or plants with the pipette, dispense 3 mL of water or SNM per cone
  • Do not reuse 25-mL pipettes
• Watering schedule:
  • During the first week, water every day with 1X SNM
  • During the second week, water every other day with autoclaved dH₂O
  • During the third week, water every other day with 1X SNM
  • During the last week, water every day (except the day before you want to record your results) with dH₂0

Inoculation

Note: Inoculation day is four days after planting, so you will want to make sure that you have steps 1 and 2 done by Day 4 of the experiment

• Grow up all your bacterial strains to singles
  • This takes 2–3 days
• From those singles patch out an ~1 cm square patch
• Fill a Falcon tube with 1 mL more of dH₂O than the number of plants you will be inoculating with the same bacterial strain
• Put enough bacteria into the tube to make the solution slightly opaque
• Vortex
• Add 1 mL of the bacteria solution to each plant
  • Be sure to dig the pipetter ~0.5 cm into the turface so that it doesn’t splash
  • Make sure you keep track of where you are so you don’t inoculate the wrong plants!
• Repeat Steps 3–6 for all bacterial strains