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&mdash;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