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
Drain off all of the water and autoclave on a dry cycle for 60 minutes
- 30 min sterilize time, 30 min dry time
Remove from autoclave and immediately cover with foil to prevent contamination
Place sterile batting squares in the cones so that turface won't fall through
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

Fill the sink up with water and place 200 cones into it
- 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
Allow them to soak for one day
After they are done soaking, place them in the freshly cleaned rack so that you can place the batting squares into the cones

Cut the white batting into 200 squares (~1 square inch each)
Soak them over night in a small tub with bleach
Stuff them into the bottom of the {{Griffitts:Conetainers#Cone preparation|cones]] using sterile technique

Soak the turface well a couple times so that it doesn’t dry out
1. this may take 4 L of water
Using flamed tweezers, dig a hole in the center of each of the cones designated for each plant species
Remove parafilm from the germinated plants and add enough dH₂0 to the plates to allow all the plants to be kept moist
Using another pair of flamed tweezers plant each plant into the desired cone
1. Be sure not to force the plant into the turface, so that you don’t damage the root
Using the tweezers that are covered with turface, compact the turface around the plant
1. You want the top of the cotyledon showing and that is all
Repeat steps 4 and 5 for all the plants on the row and then steps 2-5 for all remaining rows
After you have planted the rack cover with Saran wrap and place on the dome for a day
On day two, when you are watering, remove Saran wrap and only use the dome from then on to cover them

Note: On average we set an experiment for 25 days

During the first week, water every day with 1X SNM
During the second week, water every other day with autoclaved dH₂0
During the third week, water every other day with 1X SNM
During the last ½ week, water every day (except the day before you want to record your results) with dH₂0

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
1. This takes 2-3 days
From those singles patch out an ~1 cm square patch
Fill a Falcon tube with 1 mL more of dH₂0 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
1. Be sure to dig the pipetter into the turface so that it doesn’t splash
2. 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