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

Washing turface.
Washing removes debris like old leaves and silt.
Turface is autoclaved to sterilize it.
A. Cotton which has been inserted too far. B. Cotton which is inserted correctly.
Use a sterile scoop and funnel to add turface to the conetainer rack.
Wet the turface before planting.
When planting seedlings, be careful not to pinch them with the tweezers.
Planted seedlings should only have the cotyledon showing above the wet turface.

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 one day
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

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
Soak them overnight in a small tub with 4 L dH₂O and 130 mL bleach
- Cover with aluminum foil to prevent contamination
Stuff them into the bottom of the cones using sterile technique (a sterile inoculating stick is good for this)

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

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 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

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

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 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
- 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