Griffitts:Conetainers: Difference between revisions

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(→‎Turface preparation: Added images)
(Moved pics around)
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Image:Griffitts-Turface debris.JPG|Washing removes debris like old leaves and silt.
Image:Griffitts-Turface debris.JPG|Washing removes debris like old leaves and silt.
Image:Griffitts-Autoclaving turface.JPG|Turface is autoclaved to sterilize it.
Image:Griffitts-Autoclaving turface.JPG|Turface is autoclaved to sterilize it.
Image:Griffitts-turface covered with foil.JPG|Cover autoclaved turface with heavy-duty aluminum foil to maintain sterility.  
Image:Griffitts-turface covered with foil.JPG|Cover autoclaved turface with heavy-duty aluminum foil to maintain sterility.</gallery></div>
Image:Griffitts-Inserting cotton.JPG|Use a sterile applicator stick to push sterile cotton down into the bottom of the cones.
Image:Griffitts-Good and bad cotton.JPG|'''A.''' Cotton which has been inserted too far. '''B.''' Cotton which is inserted correctly.
Image:Griffitts-Adding turface.JPG|Use a sterile scoop and funnel to add turface to the conetainer rack.
Image:Griffitts-Hydrating turface.JPG|Wet the turface before planting.
Image:Griffitts-Digging in turface.JPG|Use sterile tweezers to dig a hole in the wet turface.
Image:Griffitts-Planting.JPG|When planting seedlings, be careful not to pinch them with the tweezers.
Image:Griffitts-Planted.JPG|Planted seedlings should only have the cotyledon showing above the wet turface.
</gallery></div>


==Cone preparation==
==Cone preparation==
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==Batting square preparation==
==Batting square preparation==
<div class="noprint">[[Image:Griffits-batting sterilization.JPG|right|thumb|A plastic tub with batting squares and bleach.]]</div>
NOTE: This should be done the same day as the [[Griffitts:Conetainers#Cone preparation|cone preparation]]
NOTE: This should be done the same day as the [[Griffitts:Conetainers#Cone preparation|cone preparation]]
* Cut the white batting into 200 squares (~1 square inch each) for each conetainer being prepared
* Cut the white batting into 200 squares (~1 square inch each) for each conetainer being prepared
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** Cover with aluminum foil to prevent contamination
** Cover with aluminum foil to prevent contamination
* Stuff them into the bottom of the [[Griffitts:Conetainers#Cone preparation|cones]] using sterile technique (a sterile inoculating stick is good for this)
* Stuff them into the bottom of the [[Griffitts:Conetainers#Cone preparation|cones]] using sterile technique (a sterile inoculating stick is good for this)
<div class="noprint"><gallery>
Image:Griffits-batting sterilization.JPG|A plastic tub with batting squares and bleach.
Image:Griffitts-Inserting cotton.JPG|Use a sterile applicator stick to push sterile cotton down into the bottom of the cones.
Image:Griffitts-Good and bad cotton.JPG|'''A.''' Cotton which has been inserted too far. '''B.''' Cotton which is inserted correctly. </gallery></div>


==Rack preparation==
==Rack preparation==
<div class="noprint">[[Image:Griffitts-Adding turface.JPG|right|thumb|Use a sterile scoop and funnel to add turface to the conetainer rack.]]</div>
* Wipe down a funnel and a scoop with 70% ethanol
* Wipe down a funnel and a scoop with 70% ethanol
* Using the sterile funnel and scoop, fill each cone to the top with turface
* Using the sterile funnel and scoop, fill each cone to the top with turface
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* Cover with Saran wrap, top with the dome, and place on the light bank
* Cover with Saran wrap, top with the dome, and place on the light bank
* On the second day of [[Griffitts:Conetainers#Watering|watering]], remove Saran wrap and only use the dome from then on to cover them
* On the second day of [[Griffitts:Conetainers#Watering|watering]], remove Saran wrap and only use the dome from then on to cover them
<div class="noprint"><gallery>
Image:Griffitts-Hydrating turface.JPG|Wet the turface before planting.
Image:Griffitts-Digging in turface.JPG|Use sterile tweezers to dig a hole in the wet turface.
Image:Griffitts-Planting.JPG|When planting seedlings, be careful not to pinch them with the tweezers.
Image:Griffitts-Planted.JPG|Planted seedlings should only have the cotyledon showing above the wet turface.</gallery></div>


==Watering==
==Watering==

Revision as of 13:34, 4 August 2009


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 turface.

  • Washing removes debris like old leaves and silt.

    Washing removes debris like old leaves and silt.

  • Turface is autoclaved to sterilize it.

    Turface is autoclaved to sterilize it.

  • Cover autoclaved turface with heavy-duty aluminum foil to maintain sterility.

    Cover autoclaved turface with heavy-duty aluminum foil to maintain sterility.

Cone preparation

A conetainer rack in the sink being sterilized with bleach.
  • 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

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)
  • A plastic tub with batting squares and bleach.

    A plastic tub with batting squares and bleach.

  • Use a sterile applicator stick to push sterile cotton down into the bottom of the cones.

    Use a sterile applicator stick to push sterile cotton down into the bottom of the cones.

  • A. Cotton which has been inserted too far. B. Cotton which is inserted correctly.

    A. Cotton which has been inserted too far. B. Cotton which is inserted correctly.

Rack preparation

Use a sterile scoop and funnel to add turface to the conetainer rack.
  • 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 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
  • Wet the turface before planting.

    Wet the turface before planting.

  • Use sterile tweezers to dig a hole in the wet turface.

    Use sterile tweezers to dig a hole in the wet turface.

  • When planting seedlings, be careful not to pinch them with the tweezers.

    When planting seedlings, be careful not to pinch them with the tweezers.

  • Planted seedlings should only have the cotyledon showing above the wet turface.

    Planted seedlings should only have the cotyledon showing above the wet turface.

Watering

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

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
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