Team:Purdue/Seed plating

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==Sterile Agar Plate Preparation and Seed Placement==
 
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[[Seed sterilization link]]
 
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[[Agar prep link]]
 
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==Materials==
==Materials==
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#sterile seed in deluted agar
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*[[Team:Purdue/Seed sterilization|sterile seed]] in dilute (0.12%) agar
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#sterile agar media
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*sterile [[Team:Purdue/MS media preparation|MS media]]
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#sterile hood environment
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*sterile hood environment
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#P1000 pipeter
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*P1000 pipet (or smaller)
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#blue pipeter tips
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*blue pipet tips
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#plates
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*plates
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#parafilm tape
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*parafilm tape
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#70% ethanol solution in spray bottle
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*70% ethanol solution in spray bottle
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#grid template for desired seed alignment (to be placed under transparent media and referenced during seed placement)
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*grid template for desired seed alignment (to be placed under transparent media and referenced during seed placement)
==Methods==
==Methods==
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Pouring Agar
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===Pouring media===
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#sterilize items one by one with 70% ethanol spray, placing each item into sterile hood after being sprayed.
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#Sterilize each item with 70% ethanol spray before transferring to the hood.
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#once all are in hood, remove aluminum foil wrap from agar flask.
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#Once all items are in hood, remove aluminum foil wrap from the flask containing the sterile media.
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#remove lid off first plate and keeping the lid as a shield between you and the open plate, pour agar into plate. Fill to halfway point, carefully avoiding air bubbles in media. Place lid right-side-up on hood surface, then set poured plate on top. Allow to cool before replacing lid as to avoid condensation.
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#Fill plates to the halfway point, carefully avoiding air bubbles in media. Holding the lid in front of you as you work is a good way to avoid contamination. Place lid right-side-up on hood surface, then set poured plate on top. Allow to cool before replacing lid to avoid condensation.
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#Repeat for remaining plates.
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Seed Placement
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===Seed placement===
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#draw horizontal line across plate with a center point roughly one inch from the perimeter of plate  
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#Draw horizontal line across plate with a center point roughly one inch from the perimeter of plate.
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#place reference grid (link to pic or file) under plate, matching horizontal lines.  
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#Place reference grid (link to pic or file) under plate, matching horizontal lines.  
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#set pipeter to small volume(?) so that when fully released the seed mixture level remains within the lower angled section of the blue tip.
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#Set pipet to small volume(?) so that when fully released the seed mixture level remains within the lower angled section of the blue tip.
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#after pulling a small amount of seed (#3) into blue tip, remove tip.
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#After pulling a small amount of seed (#3) into blue tip, remove tip.  Surface tension should prevent the solution from falling out of the pipet.
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#remove lid off first plate and keeping the lid as a shield between you and the open plate.
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#Remove lid from first plate and hold the lid above the plate to act as a face shield to prevent contamination.
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#to place seed, reference the grid below the agar and gently touch surface of agar with tip, so that only one seed is placed onto the agar. (Too little seeds in tip will result in no seeds coming out of tip, and too much seed mixture will cause a group of seeds to come out of tip onto agar). If seeds do not come easily from tip, pressing one's finger into top of tube may work to create pressure and dislodge seed.
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#To place seed, reference the grid below the agar and gently touch surface of agar with tip, so that only one seed is placed onto the agar. If the pipet tip is too full, multiple seeds will rush out. If seeds do not come easily from tip, pressing one's finger into top of tube may work to create pressure and dislodge seed.
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#place seeds at desired density.
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#Place seeds at desired density.
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#replace lid and move to next plate. Repeat steps.
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#Replace lid and move to next plate. Repeat steps.
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#when all plates are finished, seal with parafilm tape.
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#When all plates are finished, seal with parafilm tape.
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#plates can be placed horizontal or vertical in growth room
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==Notes==
==Notes==
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Keep head out of hood. Contamination from hair, mouth, nose, hands, etc. will infect culture plate with mold spores.
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*Keep head out of hood. Contamination from hair, mouth, nose, hands, etc. will infect culture plate with mold spores.
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*Plates can be placed horizontally or vertically in growth room.
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Courtesy of Mike Poling, Lab Technician: Horticulture & Landscape Architecture, Purdue University.

Latest revision as of 19:37, 8 July 2010

Contents

Materials

  • sterile seed in dilute (0.12%) agar
  • sterile MS media
  • sterile hood environment
  • P1000 pipet (or smaller)
  • blue pipet tips
  • plates
  • parafilm tape
  • 70% ethanol solution in spray bottle
  • grid template for desired seed alignment (to be placed under transparent media and referenced during seed placement)

Methods

Pouring media

  1. Sterilize each item with 70% ethanol spray before transferring to the hood.
  2. Once all items are in hood, remove aluminum foil wrap from the flask containing the sterile media.
  3. Fill plates to the halfway point, carefully avoiding air bubbles in media. Holding the lid in front of you as you work is a good way to avoid contamination. Place lid right-side-up on hood surface, then set poured plate on top. Allow to cool before replacing lid to avoid condensation.

Seed placement

  1. Draw horizontal line across plate with a center point roughly one inch from the perimeter of plate.
  2. Place reference grid (link to pic or file) under plate, matching horizontal lines.
  3. Set pipet to small volume(?) so that when fully released the seed mixture level remains within the lower angled section of the blue tip.
  4. After pulling a small amount of seed (#3) into blue tip, remove tip. Surface tension should prevent the solution from falling out of the pipet.
  5. Remove lid from first plate and hold the lid above the plate to act as a face shield to prevent contamination.
  6. To place seed, reference the grid below the agar and gently touch surface of agar with tip, so that only one seed is placed onto the agar. If the pipet tip is too full, multiple seeds will rush out. If seeds do not come easily from tip, pressing one's finger into top of tube may work to create pressure and dislodge seed.
  7. Place seeds at desired density.
  8. Replace lid and move to next plate. Repeat steps.
  9. When all plates are finished, seal with parafilm tape.

Notes

  • Keep head out of hood. Contamination from hair, mouth, nose, hands, etc. will infect culture plate with mold spores.
  • Plates can be placed horizontally or vertically in growth room.


Courtesy of Mike Poling, Lab Technician: Horticulture & Landscape Architecture, Purdue University.