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Team:Purdue/Seed plating
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==Materials== | ==Materials== | ||
| - | + | *[[Team:Purdue/Seed sterilization|sterile seed]] in dilute (0.12%) agar | |
| - | + | *sterile [[Team:Purdue/MS media preparation|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== | ==Methods== | ||
| - | Pouring | + | ===Pouring media=== |
| - | # | + | #Sterilize each item with 70% ethanol spray before transferring to the hood. |
| - | # | + | #Once all items are in hood, remove aluminum foil wrap from the flask containing the sterile media. |
| - | # | + | #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 | + | ===Seed placement=== |
| - | # | + | #Draw horizontal line across plate with a center point roughly one inch from the perimeter of plate. |
| - | # | + | #Place reference grid (link to pic or file) under plate, matching horizontal lines. |
| - | # | + | #Set pipet to small volume(?) so that when fully released the seed mixture level remains within the lower angled section of the blue tip. |
| - | # | + | #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. |
| - | # | + | #Remove lid from first plate and hold the lid above the plate to act as a face shield to prevent contamination. |
| - | # | + | #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. |
| - | # | + | #Place seeds at desired density. |
| - | # | + | #Replace lid and move to next plate. Repeat steps. |
| - | # | + | #When all plates are finished, seal with parafilm tape. |
| - | + | ||
==Notes== | ==Notes== | ||
| - | Keep head out of hood. Contamination from hair, mouth, nose, hands, etc. will infect culture plate with mold spores. | + | *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. | ||
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
- Sterilize each item with 70% ethanol spray before transferring to the hood.
- Once all items are in hood, remove aluminum foil wrap from the flask containing the sterile media.
- 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
- Draw horizontal line across plate with a center point roughly one inch from the perimeter of plate.
- Place reference grid (link to pic or file) under plate, matching horizontal lines.
- Set pipet to small volume(?) so that when fully released the seed mixture level remains within the lower angled section of the blue tip.
- 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.
- Remove lid from first plate and hold the lid above the plate to act as a face shield to prevent contamination.
- 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.
- Place seeds at desired density.
- Replace lid and move to next plate. Repeat steps.
- 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.
