Culture Establishment – Inoculation or Sowing Spores

Inoculation or “Sowing” of C-Fern Spores on the agar surface.

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Figure 1. Supplies needed for sowing C-Fern spores

Materials

  • Surface sterilized C-Fern spores in a graduated spore vial.
    (Wild type (RNWT1) and mutant spores are available as pre-sterilized class sized and bulk unsterilized units. Bulk unsterilized spores and spores collected directly from sporophytes should be surface sterilized prior to inoculation.)
  • Sterile distilled water
  • Sterile transfer pipet (plastic) OR equivalent
  • Sterile spore spreader
  • Prepared Petri dishes containing C-Fern Medium (prepared in advance)

Methods

The instructor or students may sow spores. Inoculate or “sow” spores in an area that is clean and relatively free from drafts. A spore vial holder or other rack should be used to provide a stable container that helps prevents spillage and provide a place to hold the pipets. Instruct and carefully monitor all individuals on proper and careful technique for sowing and spreading spores, as well as handling and labeling dishes.

PREPARING SPORES
Before opening any spore vial, be sure that all spores are at the bottom of the vial by tapping the bottom of the vial on a hard surface (Figure 2). If using pre-sterilized spores, loosen the cap. Transfer the appropriate amount of sterile distilled water to the spore vial using a sterile transfer pipet. To maintain sterility and eliminate cross-contamination use separate and clean pipets to dispense sterile distilled water. Do NOT ever return any water to the sterile water bottle.

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Figure 2. Looking down from the top of the vial, C-Fern spores should be visible at the bottom of spore vial

The standard pre-sterilized spore unit will sow ca. 35 dishes at a standard density of 300+ spores per plate when 4 ml of sterile distilled water is added. Allow spores to wet completely by firmly reattaching the cap and inverting the vial 2-3 times. With the cap on, check the bottom of the vial to be sure that all the spores have been suspended. Return vial to the holder and loosen the cap. If using unsterilized spores, determine the appropriate amount of spores for inoculation and surface sterilize spores.

SOWING SPORES
To achieve consistent sowings, spores must be thoroughly suspended between each sowing. Suspend spores gently by drawing the liquid along with the spores in and out of the pipet (Figure 3). To sow, withdraw a small amount of the spore suspension into the pipet and immediately dispense the appropriate number of drops – not squirts! – onto the agar surface (Figure 4, 5). A standard sowing density of 300+ spores per Petri dish requires three drops of spore suspension.

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Figure 3. For each inoculation, thoroughly suspend the spores in water. Aspirate the spore suspension into a pipet. Grasp the lid of a petri dish and elevate one side of the petri dish lid to about a 45-degree angle. Keep the lid over the agar surface.

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Figure 4. Quickly but carefully dispense 3 drops – not squirts – of spore suspension onto the agar surface.

When sowing, tilt the lid of the Petri dish upward only just enough to permit access of the pipet tip. To ensure a consistent drop size, hold the pipet at a constant angle (45°) throughout the sowing. Do not touch the agar surface with the tip of the pipet. Resuspend spores between each sowing by gently squeezing and releasing the pipet bulb.

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Figure 5. Drops of the spore suspension should be evenly distributed across the agar surface. After dispensing spores, replace the Petri dish lid.

SPREADING SPORES (Figure 6)
If you are using a plastic spore spreader, it may be effectively sterilized by wiping or dipping in alcohol, e.g. 70% isopropanol or ethanol, and allowing to completely air dry. Metal wire spore spreaders may be sterilized by heating in a flame and allowing to cool. When distributing spores, allow just the tip of the L-shape (about 5 mm) of the spreader to just rest on the surface without pressure (Figure 7). Move the spore spreader rapidly but gently back and forth across the surface of the agar while rotating the Petri slowly with the other hand. The goal is to distribute spores over the surface of the medium in a somewhat ‘random’ arrangement.

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Figure 6. Use a sterile spore spreader to distribute spores randomly on the agar surface.

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Figure 7. Again, grasp the lid of a petri dish and elevate one side of the petri dish lid to about a 45-degree angle. Keep the lid over the agar surface. To distribute spores, move the spore spreader rapidly but gently back and forth across the agar surface. While moving the spreader, rotate the Petri dish to help in distributing the spores randomly.

Thomas Warne