Culture Establishment – Spore Surface Sterilization

Establish sterile cultures with a simple method for the surface sterilization of C-Fern spores.

All spore sterilization and sowing procedures should be performed on a sterile bench or laminar flow hood.

Figure 1. Array of materials needed for spore surface sterilization

Figure 2. We recommend that C-Fern spores be surface sterilized in 6 ml plastic tubes with conical bottom and screw caps and handled with sterile plastic pipets. The photographs below use glass conical centrifuge to allow easy visualization of spores.

Materials: (Figure 1)

  • Unsterilized C-Fern spores
  • Sterile tubes with conical bottom (Figure 2)
    For routine surface sterilization of spores, use pre-sterilized 6 ml plastic tubes with conical bottom and screw caps and handle spores with sterile plastic pipets.
    For sterilization of large quantities of spores, 15-250 mg, it may be more efficient to use reusable glass centrifuge tubes. Tubes should be about 12-15 mL and must have a smooth, conical bottom on the inside. They should be covered with small foil caps and sterilized either by autoclaving or dry sterilization at 120° C for 2 hours.)
  • Sterile plastic Pasteur OR sterile glass pipets (If using glass pipets, ensure that the ends are truly square cut. Jagged ends won’t seal properly and won’t allow for proper removal of liquids)
  • Sterile distilled water
  • Sterile pipet bulbs
  • Fresh Clorox® bleach OR equivalent commercial laundry bleach without additives (5.25% sodium hypochlorite solution)
  • Waste container

Methods

Figure 3. Dry C-Fern spores in a conical tube. A pipet full of liquid, either sterilization solution or sterile distilled water

WEIGH SPORES Determine amount of spores to be sterilized. Weigh out spores onto glassine weigh paper and transfer them to a sterile centrifuge tube with conical bottom (Figure 3).

Figure 4. Liquid added to C-Fern spores in conical tube

PRESOAK SPORES Add sufficient glass distilled water to completely cover spores (approximately 2 mL) and let stand for 5 min or up to 24 hours (Figure 4). Spores may be soaked for just enough time to accomplish full wetting before proceeding. Water for soaking spores does not need to be sterile.

REMOVE FLOATING SPORE DEBRIS AND PRESOAK WATER Remove floating spore debris, if any, by placing pipet tip at water meniscus and slowly drawing off the water. The debris will be sucked into the pipet along with the water. If the spores are exceptionally dirty or contaminated, wetted suspended spores should be transferred to a clean sterile centrifuge tube and care should be taken not to touch the internal sides of the tube. Remove remaining water from the spores using a sterile pipet (spores will have settled to the bottom of the tube) using the technique described below. When the presoak water is successfully removed, discard the pipet. This procedure for removing liquids from tubes will be used also in spore sterilization and rinsing.

Figure 5. Pipet is inserted into the liquid while expelling air from the pipet.

PROCEDURE FOR REMOVING LIQUIDS Select a sterile Pasteur pipet, for glass pipets choose one with a tip that is not cracked or chipped and attach a rubber bulb on the pipet. Insert pipet into the tube and suspend spores by bubbling a small amount of air into the water (Figure 5.). While air is slowly bubbling out of the pipet, gently but securely seat the pipet onto the base or lowermost conical region of the tube (Figures 6 and 7). Don’t press too hard on the pipet or you will crush the tip. Sometimes it helps to gently rotate the pipet tip. Squeeze the bulb to force additional air out of the pipet. When the bulb is released, water should enter the pipet and the spores should collect around the outside of the base of the pipet tip, provided the pipet is securely seated on tube bottom (Figure 8). If you can’t remove liquid without bringing the spores along, try another pipet.

Remember that time is critical when the sterilizing solution is in the tube! Practice this technique with water prior to sterilizing the spores. With practice, you should be able to remove liquids free of spores, in less than ten seconds.

Figure 6. Air is continuously expelled from the pipet as it is moved toward the bottom of the conical portion.

Figure 7. Pipet is seated onto the conical portion of the tube and sufficient air is expelled to allow for complete aspiration of liquid.

Figure 8. Sequence of steps for removing liquid.

A. Pipet is inserted into the liquid while expelling air.

B. As pipet moves towards bottom expelled air begins to suspend spores. Note the small pellet of spores still at the bottom.

C. As pipet nears the bottom, bubbling air has completely suspended spores and gives a cloudy appearance. Expelling air excludes spores from the end of the pipet.

D. Pipet is firmly seated on bottom.

E. Pipet bulb is released and liquid begins to aspirate, leaving spores trapped on the outside of the pipet tip.

F. Liquid is completely separated from the spores

SURFACE STERILIZE SPORES To sterilize, suspend spores in one full Pasteur pipet of 0.875% sodium hypochlorite (1 part Clorox (5.25% sodium hypochlorite) to 5 parts distilled water). Rinse down lip and sides of the tube with sodium hypochlorite solution. To ensure that the spore mass becomes evenly suspended it may be necessary to bubble air through a clean, sterile pipet (discard this pipet after use). Sterilize spores for three minutes. During the time that the sodium hypochlorite solutions in the tube check that the pipet can successfully be used to remove the liquid, leaving the spores behind. Remove sodium hypochlorite solution with a clean, sterile pipet using the technique described above. For precise timing of surface sterilization, the time needed to remove sodium hypochlorite must be factored in. Discard this pipet.

RINSE SPORES To rinse spores, add one full pipet of sterile glass distilled water. Pipet used to add sterile distilled water may be used repeatedly as long as care is taken to prevent contamination with foreign spores. Remove rinse water with a clean, sterile pipet. Rinse spores a total of three times. times. Pipet used to remove sterile distilled water may be left in the tube for use in sowing spores.

INOCULATE OR SOW SPORES Add the appropriate amount of sterile glass distilled water to sow a given number of plates. Dispense spores to plates (refer to information on SPORE SOWING DENSITY AND CALCULATIONS). Spores may be uniformly distributed across the agar surface by gently spreading with a sterile wire bent in the shape of a small coat hanger or other appropriate spreader.

Thomas Warne