2.2. Spore Purification Using Percoll Density Gradient Centrifugation

In the 1960s, biologists capitalized on the production of industrial colloidal silica (a.k.a. Ludox^®) to develop gradient centrifugation approaches for the recovery of live microbes from environmental samples [22]. Ludox^® is still used today to purify entomopathogenic algae from host insect larvae extracts [23]. Based on this foundation, we developed a purification protocol to isolate spores from sexually developing populations of Cryptococcus using colloidal silica nanoparticles coated in polyvinylpyrrolidone (a.k.a. Percoll^®). Percoll^® generates self-forming gradients during centrifugation that are non-toxic, metabolically inert, and easily washed away from purified spores [11]. Percoll^® gradients can be scaled for the number of spores needed for any given experiment and are presented here as “large scale” and “small scale” purification protocols.

Evaluate basidia on V8 plates for adequate spore production using a microscope. Spores are generally visible around the edge of each growth spot and can be seen in chains off the basidia at 200× magnification.

Prepare one 15 mL conical tube (VWR International, Radnor, PA, USA, ref no. 89039-668) per sample. One 15 mL should accommodate crosses from three to ten large (15 cm) V8 plates. Add 10 mL 75% Percoll^® (GE Health Care, Chicago, IL, USA, ref no. 17-0891-01; 100% Percoll^® diluted to 75% with 1× phosphate buffered saline-PBS) to each tube and place on ice. Use a cell scraper (Fisher Scientific, Hampton, NH, USA, ref no. 353086) to harvest the total mass of all spots off each V8 plate and resuspend in the Percoll^®. Mix thoroughly by vortexing vigorously until the cell mass is homogenous throughout the Percoll^®. It is imperative to fully homogenize the solution, as even small clumps of cells will sediment improperly during centrifugation, leading to impurities in the final spore preparations. If necessary, clumps of cells can be physically disrupted using a sterile plastic pipette tip. Keep tubes on ice throughout the harvesting process.

Centrifuge at 4 °C, at 2110× g, in a swinging bucket rotor for 25 min to generate a density gradient. Spores will sediment to the bottom of the tubes while all other cell types will remain at or near the top of the gradient. There should be a clear separation between spores and all other cell types (Figure 1C). If there is visible sedimentation of cells from the top of the gradient through and down to the bottom of the tube, the spores will likely not be pure.

Carefully remove tubes from the centrifuge and place on ice; do not disrupt the gradient.

Collect spores from the bottom of the density gradient:

Gently secure the 15 mL conical tube to a ring stand, maintaining clear access to the bottom of the tube.

Sterilize the outside of the 15 mL conical tube with 70% ethanol and dry. Remove the cap to allow the tube to vent during the next step. Venting is critical to this process; failure to do so can create a vacuum and result in sample loss.

Use a 21-gauge needle (Fisher Scientific, Hampton, NH, USA, ref no. 1484092) to carefully and gently drill a hole in the bottom of the conical tube. It is imperative that the tube be stabilized in a manner that precludes a needle hazard. Keep both hands away from the trajectory of the needle to avoid a needle stick injury. Discard needles into a biohazard sharps container; do not recap contaminated needles!

Immediately collect the first 200 µL that drip out in a sterile 1.5 mL microfuge tube (5–7 drops total).

Fill the microfuge tube to 1.5 mL with 1× PBS, vigorously pipette up and down to disperse the spores. Vortex to ensure spores are evenly suspended in solution. Centrifuge for 5 min at 2000 RPM (~370× g). Discard the top 1.3 mL of PBS, careful not to disturb the spores at the bottom. There may not be a visible pellet at this point.

Repeat step 6.

Carry out a final wash with 1.5 mL PBS and centrifuge for 1 min at maximum speed (13,200 RPM/16,100× g) to pellet spores. Discard supernatant and resuspend spore pellet in 300–700 µL PBS. Volume depends on anticipated spore yield based on visualization of spore pellet and number of V8 plates used to generate the sample. For JEC20 x JEC21 crosses after 5 days of development on V8 pH 7 agar, spore yields should be ~5 × 10⁶ spores per V8 plate.

Count spores using a hemocytometer to determine final concentration and level of purity. Spores can be stored at 4 °C but begin to adhere to each other the longer they are stored.

Safety considerations: recovering spores by inserting a needle into the bottom of the 15 mL conical tube could result in needle stick injuries if proper precautions are not taken. Extra care must be taken to prevent the needle from slipping off the tube surface as it is punctured and to always keep hands and fingers out of the way. Both 21- and 23-gauge needles have been used according to user preference to generate the minimum amount of pressure necessary to drill into the tube. Note: the pellet drips out quickly, usually 5–7 drops are sufficient to recover all of the spores. To empty the conical tube, allow the remaining Percoll^® to drain into a beaker containing 50% bleach. Alternatively, spores can be collected from the bottom of the tube by inserting a drawn-out glass pipet from the top of the gradient and drawing up the spore pellet in ~200 µL. Extreme care must be taken with this method to avoid contaminating the spores with cells from the top of the gradient.

Tips for success: the low speed centrifuge steps are to remove the Percoll^®, higher speeds will cause the Percoll^® to pellet with the spores. After each centrifugation, homogenize the spore pellet thoroughly by pipetting up and down and vortexing (the spores tend to clump). If clumping is an issue for further experiments, 0.01% Triton X-100 can be added to each wash step to prevent spores from sticking together without affecting the viability of the spores.

Crosses from some strain pairs adhere to the V8 medium more than others, which makes it more difficult to scrape crosses from the plate for spore purification. In this case, cell lifters (Corning, Corning, NY, USA, ref no. 3008) can be used more effectively due to their higher rigidity. Care must be taken to scrape off as little of the V8 agar as possible because the nutrients in the medium could potentially trigger spore germination during purification.

Mix strains of opposite mating types together in a 1:1 ratio (OD₆₀₀ = 2.0).

Pipet 30–40 10 µL spots on one 15 cm V8 pH 7.0 agar and incubate at 20–22 °C in the dark with the agar side down for 5–7 days.

Prepare one 1.5 mL microfuge tube with 1 mL 75% Percoll^® and place on ice.

Use a cell scraper to scrape the total mass of cells and resuspend in the 75% Percoll^®. Resuspend thoroughly by vigorous vortexing until the cell mass appears homogenous throughout. If any clumps are still visible, pipette up and down to remove them. Keep tubes on ice throughout harvest of spots.

Centrifuge at 4 °C at 2000× g for 25 min to generate a density gradient. Spores will sediment to the bottom of the tubes. All other cell types will remain at the top of the gradient. There will be a clear separation between spores and other cells. If there is visible sedimentation of cells from the top of the gradient down to the bottom of the tube, the spores will not be pure.

Carefully remove tubes from the centrifuge and place on ice.

Collect spores from the bottom of the gradient:

Open the lid of the 1.5 mL gradient tube. Sterilize the bottom with 70% Ethanol.

Use a 23-gauge needle to carefully and gently drill a hole in the bottom of the tube.

Immediately collect the first 50–100 μL that drips out in a sterile 1.5 mL microfuge tube (2–3 drops total).

Fill the microfuge tube to 1.5 mL with 1× PBS, pipetting up and down to disperse the spores. Vortex to ensure spores are evenly suspended in solution. Centrifuge for 5 min at 2000 RPM (~370× g). Discard the top 1.3 mL of PBS, careful not to disturb the spores at the bottom. There may not be a pellet after the first two spins.

Repeat step 8.

Carry out a final wash with 1.5 mL PBS and centrifuge for 1 min at maximum speed (13,200 RPM/16,100× g). Discard supernatant and resuspend spore pellet in ~50 µL PBS per 5 spots purified (can be adjusted according to size of spore pellet).

Count spores using a hemocytometer to determine final concentration and purity. Spore yield should be ~1 × 10⁶ per 30 spots.