Fungi Cell Disruption Methods

Fungi are not easily disrupted by treating with detergents.  Enzymatic removal of cell walls followed by detergent lysis can be effective, but the very chemically diverse composition of fungal walls between species makes any one method unreliable.  Consequently, rupturing fungal cells and mycelia by bead beating is a reliable and fast method not restricted by the species or stage of development. 

Fungi can be disrupted using a Geno/Grinder, or HT Homogenizer II.  Unfortunately, vortexing samples mixed with glass beads on basic laboratory vortexers is not that effective for disrupting cells.

As fungi are extremely diverse in size and nature, selecting the correct bead may require trial and error.  Fungi that are growing as yeasts and pseudomycelia may best be processed with 400 µm silica beads. Mycelia is very effectively disrupted with 800 µm silica beads, while dense pellicles of fungi may require a mix of beads which include a few large beads to break up clumped cells and smaller beads to lyse individual cells (pre-filled tubes with mixed beads are available). 

Samples which contain few cells may require lysing with low binding beads.  Analytes liberated from cells non-specifically adsorb to surfaces in the lysing tube, including the beads and the tube itself.  Low binding beads are treated so that less lysate is lost by adsorbing to the beads.

Protocol - General Parameters

• Bead beating fungi can be performed in microfuge tubes, deep well plates, 4 ml vials, 15 ml vials, or in customized 125 ml jars for batch processing.  Generally the container should not be more than half full of beads and sample.  Of that, 1/3 should be beads and 2/3 sample.  Thus, in a 2 ml microfuge tube, roughly 350 µl should be beads and 650 µl sample.  It is important that the beads and sample have head space to move in during beating, thus no detergents should be added during processing.
• Depending upon the application, fungi can be homogenized in culture medium or centrifuged and resuspended in a suitable homogenization/lysis buffer.  Prepare cells and add to pre-filled microfuge tubes or other tubes or plates containing beads.  Tubes and plates should be "loaded" with beads before the sample is added.  A small scoop which is approximately 300 µl can be used to load tubes quickly.  Plates can be loaded using a scoop and small funnel to load individual wells.  Custom tube and plate fillers are also available to load racks of tubes and plates more rapidly.
• Cap the tubes/plates with closures that do not leak.  For screw cap microfuge tubes, be certain to use caps with "O" rings.  For plates, tight fitting polypropylene strip caps are recommended.  With strip caps, each well is sealed with an individual cap.  Silicone press on mats have been found not to work well with high throughput bead beating as they tend to leak and cause cross contamination.

• Individual tubes can be processed in a Pulsing Vortex Mixer effectively.  For full racks of tubes or plates, then a high throughput homogenizer is needed.  Both the Geno/Grinder and HT Homogenizer II are very effective at homogenizing large number of microorganism samples.  For a basic homogenization procedure, place the samples in the homogenizer and process for 6 minutes on high speed.  Generally yeasts are more resilient than bacteria so processing may be longer.

• If a microscope is available, examine unprocessed and processed samples under 10X or 20X.  Disrupted cells, pseudomycelia and mycelia will appear as gray "ghost" cells while intact cells are refractile (i.e., bright).  Samples which are thoroughly homogenized will appear grainy in texture.  If the proportion of disrupted fungal cells is low, continue the processing and check again microscopically.

• Optimizing the homogenization process requires beating the sample for short durations and then assessing the degree of disruption at each time point.  The release of the enzyme lactate dehydrogenase from disrupted cells is a parameter successfully used to measure homogenization efficiency in many different biological samples.  For yeasts, prepare at least six microfuge tubes with cells and beads (ideally it would best to run this test in triplicate, thus 18 tubes would be better if space allows in the homogenizer).  Start by removing a tube before processing and labeling it 0 min.  Run the homogenizer for 3 minutes, stop and remove a sample(s) and label.  Restart the homogenizer and repeat the process removing samples at 6, 9, 12, and 15 minutes.  Label all tubes accordingly.

• Pellet undisrupted cells and debris by centrifugation.  Test the supernatant for lactate dehydrogenase (LDH) activity using a commercially available test kit or standard LDH assay protocol.  The tube with the highest enzyme activity or where the activity peaks with the least processing represents the optimal processing time.  Note that LDH is only one of many possible cellular enzymes that can be used for assessing cell disruption.

References

Mideros, Santiago X., Gary L. Windham and W. Paul Williams 2009.  Aspergillus flavus Biomass in Maize Estimated by Quantitative Real-Time Polymerase Chain Reaction Is Strongly Correlated with Aflatoxin Concentration. Plant Disease 93(11):1163-1170.