Bacterial/Prokaryote Cell Disruption by Bead Beating

Some bacteria can be easily disrupted by treatment with enzymes and detergents, as with E. coli during plasmid isolation. However, many microorganisms, especially those in environmental samples, require mechanical methods for disruption. For rapid isolation of nucleic acids and proteins, bead beating is a simple and cost-effective method for lysing cells. Sonication is an alternative technique to disrupt bacteria that is quick, but it is low throughput and can generate aerosols that can be dangerous.

To bead beat any sample, the minimum requirements are beads, which can be obtained in bulk bottles or pre-filled into disruption tubes and plates, plastic tubes, a lysis buffer and a mixer mill (commonly referred to as a bead beater) that rapidly oscillates tubes with beads and sample. For prokaryotes, small beads are the most effective for cracking bacteria. Typically, 100 µm zirconium or silica beadsare used for disrupting bacteria. For dilute bacteria, the option of using low binding beads can help to minimize the loss of analytes due to non-specific adsorption that can occur during processing.

The beads used for rupturing bacteria are available in different grades. Raw beads contain significant amounts of dust and debris and should be acid washed prior to use. Alternatively, beads are readily available that are acid washed, but also come contaminant free as molecular biology grade beads (free from all biomolecules) or as low binding beads, useful for samples with few bacteria. Disruption tubes can be filled manually, with a small scoop, or obtained pre-filled. For smaller tubes, i.e., 2 ml, most homogenizers are designed to hold screw capped tubes.

Protocol - General Parameters

1. Bead beating bacteria can be performed in microfuge tubes, deep well plates, 4 ml vials, 15 ml vials, or in customized 125 ml jars for batch processing.  The choice of container will be limited by the homogenizer and designed based on the application. For instance, the liberation of DNA for PCR from a bacterial culture requires a small volume while isolation of recombinant protein might require larger disruption containers.
2. Generally, the container should not be more than half full of beads, buffer and sample. Regardless of the container, it is important to not to overfill as beads need room to move and crash around during processing. A working rule is that 1/3 volume should be beads and 2/3 volume sample and buffer. Thus, in a 2 ml microfuge tube, roughly 350 µl should be beads and 650 µl sample.  As noted, it is important that the beads and bacteria have head space to move in during beating, thus no detergents should be added during processing as foaming will impede the motion of the beads.
3. Depending upon the application, bacteria can be homogenized in culture medium or centrifuged and resuspended in a suitable homogenization/lysis buffer.  When using pre-filled microfuge, simply add the sample/buffer and cap. When preparing materials manually, 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. We find that small variations in mass do not drastically impact the homogenization efficiency. Plates can be loaded using a scoop and small funnel to load individual wells. For bead masses and mixes (more than one bead type) that are not available off-the-shelf, custom filling is available, and often at costs equal to standard products.
4. Once filled, 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, especially for square well plates, have been found not to work well with high throughput bead beating as they tend to leak and cause cross contamination. Heat sealing with foil is also effective but requires specialized equipment.
5. For 2 ml and 5 ml tubes, a benchtop homogenizer, such as the HT6, will work well. Samples can be processed in 1-2 minutes on high. For full racks of tubes or plates, then a high throughput homogenizer is needed. The Geno/Grinder is very effective at homogenizing large number of bacterial samples. This homogenizer is slower, so processing for 5 minutes on high speed should be effective. In some instances, resilient microbes may require longer processing times.
6. Optimization: When optimizing the homogenization process, beating the sample for short durations and then assessing the degree of disruption at each time point is practical. An easy to assay marker, like 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 bacteria, 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). Add bacteria and a non-denaturing buffer to the tubes with beads. Start by removing a tube before processing and labeling it 0 min. Run the homogenizer for 2 minutes, stop and remove a tube and label. Restart the homogenizer and repeat the process removing samples at 4, 6, 8, and 10 minutes. Label all tubes accordingly.
7. Pellet the bacterial cells and debris by centrifugation.  Test the supernatant for lactate dehydrogenase (LDH) activity using a commercially available test kit or standard LDH assay protocol (link).  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.