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Bead Beating: A Primer (continued)

Homogenization Parameters

In order to thoroughly homogenize a sample, the nature of the sample and its mass (size) must be matched with both a suitable vessel (vial, plate, tube) and the appropriate grinding media. Bead beating can be done in a variety of vessels, including disruption (microcentrifuge) tubes, grinding vials and microwell plates. Even square 125 ml plastic serum bottles can be used for very large samples on any one of the high throughput homogenizers. Whatever the format, sample vessels must be able to withstand the impact of grinding beads and balls. This ability is determined by the mass of the grinding beads/balls and the type of plastic used to make the vessel. Polycarbonate is the best plastic for bead beating because it is durable, clear and impact resistant, even at cryogenic temperatures. Due to the fact that many organic solvents are incompatible with polycarbonate, durable polyethylene can be used instead. Polypropylene disruption tubes and deep well plates work well with small beads but will often crack when used with metal grinding balls.

The size (mass and volume) and type of sample must be matched with a homogenization vessel of an appropriate volume as well as grinding media that can effectively homogenize the sample. Grinding media must be able to move freely to impact the sample; therefore, homogenization vessels must not be overfilled with sample, beads and/or buffer. Generally, the samples should take up no more than a sixth of the volume of the vessel, and grinding media should take up no more than a third of the volume of the vessel. As well as overfilling vessels, processing very small samples in larger grinding vials can also lead to inefficient homogenization. In such instances, it may be necessary to process the samples for a longer period of time.

Grinding media used during bead beating can be divided into beads, grinding balls, ceramic satellites and/or grinding resins. Grinding beads are a pool of beads that fall within a specific size range, while grinding balls are spherical and precision ground with a specific diameter. Beads with smaller diameters are best for disrupting microorganisms like bacteria and yeast, while larger beads or grinding balls are best for homogenizing animal and plant samples. Metal balls are used to grind resilient and fibrous samples. Because stainless steel balls may oxidize in the presence of phenolic compounds and interfere with subsequent processing, cylindrical or Saturn-shaped ceramic satellites are a useful alternative. Grinding resins are sharp, irregular shaped composites, such as garnet, that can also be effective at shredding resilient or fibrous samples.

The inherent properties and composition of grinding media affect sample homogenization (Table 3). Zirconium, silica and stainless steel (an alloy containing iron, carbon and chromium) are widely used as grinding media. Higher density grinding media (i.e. stainless steel and zirconium compounds) are generally more effective than silica at homogenization. Stainless steel and zirconium media, however, generate significant heat during processing. Stainless steel can also oxidize in extraction buffers containing phenol, which can potentially interfere with downstream applications. Additionally, silicate and chromium sheared from beads during bead beating can inhibit some enzyme reactions. Hence, no one media is good for all applications.

Table 3. Properties of common grinding media. (click on table for larger image)

Based on the size of the grinding media and composition of the homogenization vessel, not all permutations of media and vessels are compatible (Table 4). Generally, small beads can be used with any vessel, but the same cannot be said for larger grinding balls. Polypropylene deep well plates are sometimes manufactured with very thin walls, which can crack when impacted by grinding balls. Stainless steel 5/32” balls are routinely used with plates, but in many circumstances can punch through the bottom of the wells. Also, larger balls are physically too wide for many plates and disruption tubes.

Table 4. Grinding media compatibility with tubes, plates, and vials.  (click on table for larger image)

In general, grinding beads, balls, or satellites (non-spherical precision ceramic balls) should be cleaned prior to use to remove contaminants and inhibitors because some grinding media can be flush with impurities[1]. Acid washing beads is the most common method to remove contaminants. Additionally, heat treating beads eliminates them as a potential source of nuclease or nucleic acid contamination. Grinding balls must be degreased prior to use by repeatedly washing in organic solvents.

When extremely small tissue samples or a limited number of cells are disrupted, there is a chance that the desired analyte (e.g., specific DNA sequence or protein species) may be lost due to non-specific adsorption to the beads. Small glass and zirconium beads (100 to 800 micron) have a relatively large surface area that can bind analytes, effectively lowering detection limits on assays. Thus, low binding beads were developed to reduce non-specific binding. The beads allow more analyte to remain in solution and have been shown to aid in increasing assay detection limits.

Cryogenic Homogenization

The labile and/or resilient nature of some samples requires that they be pulverized cryogenically. Traditional methods use a mortar and pestle chilled with liquid nitrogen for cryogenic grinding of larger samples, but this is impractical for high-throughput processing of small samples. High throughput homogenizers, such as the Mini G™ and GenoGrinder®, are capable of processing samples cryogenically using polycarbonate vials and grinding balls. While many other plastics can become very brittle, polycarbonate can withstand cryogenic temperatures. The 4 ml and 15 ml polycarbonate vials are designed with reinforced bottoms so that they will withstand the impact of metal grinding balls. Polypropylene plates, tubes, and vials can crack if used for cryogenic grinding.

Cryogenic grinding is a relatively straight forward process. A vial containing the sample and grinding ball is chilled with liquid nitrogen by placing it in a bath prior to homogenization. The vial should be chilled without a cap, as caps are typically polypropylene and will crack if chilled. Using this simple method, chilled vials are removed from the liquid nitrogen bath, rapidly capped, racked, and placed in a bead beater for processing. While this is a useful homogenization method when working with resilient samples that are made brittle by the cold, it is not ideal for samples that contain heat labile molecules because samples will warm during processing. To offset generated heat, vials can be placed in a liquid nitrogen chilled Cryoblock (a machined aluminum block) to keep the vials and sample at cryogenic temperatures throughout processing. Cryoblocks can only be used with the 1600 Mini G™ and 2010 GenoGrinder®, as these two homogenizers are designed to carry the additional weight. The 1600 Mini G™ can accommodate one Cryoblock while the 2010 GenoGrinder® can hold two Cryoblocks.

For more details on cryogenic homogenization, see Appendix B.

[1] All grinding media manufactured by OPS Diagnostics is cleaned, treated, and ready-to-use.