Homogenization/Grinding Application Table

BT&C, Inc., Offering creative and innovative solutions to our customers for over 23 years.

 

BT&C Inc.

291 Route 22 East

Building 6

Lebanon, NJ 08833

TEL: (908) 253-3444

FAX: (908) 575-1660

Email:info@btc-bti.com

We are active scientists and homogenize samples constantly.  Many of our approaches are listed below.  If you need ideas on how to homogenize your sample, call or email us.  We'll be glad to talk with you.

The following table, compiled from experiments performed at OPS Diagnostics, is a general guide as all disruption procedures require optimization.  Please refer to the links below for additional information on methods.  Much of our application data has come from scientists who have contacted us with problems, so feel free to call us at (908) 253-3444 to discuss your options for homogenizing samples.

† Key to grinding method: v = Vortexer or Multitube Vortexer, s = High Speed Microplate Shaker, m = mixer mill (e.g.., HT Homogenizer), f = freezer mill

  Sample (method)† Method and Format Comments

Microorganisms

Bacteria (v, s, m)

Bacteria can be disrupted by vortexing, shaking, or milling with 200 μm Zirconium Beads.  Lysis efficiency will depend upon the size of the bacteria and toughness of the cells.

Grinding beads are available in three grades.  The applications for these grades are the same for microorganisms and pollen.

 

Yeast (v, s, m)

Yeast can be readily cracked using 400 μm Silica Beads in tubes, microfuge tubes and standard 96 well plates.  Best results are obtained when an equal volume of yeast and beads are combined for grinding. [Application Note]

· Acid Washed Beads have been treated to remove fine particles and contaminants.  These beads are suitable for most basic applications.

 

Filamentous Fungi (v, s, m)

Fungal filaments can be disrupted with 800 μm Silica Beads.  Fruiting bodies, such as conidia, are also disrupted during the processing. [Homogenized Fungi]

· Molecular Biology Beads are treated to inactivate contaminating enzymes and are tested accordingly.

Plants

Pollen (m)

Pollen can be difficult to homogenize, but it will crack with 800 μm Silica Beads using a vertical motion mixer mill (e.g., GenoGrinder). 

· Low Binding Beads are coated to reduce non-specific binding of proteins and nucleic acids.  Low Binding Beads are used for lysing dilute samples of cells.

 

Leaves (m)

Soft plant tissue is efficiently homogenized using a mixer mill.  Options:

·    Leaf punches are easily homogenized in polypropylene deep well plates using 5/32" grinding balls.  For genetic studies, plates should be heat-sealed.

·    Up to 300 mg of tissue can be homogenized per vial in 24 Well Vial Sets.

·    Over 1 gm of leaf tissue can be processed per vial in 15 ml Vial Sets.

Leaves are easy to homogenize.  Sample size will define the format (i.e., punches vs. grams).  For procedures requiring organic extractions, polypropylene plates or 24 Well PE (polyethylene) Vial Sets should be used. 

 

Bark/Root (m, f)

Hard plant material is best ground in 24 Well PC Vial Sets using a mixer mill.  The PC (polycarbonate) vials are best suited for very hard samples. [see note]

Arabidopsis roots can be homogenized with 5/32" balls using a deep well plate.

These are difficult samples and may require that water be removed prior to processing by lyophilization.

 
 

Seeds (m)

Virtually all seeds must be disrupted with the larger grinding balls used with the 24 Well Vial Sets and 15 ml Vial Sets.  Examples are:

·    Soybeans can be processed individually in 24 Well Vial Sets (one per vial). [Application Note]

·    Up to 20 grains (400 mg) of rice can be ground per vial using 24 Well Vial Sets or as much as 3 gm with the 15 ml Vial Sets.

·    Corn kernels can be individually disrupted using polycarbonate vials.  Pooling up to 15 kernels can be processed per vial using the 15 ml Vial Sets. [Application Note]

Using Vial Sets with the GenoGrinder allows for dry grinding of seeds.  Harder seeds, such as kernels, require the polycarbonate vials.  Pooled samples from field trials can be processed using the 15 ml Vial Sets.  Vial Sets should be treated for molecular biology procedures.
Animal Tissue

Insects (m)

Small insects, such as Drosophila, can be homogenized with lysis buffer in standard 96 well plates.  Larger insects can be treated in deep well plates or 24 Well Vial Sets.

For PCR analysis, we suggest heat sealing plates or using vials with individual caps to prevent cross-contamination.
 

Soft Tissue (m)

Samples <150 mg can be homogenized in deep well plates using an equivalent volume of lysis buffer and 5/32” balls.  Larger samples require 24 Well Vial Sets.

24 Well Vial Sets should be treated for RNA isolation procedures.
 

Bone (m)

Bone requires 24 Well PC Vial Sets that uses large 3/8" grinding balls to effectively disrupt 200-300 mg samples.

Vial Sets are available in polyethylene (PE) and polycarbonate (PC), with PC Sets being used for the more difficult homogenization steps.

Notes from Application Lab:

Roots/Bark: Small pieces of bark can be homogenized directly without drying, however it is very dependent upon the characteristics of the sample.  Roots can be particularly difficult and may first require removing water.  For very tough samples, a freezer mill may be necessary.  Freezer mills are effective in disrupting hard materials as the samples are submersed in liquid nitrogen and then smashed with a steel piston.  It is similar to grinding a sample with a mortar and pestle using liquid nitrogen, but the action is machine driven.  It is effective, but generally slow compared to high throughput methods. [return]