|
Home
Products
Online Store
How to Order
International
Distributors
Contract
Services
About Us
Contact Us
Links
Sales Opportunities
OPS Diagnostics, LLC
291 Route 22 East
Salem Industrial Park
Building 6
Lebanon, NJ 08833
TEL: (908) 253-3444
FAX: (908) 575-1660
Email:
info@opsdiagnostics.com
Distribution Opportunities
|
Home >
Application Notes >Application
Table
Bacteria can
be easily disrupted by treatment with enzymes and detergents. For gram
positive organisms, removing the cell wall and then lysis by the
addition of SDS or similar detergent are suitably early steps in
isolating nucleic acids. Gram negative organisms, such as E. coli,
simply require exposure to SDS to lysis. Enzymes and/or detergents are
useful if nucleic acids are the target. If proteins or cellular
components are sought, then alternative methods must be used. Bead
beating and sonication are both proven alternative techniques to
disrupt bacteria.
Small beads
are the most effective for cracking bacteria. Typically
100 um zirconium
or silica beads are the most effective for disrupting bacteria. For
dilute bacteria, low binding beads will provide high yields of analytes,
thus improving assay sensitivity.
Protocol -
General Parameters
-
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. Regardless of the container, it is important to
not to overfill with both beads and culture. Generally the container
should not be more than half full of beads and bacterial sample. Of
that, 1/3 should be beads and 2/3 sample. Thus, in a 2 ml microfuge
tube, roughly 350 ul should be beads and 650
ul sample. It is important that the beads and bacteria have head space
to move in during beating, thus no detergents should be added during
processing.
-
Depending upon the application, bacteria 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 ul 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
tubs, 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 are very effective
at homogenizing large number of bacterial samples. For a basic
homogenization procedure, place the samples in the homogenizer and
process for 5 minutes on high speed.
-
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 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). Start by removing a tube before processing
and labeling it 0 min. Run the homogenizer for 2 minutes, stop and
remove a sample(s) and label. Restart the homogenizer and repeat the
process removing samples at 4, 6, 8, and 10 minutes. Label all tubes
accordingly.
-
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.
|
