Sonics VC100 100-watt (1992) - Manual (Page 15)

15
Microorganisms differ greatly in their sensitivity to ultrasonic disintegration. For
example, the most readily disintegrated are the rod- like forms (bacilli), while the
spherical organisms (cocci) are much more resistant. The group Mycobacteria, to which
the tuberculosis organism belongs, is particularly difficult to disrupt. Generally, animal
cells are more easily disintegrated that plant cells, and red blood cells are more readily
disintegrated than muscle cells because they lack a protective cell wall.
Cellular disruption is the first step in RNA isolation and one of the most critical steps
affecting yield and quality of the isolated RNA. Typically, cell disruption needs to be fast
and thorough. Slow disruption, for example placing cells or tissue in guanidinium
isothiocyanate (GITC) lysis solution for a long time prior to sonication, may result in
RNA degradation by endogenous RNases released internally. This is especially a concern
when working with tissues high in endogenous RNase such as spleen and pancreas.
Disrupting frozen tissue is more time consuming and cumbersome that processing fresh
tissue, but freezing samples is sometimes necessary. Samples are usually frozen when, 1)
they are collected over a period of time and thus, cannot be processed simultaneously; 2)
there are many samples, 3) samples are collected in the field, or 4) mechanical processing
of fresh samples is insufficient for thorough disruption. A mortar and pestle or bag and
hammer are typically used when the starting material is frozen. RNA will remain intact in
tissues for a day at 37ºC, a week at 25ºC a month at 4ºC and indefinitely at subzero
temperatures.
Ultrasonic processing will typically cause the temperature of the sample to increase
especially with small volumes. Since high temperatures inhibit cavitation, the sample
temperature should be kept as low as possible - preferably just above its freezing point.
This can be accomplished by immersing the sample vessel in an ice-salt-water bath.
Temperature elevation can also be minimized by using the pulser.
Increasing hydrostatic pressure (typically 15-60 psi) and viscosity can enhance cell
disruption. For microorganisms, the addition of glass beads in the 0.5 to 1mm size range
promotes cell disruption. Beads are almost a prerequisite when working with spores and
yeast. A good ratio is one volume of beads to two volumes of liquid. Glass beads are
available from Cataphote, Inc. P.O. Box 2369, Jackson, Mississippi 39225-2369 USA,
phone (800) 221-2574 or (601) 939-4612, FAX (601) 932-5339, Jayco Inc. 675 Rahway
Ave., Union NJ 07083 USA, phone (908) 688-3600, FAX (908) 688-6060 or Sigmund
Lindner GmbH. P.O. Box 29. D-95483 Warmensteinach, Germany. Phone (49) 0 92 77 9
94 10, FAX (49) 0 92 77 9 94 99.
When processing difficult cells such as yeast, pretreatment with an enzyme is beneficial.
Lysozyme, byaluronidase, glycosidase, glucalase, lyticase, zymolase and lysostaphin
digestion are among the enzymatic methods frequently used with yeast and Lysozyme
with bacteria. Enzymatic treatment is usually followed by sonication in a GITC lysis
buffer. Collogenase may be used with collogen, lysostaphin with staphylococcus, and
trypsin hyaluronidase with liver and kidney.