Sonics VC100 100-watt (1992) User Manual download pdf (Page 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.

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