Freeze/thaw is also a method believed to rupture cells, used in various combinations with SDS and enzymes. Ice crystal formation during freezing is well known to cause cell rupture (hence glycerol is added when cultures are preserved by freezing) but the effect on microbial cells in soil appears moderate. Smalla et al. (1993) found bead beating to be substantially more efficient than freeze/thaw (both conducted after lysozyme and cold SDS treatment). Both Kauffmann et al. (2004) and Zhou et al. (1996) concluded that freeze/thaw in combination with SDS may be sufficient to lyse Gramnegative cells but not Gram-positive ones. The main effect of the freeze/thaw may be to release DNA from already lysed cells (Tsai and Olson 1991). Thus freeze/thaw treatment has often been used subsequent to lysozyme treatment and in combination with SDS (Rochelle et al. 1992; Lee et al. 1996), or subsequent to bead beating with SDS (Moré et al. 1994; Ogram et al. 1995).
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