Methods of Cell Lysis

Lysis Method Instrument Requirements Mode of Lysis Mechanism 

Pressure shearing Required, moderate cost Harsh Shear forces 

Ultrasonic disintegration Required, moderate cost Moderate Shear forces 

Bead milling Required, inexpensive Harsh Shear forces 

Enzymatic Not required, cost of enzyme (moderate) Gentle Breaks covalent bonds— specific to cells 

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There are several methods of cell lysis (Table 8.2) [1,2], but there is none that works with cells of all biological origins. Each technique has its advantages and disadvantages, and the specific method of choice depends on the cell characteristics, the cell type, and the final application. A combination of more than one method may also be used. For example, enzymatic lysis uses specific enzymes to target the cell wall. However, to disrupt the cytoplasmic... 

Nonmechanical Methods of Cell Lysis Enzymatic Lysis... 

It is important to note that no single method of cell lysis or DNA purification will be appropriate for all environmental samples. Therefore, researchers should consult protocols that may apply to their systems of study in order to optimize DNA recovery from novel environmental samples. 

In addition to the foregoing more general concerns are questions concerning the localization of an enzyme activity. The location of an enzyme can determine the type of cell lysis, since it could be more advantageous to lyse the cell completely or in such a manner that the organelles are left intact. For example, some lysis methods such as sonication completely disrupt mitochondria, nuclei, and Golgi systems. If an activity is localized in an organelle such as a mitochondrion, it would seem sensible to adopt a method that leaves these structures intact, to facilitate their separation from the rest of the cellular debris. Thus, for the isolation of mitochondrial enzymes, sonication is not the method of choice for cell lysis. 

Although extended reaction time was also used by Selsted and Martinez (1980) in their turbidimetric method, they did not stress the importance of the kinetic order of cell lysis. McKenzie and White (1986) showed that, while the reaction is biphasic, simple kinetics are obeyed over a sufficiently long period that this property can be exploited in the determination. The kinetic order must be considered in the quantitative treatment of the results. The limit of detection was found to be 100 pg ml of reaction mixture, equivalent in the case of bovine milk lysozyme to 6 ng ml of skim milk, for a 50-/xl sample. 

Information gained from assays and future potential The application of esterases assays have provided the first direct evidence that cell lysis (as distinguished from loss terms such as grazing and sedimentation) may be an important factor in marine systems (see Kirchman, 1999). While the meaning of cell lysis, its incidence and ecological importance remain unclear (see Franklin et al, 2006), and there are concerns about the assay, we currently lack objective, independent methods with which to measure lysis (cf, Agusti and Duarte, 2002). In future, it may be possible to identify particular esterases that are truly found only within cells and choose substrates that are more specific for them. 

In terms of cell lysis on-chip, Li and Harrison [39] were the first to report cell lysis through combination of a chemical method using SDS and electrical techniques. Since then, other on-chip lysis methods have appeared, including mechanical [40], thermal [41], ultrasonic [42], and electrophoretic [43, 44]. 

The choice of cell lysis method depends to a large extent on sample type. Mammahan cells, bacteria, and yeast all have different requirements for lysis depending on the presence or absence of a cell wall. In some cases, a combination of chemical and mechanical disruption may yield best results. Another important factor in the choice of lysis method is the sample size of cells to be dismpted. If only a very small volume of sample is available, care must be taken to reduce loss and avoid cross-contamination. Consideration should also be given to the compatibility of the chosen method with downstream applications. 

Here, we discuss an optimized DNA extraction method specifically for electrode-adhered biofilm communities. We describe the use of a biofilm DNA extraction kit from MoBio Laboratories Inc. to extract DNA from electrode-adhered communities (PowerBiofilm DNA Isolation Kit, www.mobio.com). Here, we included a slightly modified alternative for cell lysis to include less meehanical bead-beating and multiple heat treatments. This method for cell lysis has been found to be successful for electrogenic community analyses [18, 21], We do not endorse MoBio Laboratories. This protocol is solely meant to be a starting point for researchers. 

In addition to these various chemical treatment methods, a number of physical methods of cell disruption can be used in a chip-based system. These physical methods include osmotic shock, which occurs when cells are suspended in a hypotonic solution shearing and fracturing of cells walls and membranes using microfabricated needles or spherical particles (beads) application of an electric field that causes electroporation ultrasonication of the cell sample and thermal lysis. 

T6 plus CN (84). To assure that the intracellular growth curves could be duplicated by an independent method, Anderson and Doermann (21) investigated sonic disruption of the infected cells. This was applicable only to a phage such as T3, which is resistant to sonic vibration. In this procedure metabolism is arrested by chilling, and the cells are treated sonically for 5 minutes. From Fig. 5 it can be seen that the two methods yield about the same amount of intracellular virus. The cyanide method of inducing lysis has proved of great value in isotopic experiments on the kinetics of the assimilation of intermediates used for phage synthesis. 

The previous biomarkers relate to phenotypic assessments of microbial diversity and most will probably measure a restricted part of the total microbial pool, since not alt markers will be expressed uniformly by every cell. In contrast, methods involving the detection of nucleic acids may be directly applicable to all microorganisms provided that the complete extraction of DNA (lysis of cells) or permea-bilization of cells can be achieved. 

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