Removal of extracellular material via chelation washing

Archae and Bacteria frequently produce a matrix of extracellular material when they grow. Unfortunately, this substance interferes with routine laboratory manipulations, and in many cases the volume of extracellular substance can equal or exceed the volume of cells present. Thus, removing any extracellular materials, ideally gently enough to maintain cell viability, is crucial for effective laboratory manipulation.

Strategy

Extracellular materials can be composed of many different types of molecules. To disrupt this structure without damaging the cells, a washing technique is employed using a small amount of non-ionic detergent and a chelator, which works to solubilize the bulk material of the matix while removing crosslinking by chelating divalent cations (e.g., Mg²⁺, Ca²⁺).

Sharpea azabuensis

Salt Buffer Formulation

Component	Concentration
Tris HCl	10 mM
EDTA, pH 8.0	100 mM
Triton X-100	0.1% (v/v)

S. azabuensis responds well to Salt Buffer (see table), which is made days prior and allowed to deoxygenate in an anaerobic chamber. 50 mL of culture is spun down in a 50-mL conical vial at 5000 g for 10 minutes at room temperature. The supernatant was discarded, 30 mL of Salt Buffer was added, the cell pellet together with the matrix are resuspended, then the vial was centrifuged with the same conditions as before; this is repeated for a total of 2 times. The extracellular matrix should be greatly reduced with each wash, and the wash supernatant after each spin should be non-viscous. Proceed with transformation or DNA extraction from this point with a bare S. azabuensis cell pellet.