SPADA Draft Documents

persist for days to months in soil (4). A time-course study of pathogens in soil can be 206 performed using RNA (or messenger RNA) analysis or by culture (if culturable). 207 If nucleic acid preservation is desired, a commercial preservation solution with a 208 validated expiration date is recommended. The preservative should be evaluated before 209 use for compatibility with the method or system under evaluation. Likewise, commercial 210 nucleic acid extraction kits intended for use with soil are recommended. Appropriate 211 bacterial controls should be used. 212 Most naturally occurring microorganisms do not grow on common culture media (5). 213 However, culturing microbes does provide an indication of the diversity and types of 214 microbes occurring in the soil. Culturing can also be used to indicate whether a soil was 215 adequately sterilized. A general-purpose solid culture medium for soil samples is R2A 216 Agar, which is a low-nutrient agar formulated to promote growth of stressed or slow- 217 growing bacterial cells. To culture soil bacteria, first make a 10-fold dilution by 218 suspending 3 g of soil sample in 27 mL of 0.1% (by mass) peptone solution. Vortex the 219 soil suspension for 30 sec, shake 25 times in a wide arc, and allow the tube to sit for 220 about 5 min until the coarse particles have settled. Finally, spread-plate 0.1 mL of the 221 supernatant solution onto an R2A agar plate. It is recommended that serial 10-fold 222 dilutions in 0.1% peptone be prepared and plated in order to obtain isolated bacterial 223 colonies. Dilutions of 10 -6 through 10 -8 should produce a few plates with 20-200 isolated 224 colonies for most soils. Incubate the plates at 20 – 28 °C for 10 days. Fast growing 225 bacteria will appear in about 2 days while slow growing bacterial and other microbial 226 colonies do not appear until 8-10 days of incubation (6).

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