Soil samples were taken for organism enumeration and moisture content analysis as follows: Sampling bags were prepared with one sterile 'Whirlpak' bag and clean plastic scoop per sample.  Centers of 8 polygons were found and labelled "A" to "H".  Samples were taken from within a 10 cm diameter circular area of each plot.  The location of the sampling was recorded each year so that areas were not re-sampled.  A hole approximately 20 cm deep was dug at the center of each polygon.  Samples were taken at depths 0-2.5 cm, 2.5-5 cm, 5-10 cm and 10-20 cm by digging in from the sides.  Very large  rocks (>20 mm diameter) were excluded from the sample.  The soil was shoveled into the 'Whirlpak' bag until three quarters full (about 1.5 kg soil).  The soil was mixed well in the bag, then the bag was closed tightly, expelling as much air as possible.  The soil samples were stored in a cooler for transportation.  On return to the laboratory (within 8 hours of sampling), the soils were stored at -8 degrees C until further processing.

 

In the laboratory, soil samples were handled in a laminar flow hood to prevent contamination.  The Whirlpak bags of soil were mixed thoroughly prior to opening.  The remaining soil in the plastic beaker was weighed.  100g of soil (less rocks) were measured into a beaker and brought up to 250 mL with a 1.25M sucrose solution.  Mixture was star stirred using a spatula for 30 seconds.  Immediately the liquid was poured into screens-a stack of 40 mesh on top of a 400 mesh, prewetted with 1.25M sucrose solution.  The screens were rinsed gently with 1.25M sucrose solution (from a wash bottle) through the top of the stack, keeping the screens at an angle as the solution filtered through.  The solution was kept on ice at all times.  The top screen was removed, and the lower screen rinsed top down, never directly on top of the soil, but at the top of the screen and from behind.  The sucrose solution was allowed to cascade down and carry the particles into the bottom wedge of the angled screen.  The side of the screen was tapped gently to filter all the solution through.  The suspension was rinsed from the front and the back, keeping the screen at an angle and not allowing the solution to overflow the edge of the screen.  The bottom screen was then rinsed into a 150 mL beaker with 1.25M sucrose, from the front, using a funnel.  The sugar and sediment were then slowly pipetted, using an automatic pipetter, from the beaker into a centrifuge tube containing 10 mL of 2M sugar on ice, at an angle to retain the boundary between sugar layers.  The beaker was rinsed with 1.25 sucrose and the dregs were pipetted as well.  As many centrifuge tubes as was necessary per sample were used. Each tube was evened off with 1.25M sucrose and centifuged for 5 minutes at 1744 RPM. The supernatant was then poured through a prewetted (with 1.25M sucrose solution) 500 mesh screen, stopping before the sediment came out and rinsed well with ice cold 1.25M sucrose and backwashed into a centrifuge tube.  Samples were refrigerated at 5 degrees C until counted.  

 

Samples were washed with 1.25M sucrose into a counting dish and examined under a microscope at x10 or x20 magnification.  Nematodes were identified as coiled or uncoiled.  Total numbers in each sample were recorded on data sheets.  Data were entered into Excel files, printed, and checked for errors.