ESTCP Project #1011, Rhizosphere
Final Report
In autoclave bags, autoclave clean glass spreading bars. Flame with alcohol
and store in sterile glass bowl.
Procedure:
Weigh out 10 g of field moist soil to be plated. Place soil in -1 dilution bottle.
Shake dilution bottle on horizontal shaker table for 5 minutes. Remove from shaker.
Open -2 bottle and sterilize bottle mouth and cap in flame from alcohol lamp or gas
burner.
Shake -1 dilution bottle 50 times by hand (full 90-degree arc).
Pipet 10 mL from -1 bottle into -2 bottle. Cap -2 bottle. Dispose of 10 mL pipet in
waste container.
Shake -2 dilution bottle on horizontal shaker table for 5 minutes. Remove from
shaker.
Shake -2 dilution bottle 50 times by hand. If desired, plate -2 dilution on appropriate
media plates.
Place pipet tip on 100 l Eppendorf pipet.
Rinse pipet tip with solution from -2 bottle 3 times. Dispense 100 l solution onto
media plates. Dispose of pipet tip in waste container.
Place plate, without lid, on inoculation turntable.
Holding sterile spreading bar lightly on surface of media, spin inoculation turntable,
making sure to spread suspension evenly. Place used glass spreader bar in unused
glass bowl (or other suitable container).
Place lids on plates and incubate plates upside down at 25C.
Open -3 dilution bottle and sterilize bottle mouth and lid in flame from alcohol lamp
or gas burner.
Shake -2 dilution bottle 50 times by hand.
Pipet 5 mL from -2 bottle into -3 bottle (45 mL dilution bottle). Cap -2 bottle.
Dispose of 5 mL pipet in waste container.
Shake -3 dilution bottle 50 times by hand.
Plate -3 dilution, or dilute to -4.
Continue in this manner, plating where appropriate to media.
Notes:
Only -1 and -2 dilution bottles get shaken on shaker table.
Only use sterile pipets or pipet tips. Do not forget to flame cap and bottle each time
it is opened.
Soil Moisture Determination
1. Determine moisture content of soil by drying a known amount of soil @ 105C to
a constant weight.
47
Previous Page
