ESTCP Project #1011, Rhizosphere
Final Report
Agar Preparation
1. Prepare the Martin's Medium using the recipe above. (You will need 2 Erlenmeyer flasks
for autoclaving each liter of media.) Add 500 mL media to a 1000-mL Erlenmeyer flask.
Autoclave @ 121C for 15 min. After cooling to approximately 45C, add 15 mg
streptomycin sulfate to each 500 mL of media. Pour media (approx. 20 mL) into petri
dishes. (500 mL of medium/sample.)
Prepare the 0.1 X TSA broth using the recipe on the media page. Add 500 mL of media to a 1000
mL Erlenmeyer flask. Autoclave @ 121C for 15 min. After cooling to approx. 45C, add 0.5
mL of 100 mg (0.1 g) cycloheximide in 1 mL MeOH solution to each 500 mL of media. Pour
media into petri dishes.
Prepare Starch Casein medium using the recipe on the media page. Add 500 mL of media to a
1000 mL Erlenmeyer flask. After agar has boiled, allow to cool slightly and adjust to pH 7 with
HCl or NaOH. Autoclave @ 121C for 15 min. After cooling to approx. 45C, add 0.5 mL of
100 mg (0.1 g) cycloheximide in 1 mL MeOH solution to each 500 mL of media. Pour media
into petri dishes.
Procedure for Plating Soil
Materials:
dilution bottles (95 mL, 90 mL, and 45 mL volumes as needed)
top loading balance
weighing boats
shaker table
10 mL disposable glass pipets
pipet bulb
alcohol lamp (or gas burner)
100 l Eppendorf pipet
pipet tips
glass spreading bars (or disposable hockey sticks (Midwest Scientific #LLS-50))
glass bowl (2)
inoculation turntable
plates w/media
matches
Preparation:
Dilution Bottles:
To allow for volume loss during autoclaving, initial dilution volumes should
be measured to 97, 92, and 47 mL to achieve final volumes of 95, 90, and 45
mL of buffer, respectively. For -1 (95 mL) dilution bottles, add 3 to 5 glass
beads and two drops (0.1 mL) Tween 80. For -2 (90 mL) and -3 and higher
(45mL) dilution bottles use MPP Buffer without Tween 80. Cap all dilution
bottles loosely and autoclave @ 121C for 15 min.
Glass Spreading Bars:
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