Wet the soil well to create a saturated paste (see the protocol for isolating from soil by baiting above and Richards, L. A. and Staff, U. S. S. L. 1954. Diagnosis and improvement of saline and alkali soils. U. S. Department of Agriculture handbook 60:8-9) and put 1-2 ml (about ½ tablespoon) on each of several plates of selective medium (PARP or NARF are preferred by Moorman; see Part 1: Media for Pythium Culture).
Incubate the plates for about 24 hours.
Gently wash the soil off the agar surface using cold tap water.
Gently shake off excess water.
Incubate the plates about 24 hours.
Using a light microscope, examine the hyphae.
Transfer coenocytic hyphae to a new plate of water agar or selective medium. Do not throw away the original plate until you are certain you successfully transferred the organism and it is definitely growing on the new plate.
Isolating from water by filtering
Filter water through a membrane that will retain 5.0 µm particles or larger.
(Very good filters for this purpose are: www.millipore.com; Cat. No. SVLP04700; 5.0 µm, White Hydrophilic Durapore. These are 47 mm diameter. For a Buchner funnel, use Cat. No. SVLP09050; 5.0 µm, White Hydrophilic Durapore. These are 90 mm diameter.)
Invert the filter onto water agar or a selective medium (see Part 1: Media for Pythium Culture).
Incubate the plates about 24 hours.
Discard the filter.
Using a light microscope, examine the hyphae.
If no hyphae are obvious, incubate the plate for another 24 hrs and then examine them.
Transfer coenocytic hyphae to a new plate of water agar or selective medium. Do not throw away the original plate until you are certain you successfully transferred the organism and it is definitely growing on the new plate.
Examine the water agar for structures characteristic of Pythium (oogonia, sporangia, appressorium-like structures where the hyphae contact the plastic dish. Look both close to the initial inoculum block AND far from the inoculum block.)
Transfer isolates suspected of being Pythium to a selective medium and water agar.
