This
is an outline and modification of methods developed by Jaffee et al. (1996):
1. Prepare quarter-strength cornmeal agar
(CMA/4)1 amended with 0.1 g/L of streptomycine sulfate after the
CMA/4 is cooled down to approximately 50°C (does not burn your hand). Pour the agar medium into
9-cm-diameter or 6-cm-diameter petri dishes. Prepare 15 plates of CMA/4 for
each sample.
2. Subsample 100 g of soil from each
sample, placed in a 250-mL beaker, and add 200 mL sterile distilled water. Stir
the suspension vigorously for 3 minutes.
3. Conduct 10-fold dilution of the
well-mixed soil suspension twice. This will provide three soil dilutions: 0.5,
0.05, and 0.005g of soil/mL H2O.
4. Each dilution will be plated on 5
replicated petri dishes. Each petri dish will receive 100 ml of soil dilutions. Thus, a total of 15
plates for each sample with 5 plates each of 0.05, 0.005, and 0.0005 g of soil
/ plate.
5. Add approximately 1000 Steinernema
glaseri (a large and long-lived entomophilic nematode) in 0.1 mL distilled
water to each plate as a bait for nematode-trapping fungi. This will increase
the chances of detecting nematode-trapping fungi.
6. Infective juveniles of S. glaseri
can be obtained from infected Galleria mellonella according to protocol by Woodring and Kaya (1988)
and stored at 10°C.
7. Observe the entire surface of each petri
dish 3 weeks after plating under a dissecting microscope with 50-140 ´ magnification.
8. Record the presence and absence of
nematode-trapping fungi in species using the Cooke and Godfrey key (1964) for
each petri dish.
9. Use a most-probable number table
(Alexander, 1982) or see the Most Probable Number Table below to estimate the
number of fungal propagules per g soil.
_______________________________________
1 Prepare CMA/4 by mixing quarter strength of Corn Meal Agar (Becton
Dickinson and Company, Sparks, MD), i.e. 4.25 g/L H2O with three quarter
strength of Bacto Agar (Difco Laboratories, Detroit, MI) i.e. 8.25 g/L H2O.
Most
Probable Number (MPN) Table
Base of dilution ratio = 10
No plates/dilution = 5
Inoculation vol. (in mL) =
0.1
Initial dilution is 1 to = 2
|
|
No. of
positive plates on dilutions |
|
|
|
||
|
0.05 |
0.005 |
0.0005 |
MPN |
P(0.95) |
||
|
0 |
0 |
0 |
0 |
- |
||
|
0 |
1 |
0 |
3.6328 |
1.1002-11.9952 |
||
|
1 |
0 |
0 |
3.9690 |
1.2020-13.1053 |
||
|
1 |
1 |
0 |
8.0454 |
2.4366-26.5653 |
||
|
2 |
0 |
0 |
8.9257 |
2.7032-29.4719 |
||
|
2 |
1 |
0 |
13.6612 |
4.1373-45.1079 |
||
|
3 |
0 |
0 |
15.5307 |
4.7035-51.2812 |
||
|
3 |
1 |
0 |
21.3767 |
6.4740-70.5844 |
||
|
3 |
2 |
0 |
27.6370 |
8.3699-91.2553 |
||
|
4 |
0 |
0 |
25.4592 |
7.7104-84.0646 |
||
|
4 |
1 |
0 |
33.6994 |
10.2060-111.2728 |
||
|
4 |
2 |
0 |
43.1111 |
13.0564-142.3500 |
||
|
4 |
3 |
0 |
54.0130 |
16.3580-178.3470 |
||
|
5 |
1 |
0 |
65.4321 |
19.8163-216.0520 |
||
|
5 |
2 |
0 |
97.7203 |
29.5949-322.6654 |
||
|
5 |
3 |
0 |
156.1747 |
47.2980-515.6776 |
||
|
5 |
4 |
0 |
254.5840 |
77.1016-840.6184 |
||