Media for the Growth of Bactertial Symbionts for Entomopatho

Friday, October 24, 2003

Description
Exact preparation of plates with lawns of bacteria for the in vitro growth of entomopahtogenic nematodes belong to Heterorhabditis and Steinernema

Procedure
Here's the protocol for raising nematodes on bacterial lawns:

Protocol for raising Steinernema carpocapsae on bacterial lawns of
Xenorhabdus nematophila

Day 1
1. Using a single bacterial colony, inoculate 2 ml of LB broth containing 50 ug/ml ampicillin and grow bacteria at 29-30o C for 18 hrs. with shaking at 150 rpm

Day 2
2. Put 5 ml of LB broth in a 200-250 ml sterile flask. Inoculate the 5 ml of LB broth with 100 ul of the 18 hr culture (above). Record initial Klett reading or OD600 reading.
3. Shake culture at 29-30oC with shaking at 150 rpm. Monitor cell growth. When the Klett reading is approximately 100-150 (midlog phase) transfer the bacterial culture to room temperature and determine the OD 600 value. We are shooting for an OD value between 1.0 to 1.5. If you are growing different strains, make sure that they are all at identical OD600 values. Correct for differences by diluting the cultures with high cell density to match the one(s) with lower cell density.
4. Aliquot 100 ul of the bacterial culture onto the middle of the lipid agar plate making a puddle. Use a sterile loop to spread the culture over the surface.
5. Incubate the lipid agar plate at at 29-30oC for 24 hrs.

Day 3
6. Incubate the lipid agar plate an additional 24 hrs at room temperature in the dark under moist conditions (a drawer with wet paper towels).

Day 4
7. Application of nematodes onto the bacterial lawn.
A. Depending on the density of the IJs in the water trap, take enough volume to make a small nematode pellet after a quick spin. Usually 1 ml is sufficient.
B. Spin the nematodes for 10 secs making a nematode pellet. Remove the supernatant
C. Add 1 ml of 2% bleach (dilute 1 part Chlorox (6%bleach) with 2 parts distilled water). Mix and let nematodes sit a room temperature for 2 min.
D. Spin for 10 secs and remove the bleach. Wash the nematodes 6 times with 1 ml sterile distilled water each time.
E. Depending on the size of the nematode pellet, add 0.5-1.0 ml of sterile distilled water. We are shooting for a nematode concentration of 15 nematodes/ul
F. Determine the nematode concentration by making 1 ul spots on a glass slide. Make 6 spots and calculate the average number. Make sure you mix the nematodes after each spotting.
G. Determine the volume necessary to spot 300 nematodes (approximately 20 ul) around the bacterial lawn.
H. Place the lipid agar plate in a drawer with moist towels. Observe the development of the nematodes daily. It will take between 10-14 days before the lipid agar plate is fully covered with nematodes. At this point you will place the lipid agar plate into a water trap.

Recipes
Standard Nutrient Broth with 40ug/ml ampicilling and 5% Karo Syrup as a lipid source

Supplies
Sterile Petri dishes (3mm)
Sterile disposable 1 ml pipetters

Tips

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Media for the Growth of Bactertial Symbionts for Entomopatho