Nematode assay
(modified from Hallem et al. 2007)
96 well plate setting:
- tissue paper approx. 1cm2
- 25 Us in 10pl PBS/ well / 29°C
- 100 Us in 10pl PBS / well / 25°C
- 100 Us in 10pl PBS / well / 22°C
- Drosophila larva in each well
- mortality counting after 24, 48 hours
- 48 larvae / group, in triplicates
Counting of mortality
Live pupa
Live larva
Mishandled larva (melanization)
Optimization - media (w1118,\ab. temp. 100 IJ/w):
Different media H.b., 100 IJ/well, n=3x96
	100 -1
	80 -
	
	60 -
	
	40 -
O	
E	20 -
	0 -
24 hours
48 hours
□ agarose
□ agar ■ paper
72 hours
100 90 80 S? 70 > 60 I 50 £ 40 E 30 20 10 0
Different media S.f., 100 IJ/well
T _JL
24 hours
48 hours
72 hours
□ agarose
□ agar ■ paper
Mortality of D. melanogasterlarvae after invasion of H. bacteriophora or S. feltiae in different media: agarose, agar or
moistured tissue paper. The dose used is 100 IJs/1 larva (mean ± S.D.).
Mortality of controls
	100 -i
	80 -
Co*	
	
>.	60 -
re	
o	40 -
E	
	20 -
	0 -
□ agarose
□ agar ■ paper
24 hours
48 hours
72 hours
Mortality of control D. melanogaster larvae in different media: agarose, agar or moistured tissue paper (mean ± S.D.).
Optimization - dose (w1118,\ab. temp. 100 or 1000 IJ/w):
100 Co" 80
o E
40 20 0
1000 IJ/well, n=3x96
I
21
24 hours
I
48 hours
72 hours
□ S. f eltiae
□ H.bacteri ophora
Mortality of D. melanogasterlarvae after invasion of S. feltiae or H. bacteriophora in tissue paper using the dose 100 or 1000 Us per larva (mean ±S.D.).
H. bacteriophora
-i—•
o
100 -1 80 -60 -40 -20 -0 -
1
24 hours
48 hours
4
72 hours
□ 100 IJ/well
□ 1000 IJ/well
(25°C, 100 IJ/w):
Scale down of nematode assay using plastic bags
only one layer of tissue paper should be used.
D.m. larvae should be washed out from the flyfood (using a sieve) and than by soft forceps transported into the bags
nematode suspension in PBS
wet chambers for incubation in termostate
mortality counting under fluorescence microscope - day light, GFP plant, merged
Plastic baas settings:
- folded tissue paper
- Heterorhabditis bactehophora TT01
- 25 Us / 1 Drosophila larva / 29°C (25 Us in 10|jl PBS/ 500 pi per bag)
- 50 Drosophila larvae / bag
- only 48 hrs mortality counting under fluorescence microscope
- in triplicates
□ W1118 DBclmd
12,5 I J/1 Oul    25 I J/1 Oul      50 I J/1 Oul