Worm Breeder's Gazette 10(1): 138

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Protocol for Testing Water Insoluble Compounds on Caenorhabditis spp.

A. Fodor and E. Kulcsar

Nematodes belonging to the Caenorhabditis genus can live only in wet 
conditions, like the surface of an NGM plate.  Majority of the drugs 
and chemicals which should be tested on nematodes because of one 
reason or another are water insoluble and hydrophobic.  When testing 
precocenes on nematodes we used to face this problem.
Now, we present a method here which we found applicable to the 
majority of hydrophobic compounds.  The idea was to attach water 
insoluble compounds to liposomes.  We decided to try to use milk as a 
natural and universal liposome.  The protocol we used was the 

(1)  Compound under test was dissolved in its proper organic solvent 
(acetone or ethanol).  A stock solution of 100 mg/ml (I) was made up.
(2)  A dilution series (II) of 50, 25, 12.5, 6.05 and 31.25 mg/ml 
would then be prepared.
(3)  0.1 ml of each II solution was mixed with 0.9 ml of sterile 
milk (10 mg milk powder/ml of M9) to obtain homogeneous suspensions (
III) of 10, 5, 2.5, 1.25, 0.625, 0.3125 mg/ml concentrations, 
respectively.  Majority of the compounds (including 121 precocenes and 
89 other compounds) we examined. Furthermore, avermectin (as a 
positive control) behaved as if it had been coupled to liposomes and 
could be mixed up with NGM agar homogeneously.
(4)  The total (of 1 ml) volume of each III 'solution' was mixed up 
with 9 ml of melted NGM agar, (IV). 4X1 ml samples of each IV solution 
were pipetted into the wells of LINBRO embryo plates and let become 
solidified.  In single LINBRO plates we had four repeats of 1000, 500, 
250, 125, 62.5, 31.25  g/ml NGM concentrations; (e.g., 24 small 'Petri 
plates').  In case of more effective molecules, further dilutions were 
made up.
(5)  Eggs were prepared from gravid female (hermaphrodite) worms and 
a 10-15 eggs/ l suspension were made.  5  l of this suspension was 
pipetted to the agar surface and dried for five minutes in laminal box.

(6)  The eggs were to be counted immediately.
(7)  5  l of E. coli (OP50) suspension was then pipetted on the agar 
surface and dried for five minutes in laminal box.
(8)  The LINBRO plates were then covered and placed into the 
thermostate.  Our test animals were C.  remanei and were incubated at 
27.5 C for 36 hours.
(9)  During this time, in the control plates the animals grew to 
adults but just started to lay eggs.  (The eggs did not hatch within 
the next nine hours, if there was about 20 C in the lab.)  The animals 
were counted and scored during this time and the next generation was 
not disturbed.
This method proved good for studying both toxic and developmental 
effects of compounds we have tested so far.  It gave reproducible and 
quantitative data.