Endpoint for Bacterial Pathogen Feeding Assay

Hello All

I know this topic was asked already but I have some more questions:

We are feeding Campylobacter in a liquid state Nematode assay but we are having a hard time finding a relatively non-subjective endpoint for the assay.

Healthy worms are motile and wiggling and the worms go through various stages after the feed

  1. Non-motile but wiggling
  2. Non-motile and occasionally wiggling
  3. Non-motile and rarely wiggling
  4. Non-motile and non- wiggling and bent
  5. Non-motile and non- wiggling and straight but still moves if poked
  6. Non-motile and non- wiggling and straight and non-responsive (this takes about 2 hours in our assay)

Interestingly enough, the worms still score as alive even at stage 6 when stained with SY-TOX Green and observed with a fluor microscope.

It has been said that Nematodes die from the inside out and since the worms are so small it is conceivable that some parts of the worn could take a long time to die.

Which of the stages are the most meaningful WRT toxicity of a feed? Stages 2-6 are all absent in the media and dead feed controls so they all may have some meaning.

We are considering mass screening worms using cytox green and a fluor plate reader using the method described in :

DNA methylation modulates Salmonella enterica serovar Typhimurium virulence in Caenorhabditis elegans by Javin P. Oza, Jimmy B. Yeh and Norbert O. Reich

but there is the problem with the worms looking dead but not allowing dye permeation. Does anyone know about the death stages of nematodes and how far along they have to bee to allow permeation.