Worm recovery from cups

Materials:

The cup is filled with water (200 mL)
One half of a petri dish is placed on top. The cup with the petri dish is then turned upside down
The cup is left at room temperature ON, to allow the dry material to soak water and deposit on the bottom
The following day the cup with the petri dish are tilted to recover the liquid, which contains the worms
The worm solution is left in the fridge for 3 hours, to allow a complete deposit of the worms
Most of the supernatant is removed, and the worm solution can be stored

Cotton is added to a Pasteur pipette to obtain a filter around 1 cm wide
Some drops of physiological solution are added to each pipette, to soak the filter
One worm solution deriving from each isolation protocol is added slowly making sure it doesn't flow through
The solution is left in contact with the filter for 30 min
Around 5 mL of physiological solution are added on top of the filter, until all the liquid has run through. Lifting the pipette from the liquid in the bottom of the falcon can help in this process
Purified worm solutions are stored at 4°C to leave the nematodes to settle. When sample is needed, supernatant is removed to concentrate the samples

The physiological solution, the sample and the bleach solution are stored in ice, to maintain the cold temperature, which favours the formation of the worm pellets
Physiological solution and bleach solution (1%) are added to a final volume of 5mL
The sample is centrifuged at 2000 rpm at 4°C, for 2 min
Most of the supernatant is removed, and the pellet is resuspended in physiological solution
Steps 4 and 5 are repeated for a total of 4 times
The sample in physiological solution is stored in the fridge until needed
100 uL of each sample is incubated in 10 mL of LB in a 50 mL flask
Flask is incubated at 37 °C ON
Visual inspection of the flasks allows the determination of contamination presence