Nematode recovery from cups

Introduction

This protocol described the steps necessary to successfully hatch the strongyles eggs present in horse faeces and the successful recovery of strongyles larvae in the third larval stage.

Materials

• Plastic ups
• Faeces
• Physiological solution (0.9% NaCl)
• Tap water

Procedure

Incubation of faeces

• Around 20 g of faeces are added to a cup
• The faeces are stirred with tweezers to make the sample less compact
• The cup is incubated at 29 °C for 1 week

Nematode recovery

• 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

Worm purification

Introduction

This protocol described the procedure used to remove faecal debris from strongyles samples, that would otherwise compromise the nematode sterilization procedure.

Materials

• Loose cotton
• Pasteur pipettes
• 15 mL falcon tubes
• Physiological solution (0.9% NaCl)

Procedure

Debris removal

• 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

Worm sterilization

Introduction

This protocol described the procedure used to sterilize the purified nematode samples. An initial high nematode concentration is not suggested since it leads to a reduced efficiency of strongyles sterilization. For a single co-culturing experiment between 2 and 4 purification samples are used, to reach, in the end, a nematode concentration high enough.

Materials

• Bleach 2%
• Sterile physiological solution (0.9% NaCl)

Procedure

• 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

Co-culturing

Introduction

This protocol described the steps necessary to perform a successful co-culturing of E. coli with nematodes, necessary for inducing the genetic response in E. coli.

Materials

• Sterile nematodes
• M9 media
• RNA protect (Qiagen)
• Whatman n°1 filters
• Buchner flask
• Vacuum source
• E. coli MG1655 ON culture

Procedure

Culture preparation and co-culturing

• A colony of E. coli is dissolved from a plate in 10 mL of M9 media
• Culture is incubated at 37°C ON
• The day after, the OD of the ON culture is measured
• The supernatant of the sterile worm pellets, taken from the fridge, is recovered. The pellets are joined in a single falcon and washed once with sterile physiological solution. (centrifugation at 2000 rpm for 2 min at 4°C). Supernatant is discarded
• 2 cultures are prepared: the control, with Vf=10 mL and the worm sample, with Vf=6 mL in M9 media
• ON culture and fresh M9 are mixed in the calculated volumes to obtain an OD of 0.05
• 500 uL of the initially recovered supernatant are added to the control sample, and the concentrated worm pellet is added to the worm sample
• The initial OD600 of the samples is determined, using a 1:2 dilution in new M9
• The samples are incubated at 37 °C in a shaker. A horizontal disposition of the falcon is suggested to increase the movement of the liquid and of the worms, allowing a better contact with the bacteria
• The OD600 is measured every hour in the control sample, until an OD of 0.8 is reached
• The OD600 of the worm sample is determined as well (1:10 dilution), to ensure that the sample has grown
• RNA protect is added to both samples with a volume of 2 volumes of RNA protect per volume of culture left in the samples
• The worms are removed through vacuum filtration
• A Buchner flask is set up with a Whatman n°1 filter on top
• Vacuum is connected to the flask
• Co-culturing solution is placed inside the filter. The vacuum is opened and the filtered solution is recovered and used for further experiments

Worm separation system

Introduction

This protocol describes the steps necessary for the realization of a microfluidics chip and the protocol for the separation of large from small strongyles.

Materials

• Polydimethylsiloxane (PDMS)
• Optical microscope
• Syringe pump
• 2 mL syringes
• 1 mm tubing
• Ethanol 70%
• Corona discharger
• PDMS prepolymer curing agent
• Isopropyl alcohol 70%
• Physiological solution (0.9% NaCl)

Procedure

Chip creation

• The mold for the microfluidic chip has been designed through the use of a 3D design software
• The mold has been printed with a 3D printing machine
• The printed mold has been cleaned with isopropyl alcohol, to remove any resin residue from the 3D printing
• PDMS activated with the prepolymer curing agent with a ratio of 10:1 has been poured in the mold
• The air bubbles have been removed through a 30 min incubation in a vacuum chamber
• The chip is left curing at 60 °C for 1h
• The polymerized chip is removed from the mold. If occluded, the holes are punched where the tubing has to be connected
• The chip and a glass microscope slide are oxidized through a 30 sec treatment with a corona discharger
• The chip is placed on the glass slide to obtain the complete microfluidics chip

Large strongyles separation

• The separation is performed every time on one sample deriving from the worm purification
• The system is cleaned with ethanol 70% followed by physiological solution, to equilibrate the chip and remove any residue of alcohol
• The sample is collected in a 2 mL syringe
• The syringe is placed in a syringe pump
• The solution is loaded at a higher pressure until it reaches the chip
• Once the separation starts, a flow between 1 and 2 mL/h is used, depending on the worm concentration
• The flow is directed with the valves at the end of the system, to channel all the large strongyles in one collection tube
• Once all the sample has run through, the system is cleaned with ethanol 70%, which is left in the chip for future use