Difference between revisions of "Team:Uppsala/Protocol"
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<h1>Worm recovery from cups</h1> | <h1>Worm recovery from cups</h1> | ||
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<li>Cups</li> | <li>Cups</li> | ||
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<h1>Worm purification</h1> | <h1>Worm purification</h1> | ||
| − | <h2>Materials | + | <h2>Materials</h2><br> |
<ul> | <ul> | ||
<li>Loose cotton</li> | <li>Loose cotton</li> | ||
Revision as of 14:23, 16 October 2018
Worm recovery from cups
Materials
- Cups
- 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
Worm 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
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
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