NA Incubation: Simplest procedure for lots of larvae
This page is still being developed, so please call back later to read further details as they are added.
Safety
Essential safety requirements
- Use a dedicated work area that you will be able to thoroughly clean after processing.
- Restrict access to the work area: no children, pets, or careless friends.
- Wear appropriate PPE, including gloves at a minimum.
- Disinfect all materials and work surfaces after processing.
General safety considerations
- Since a NA incubation period is generally 5-10 days, you can easily schedule processing for an appropriate time when you will not be interrupted.
- Review and understand this procedure before you start.
- Prepare your work area ahead of time with all the supplies you will need.
- If you need to leave the work area, remove gloves and wash hands.
- Clean and disinfect all areas exposed during processing. Sterilize any leftover sample materials and dispose of them properly. Wash or dispose of any PPE used.
The most effective sterilization technique is boiling for 5 minutes, or texting for 24 hours. If you use a microwave to sterilize your equipment after use, it must be in boiling water in the microwave. 70% (140 proof) alcohol is also effective and it's analgesics as isopropyl alcohol in stores. See this page: ******** link to safety procedures **********
The method
Incubation
1. Smear a small amount (½-1 tsp, 3-5 ml) of feces in the inside top of a cone coffee filter. You do not have to process the feces unless it is VERY hard. As long as you can smear it on the paper, it's good. Cone filters are stiffer than basket filters and will not collapse. This keeps the water clean.
2. Place the filter in the a quart (liter size) container (like for yogurt or store cream). Add enough water to wet the filter and leave about ¼" (0.5 cm) of water in the bottom of the container. Put the lid on. Label the lid with the date. Keep it out of direct light in room temperature for 7-10 days.
3. To harvest, pour the water into a conical bottom container and let it settle a few minutes. NA larvae settle at 0.39 m/h, so a few minutia is plenty of time.
4. Once settled, pull a drop of water from the conical bottom and place on the slide. Examine at 40X for larvae. If too many, take part of that dip and make a smaller drop, continuing until you have the desired number of larvae in a drop.
5. Put that drop in a storage container, add more water to the slide and agitate gently to loosen any missed larvae while you pull the rinse water into the disposable pipette, and add that to your storage container. This will captor any larvae that were missed the first time.
Notes: .This writer believes that larvae die when stored any length of time in water that is too deep. She finds that 2", 5cm is safe. . This method prevents the common issue of confusing lint and other things with larvae, but it doesn't affect the fact that many new incubators have a hard time seeing larvae at first. Patience, practice, and trying different techniques such as focus work. If your see movement, stop and focus. . This author has seen that larvae literally disappear with minutes once they die. Healthy larvae don't necessarily move. They conserve their energy.
Harvest
4. To harvest, pour out the water, settle in a conical bottom glass, and scope the liquid from the bottom of the glass, where the larvae have settled. I add more water to the original container if I want more harvests from the culture. Can apparently harvest for weeks.
Note: one of my incubation problems was water level when settling larvae. Apparently they cannot take water more than 1" (2 cm) deep for any length of time. I believe the water pressure kills them. Or perhaps it's the oxygen saturation as Forrest suggests.