NA incubation: super-simple method by Laurel

⚭Home>Incubation>NA incubation: super-simple method by Laurel

Method

I use a toothpick so I can throw it away, although it isn't the most convenient applicator.

Ready to cap and label

Pouring into conical bottom container.

Benefits of this method

* Perfectly clear water free of lint and other confounding items.

* No need to ever touch feces or larvae (until you apply the larvae to your skin).

* Entire process takes 5 minutes to prepare, and 5 minutes to harvest.

Summary

1. Collect sample and smear a small amount on a coffee filter.

2. Place filter in container, add water and close lid. Label with date, and wait about a week.

3. Pour the water from the incubation container into a conical bottomed container and let it settle.

4. Microscope for larvae.

Sample collection (no-touch method)

1. Line a colander with toilet paper and place under the toilet seat.

2. Take a sample from more than one area of the feces in case the eggs are not distributed evenly.

3. Smear ½-1 teaspoonful (3-5 ml) of feces around the inside top of a paper cone coffee filter. There is no need to process the feces unless they are VERY hard. As long as they stick to the paper, it's good. Cone filters are stiffer than basket filters and will not collapse. This keeps the water clean.

4. Dump the remaining contents of the colandar in the toilet and flush.

Incubation

1. Place the filter in a quart (liter size) container (like the ones used 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 and label this with the date.

2. Keep the container at room temperature and out of direct sunlight for 7-10 days. (See more about incubation temperature here.)

Harvest

1. After at least 10 days, pour the water from the sample into a conical-bottomed container such as a wine glass, silicone snow cone cup, or glass test/centrifuge tube and let it settle for a few minutes. Since NA larvae settle at 39 cm/h (15" an hour), a few minutes is plenty of time. Laurel feels this purchase was the very best one she made for incubation, and considering there are no other supplies to be purchased, it is well worth the convenience it provides.

2. Once settled, use a pipette to pull a drop of water from the conical bottom and place this on a microscope slide. Scan this for larvae at 40X magnification. If there are too many larvae, take part of that drop to make a smaller drop, and continue to do this until you have the desired number of larvae in a single drop.

3. If this drop is to be used as a dose, place it directly onto a bandage / dressing to be applied to the self-treater's skin. (See more about inoculation here.)

4. Add more water to the slide and agitate this gently to loosen any remaining larvae, then draw this rinse water into the pipette and add it to a storage container. This will capture any larvae that were missed previously.

5. Further harvests can be made over several weeks from the same incubation, but the larvae do become progressively weaker with time as they rely on their own finite fat stores at this stage in their development. (See more about hookworm larvae storage here.) Please note that this was added by a site admin; Laurel does not see any diminution of viability for several months, when applied directly (not shipped).

Cleanup

When finished with incubation supplies (tub, tube, slide), I freeze them at least 24 hours, then wash them as I would any dish. I dispose of the contents of the tubs (after they thaw) in the toilet before I wash them.

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 personal protective equipment as needed.
• Disinfect all equipment, work surfaces and materials after processing.

General safety considerations

• Review and understand this procedure before you start.
• Ask any questions in one of the helminthic therapy support groups, ideally in the Helminth Incubation group on Facebook.
• Prepare your work area ahead of time with all the supplies you will need.
• Since an NA incubation period is generally 5-10 days, you can easily schedule processing for an appropriate time when you will not be interrupted. I personally harvest up to 2 months later, so there is no rush as far as scheduling is concerned. I personally leave them for many weeks, until I need more larvae.
• If you need to leave the work area, remove gloves and wash hands.
• After processing, clean and disinfect all areas that have been exposed to larvae, and wash, or dispose of, any PPE used. Sterilize any leftover sample materials and dispose of them appropriately. The most effective sterilization technique is boiling for 5 minutes or freezing. If a microwave is used to sterilize equipment after use, the items must be in boiling water inside the microwave. 70% alcohol (140 proof) is also effective and it's sold as isopropyl alcohol in stores, but note that 90% isopropyl is not as effective for sterilization. (See further details about safety, here.) However, larvae will not survive more than an hour after drying out. It is the ova that can survive longer.

Supplies

Disposable pipettes

50 ml centrifuge tubes with stand

microcentrifugal tubes. Optional for home use

for incubation

• A 1 liter/ 1 quart container with a lid.
• A cone coffee filter. You can put 2 in one container if you like.
• Water. I now use tap water, which is chlorinated, but not strongly so. I used to use dechlorinated.
• A secure location to store the container until you harvest. I use a high shelf, now that I don't have kids.

for harvesting

• Microscope. Selection of a suitable microscope is discussed here. Personally, the only feature I consider essential is a movable stage. I don't even use the binocular function.
• Slides. I only use (and reuse) a couple of dozen slides, but you have to buy them in large boxes. There is no need for slide covers.
• Disposable pipettes. I use 5.0 ml and 0.5 ml.

• Centrifugal tubes. These are not essential, but I consider centrifugal tubes with a stand to be my best incubation purchase by far.

for storage

• You can store the larvae in any food-safe container with an airtight lid. The main thing is to keep the water level low. I just store in the incubabion container or the conical centrifuge container until I am ready to count some out.
• Microcentrifuge tubes are not essential and I only use them when shipping larvae to a friend.

I prefer to store my larvae uncounted, and count them when I am going to apply them. I find this the most flexible and dependable method.

Notes

• Many new incubators have difficulty in actually seeing any larvae. Patience, practice and trying different techniques - such as adjusting the microscope's focus - are needed initially. If you see movement, stop and re-focus, but bear in mind that:

• Healthy larvae do not move constantly because they need to conserve their energy.
• Larvae die when stored for any length of time in water that is too deep. This may be a result of the water pressure, or perhpas it's a matter of oxygen saturation. Anyting less than two inches (5cm) appeaars to be safe. (Note that some incubators disagree with me and do not have this issue. You will find your own path.)
• Larvae can literally disappear within minutes once they die. At least that's been the experience of this author. So if they look healthy, they are. Whether they are moving or not.

• Do not use distilled water for incubating. The larvae will die. To dechlorinate your tap water, put it in an open container for 24 hours. Alternately, you can purchase spring water or drinking water with added minerals.

Other NA incubation methods

• Alternative NA incubation methods

the human hookworm, Necator americanus

The active immature form of an insect, or an animal such as a helminth, which develops from an egg and eventually transforms again into its adult state.

Someone who treats their own disease or condition without medical assistance.

The introduction of an infectious agent into an organism. Helminth inoculation

A helminth that lives in the small intestine. Necator americanus (NA) is the only hookworm species used in helminthic therapy. Its microscopic larvae are applied periodically to the skin.