NA incubation: simple method by Anon
(Originally titled: Necator Americanus Larvae Recipe v1.3. Author unknown.)
Always use universal precautions when handling infectious or potentially infectious material.
- One fresh poop from a transmittable-disease free person who inoculated with a pure colony of Necator Americanus between two months and four years ago.
- 3 cups potting soil, vermiculite, or other additive-free grow medium (no Miracle Grow)
- One reptile or bird incubator that is capable of maintaining a temperature under 96 degrees F (35.6 degrees Celcius) (optional)
- Gauze pads
- Cotton balls
- Coffee filter, preferably not bleached
- Martini glass
- Household bleach
- Filtered or distilled water
- Clean plastic container around quart-sized, such as a large clean yoghurt or sour cream container with a lid wider than its base
- Microscope and slides
- Test tubes with stoppers, microcentrifuge tubes, or other leak-free small container
- Box of disposable latex-type gloves
- Disposable plastic pipettes, 7ml or less
- Waterproof, small garbage bags
Boil 2-3 cups of grow medium (soil or other) in a stove pot for at least ten minutes to kill any organisms. Drain excess water so that medium is wet but not soupy. Let it cool to room temperature.
1. Poke a few holes in the bottom of yoghurt container. If you will be incubating your sample, cut the container to a height that will fit in your incubator, if needed. Spread a one inch layer of grow medium on the bottom of the container. Wearing gloves, spread about a ½ to 1 inch layer of poop over the medium, then add another 1/2 inch layer of medium. Spray the surface with water if needed to keep soil very moist but with no standing water or water dripping through the drain holes. Use the container lid (or plastic plate) under the container to catch excess moisture. Remove gloves inside out and dispose of them in waterproof garbage bag designated for infectious waste before touching anything clean. Put any remaining cooked medium in air-tight baggies in the freezer for future hatching.
2. Necator larvae hatch best at 88-101 degrees F. (31-38 degrees Celcius) Ancylostome larvae hatch best at room temperature. Both species, once hatched, live longest at room temperature. As an added safety precaution against hatching ancylostoma, I recommend incubating your poop lasagna for 5 days around 92-95 degrees F (33-35 degrees Celcius) if possible to create conditions that are ideal for Necator, not ancylostoma, to thrive. Fill all water chambers in your incubator and add your specimen container. Spray the top of the soil as needed (usually 2-3 times in 5 days) to keep it moist. If you are not using an incubator, store specimen in a very warm, dark place for 5-6 days, protected from flies, etc., and spray daily or as needed to keep the soil moist.
3. At the end of five days, unplug your incubator. Put on your gloves and gently press one layer of gauze over the top layer of soil. Unravel some cotton balls and saturate them in 102 degree F (39 degrees Celcius) filtered water. Put about a ¼ inch layer of wet cotton over the layer of gauze. The larvae will be attracted to the heat and moisture. Wait 12-24 hours, then spray the cotton layer with 102 degree (filtered) water again.
4. Warm a martini glass by filling it with hot filtered water. Once the glass is warm, dump out all but 1 tablespoon of the water. Set the coffee filter inside the glass, allowing the water to wick into the filter, and shape the filter around the glass so that the filter comes over the edges of the glass and the center of the filter dips down about 1 inch from the bottom of the inside of the glass. If you do this correctly, the wet filter will hold its position without needing to be secured.
5. Use tweezers to remove the cotton from the top of specimen and place it upside down in the filter in the martini glass. Poor enough 104 degree F (40 degree Celcius) filtered water over the cotton so that the bottom of the cotton is submerged slightly in water and the top is exposed to air. The larvae will move away from the cold air and towards the warm water. Set the martini glass in a 4 inch deep steaming water bath for at least two hours to encourage larvae to burrow downward through the filter and towards the warmth below. This can be done over the stove if you are careful to avoid temperature extremes that might break the glass. It will take 24 hours or more for the majority of the larvae to reach the bottom of the glass. Give them time.
6. After 24 hours, prepare a bleach/filtered water solution of 1 drop regular strength, unscented household bleach in a US or UK quart (1 litre) of filtered water and set aside. The bleach is optional, but the larvae tolerate it well and it will help kill and inhibit other contaminates, especially if you plan to store your larvae for any length of time.
7. Put on your gloves and gently slide your pipette between the filter paper and the martini glass to draw up a couple drops of water from the very bottom of the martini glass. Look at the drops under 40x magnification to find and count larvae on a slide without a cover slip (100 to 200x magnification is best for looking closely at individual larvae). The larvae may be sluggish or seemingly dead, particularly if their water is still warm. Some may be active while others are not. If they have been at room temperature for awhile, they often become active under the warm microscope. They may move quickly like sidewinders, slowly without much purpose, or even try to stand up and poke their heads through the surface of the drop. If they have debris on them, they may appear to be cleaning themselves. They may be straight or curved. It is possible, particularly as they age, that you see them shedding an outer layer. If this is the case, they may appear longer than normal. If you see anything moving around that looks different from the rest of the translucent, pink-gray-green larvae (appendix pictures under green lens do not show true color), freeze and discard everything. If you see plenty of larvae, you can now bag, freeze and then discard the feces, medium, gauze, and cotton balls. If you don’t see larvae, repeat the process starting with the application of gauze and cotton (1st paragraph of this page). If after two times you still don’t see larvae, you probably need to start over with a fresh sample.
8. Count larvae using a down-across-down-across pattern in the drop of water on your slide until you have covered the entire drop. Repeat this until you get the same number more than once. Alternatively, it may be helpful to use a fine-tip permanent marker to draw a + on the back of the slide, then place the droplet in the center so that you can count larvae in each quadrant. There are sometimes larvae trying to burrow at the sides of the drop that are hard to see so look carefully at the drop edges, refocusing if needed. Avoid killing the larvae by letting their water droplet dry out from the heat of the microscope light. They will become very frantic and active as the droplet gets shallower and smaller. If needed, pipette a drop of bleach water onto the slide before the droplet starts to evaporate. If there are too many larvae to count, add a drop of bleach water to the slide droplet. Suck part of the droplet (and larvae) back into the pipette and return them to the martini glass. Add another droplet of water to the slide if needed. Repeat this until there are a manageable number of larvae on the slide.
9. When you are ready to store your counted larvae, suck the droplet from the slide into just the tip of the pipette and transfer to test tube or other container. Then pipette up a little bleach water and use it to rinse remaining larvae off the slide so that the wash water/ larvae drip into the container. If the collection container is getting too full of water, give the larvae about an hour to settle at the bottom of the container, then pipette excess water off the top portion only. Replace the test tube stopper/lid and label your container with # of larvae, date harvested, or any other information you want to track. Stored at room temperature away from light, larvae can remain alive 8 weeks or more.
Tips and Tidbits
The following information was obtained from the book Hookworm Disease: Its Distribution, Biology, Epidemiology, Pathology, Diagnosis, Treatment and Control, by Asa C. Chandler, 1929
- Necator larvae from the stools of different individuals have a varying ability to survive under the same conditions. No correlation has been made on why this is the case, but heredity, diet, acidity of stools, and body excretions have been suggested as contributing factors.
- The minimum time required for larvae to go from entry point (skin) to trachea is two to three days. The majority of the larvae reach the trachea within five days.
- The time from infection to the excretion of eggs in the feces is approximately six weeks.
- In nature, Necator larvae have caused infestations in soil with a pH as low as 5.
- Very low degrees of infestation occur in areas where soil is heavily impregnated with salt.
- Larvae yields from soil are largely dependent on soil texture. Fine textured soil (clay) yields fewer larvae than coarse (sandy loam).
- Hookworm-related anemia appears to be due to suppression of the body’s ability to form blood, not from blood loss.
- Ancylostoma duodenale is far more injurious than Necator americanus. Infection with as few as 20 ancylostoma can cause hookworm disease (anemia, abdominal pain, pallor, fatigue, etc.).
Figure 1: Necator Americanus (NA) infective larva under green lens. Pointed tail is at the top of the picture, head is at the bottom. Approximately ¼ body length up from the head is a transverse line marking the union of the esophagus and intestine, which distinguishes it from the curved appearance of the same structure in Ancylostoma Duodenale (AD) 3rd stage larvae.
Figure 2: Head end of NA infective larva under green lens. The black line inside the tip of the head is the esophageal spear. This structure is longer and thicker-walled than the same structure in AD infective larvae. In NA, the walls widen anteriorly to give it a spear-head appearance. In contrast, the AD lumen widens anteriorly, which makes this structure in AD larvae appear hollow in comparison to NA larvae. Note outer sheath sloughing off at head.
Other NA incubation methods
- NA incubation: very simple Harada-Mori method by Sarah (The method most used by home growers and featuring supplementary details that will be useful to anyone using a different method.)
- NA incubation: very simple petri dish method by Steven (The second most popular method.)
- NA incubation: very detailed method by Alana. (This method provides information about every aspect of hookworm incubation.)