NA incubation: very simple Harada-Mori method by Sarah

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Home>Incubation>NA incubation: very simple Harada-Mori method by Sarah

Introduction

This simplified protocol will suit anyone with appropriate knowledge and experience of laboratory practice. Those without this background may benefit from first reading Alana's very detailed NA incubation method.

Preparing the mixture

If you are using a donor, have them do this step if they don’t live nearby and if it is going to take over 20 minutes to get the sample to you.

Supplies

  • wide mouth container (urine container, mason jar, etc.) with screw tight lid
  • barrier gloves (latex, nitrile, etc.)
  • gallon ziplock bag
  • waxed disposable plates (for a work surface)
  • plastic knife
  • vermiculite (with no added material)
  • charcoal (capsules are fine)
  • distilled water

Procedure

Take some feces from a hookwormA 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. host who has been infected for at least 6 weeks. (Eggs aren’t produced before this, and the quantity of eggs increases in the following weeks.) Place the feces in a covered container. A large amount of feces is not necessary, a few tablespoons will be plenty.

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Add vermiculite (one part vermiculite to one part stool) and charcoal. I use one charcoal capsule, or two if I’m doing a big batch. The charcoal kills the smell and balances the pH, and the vermiculite retains moisture so prevents the mixture drying out.

If necessary, add distilled water to get to a pudding consistency.

Use a knife to mash any large chunks and repeat until smooth.

Wash outside of container with soap and water or alcohol swabs.

Clean up by placing supplies in ziplock bags and into freezer overnight to kill any residual material.

Now you are ready to incubate.

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Incubation

Supplies for the rest of the process

  • wide mouth container (urine container, mason jar, etc.) with screw tight lid. It's also possible to use centrifuge tubes with conical bases propped up in an egg carton. In this case, the larvaeThe 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. collect conveniently in the tube’s conical base. [1]
  • barrier gloves (latex, nitrile, etc.)
  • gallon ziplock bag
  • waxed disposable paper plates (for a work surface)
  • glass slides
  • coffee filter paper
  • plastic spoon
  • distilled water
  • pipette
  • conical-bottomed container such as a wine glass, silicone snow cone cup, or glass centrifuge tube
  • microscope (capable of 100x magnification, preferably binocular compound and with a moveable stage)
  • gauze
  • tape/ bandage

Procedure

Cut a strip of filter paper to the size of the glass slide, leaving an area free so you can pick up the slide. (A wooden craft stick makes a suitable alternative to a glass slide if it is cut to size with shears so it fits inside the container. Old credit cards and sections of CDCrohn's disease, also known as regional enteritis, is an inflammatory disease of the intestines that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea (which may be bloody if inflammation is at its worst), vomiting, or weight loss, but may also cause complications outside the gastrointestinal tract such as skin rashes, arthritis, inflammation of the eye, tiredness, and lack of concentration. cases or takeaway food containers can also be used, and these will hold more stool than sticks or slides.)

Wet the paper and put it on the slide.

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Put about half a teaspoon of feces on the middle of the slide.

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Add some water to the bottom of the jar. The mature larvaeThe 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. will wriggle into this water. (Note: use only enough water to wet the bottom of the filter paper, because you’ll have to search through all this water later when you look for larvaeThe 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.!)

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If you have more prepared feces, you can set up multiple jars, which is a good idea since some incubations can fail inexplicably. (See Possible reasons for failure.)

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I've had two containers, side by side, using the same poop preparation - one has some L3s, the other has nothing. [2]

Let the container sit somewhere away from light for 1-2 weeks at room temperature. No incubator is needed if the temperature is 70ºF (21ºC) or above, and you can put it outside if the weather is warm. (Place a jar of coconut oil nearby. The oil should be liquid if warm enough.) Keep the container covered to make sure no bugs can get inside. If the ambient temperature is too low, use an egg incubator or other suitable container and heat source. [3] Or place the container in an airing cupboard. [4] For more detail about incubation temperature, see Tips and adaptations, below.

Mold may form due to the high moisture, but this doesn’t matter because it won’t affect the worms. The only problem I have with the mold is sometimes larvaeThe 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. get stuck to it when I’m trying to count them in the solution.

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My most recent batch got pretty moldy, but… the mold doesn't affect the worms, just looks unpleasant. [5]

If you use a coffee filter or cloth over the top of the container instead of a lid you will get less mold but there's more chance of it drying out. If you use cloth you will need to mist daily. See Tips and adaptations, below.

I have had the most failures due to forgetting about it and letting it dry out.

HookwormA 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. eggs do need air for development [6] but there is plenty of air for them in a jar, even when this is sealed with an airtight lid. [7] If using smaller containers, such as test tubes or urine sample bottles with airtight lids, it may help if the lids are removed briefly every day to provide an air change.

Harvest

One of the benefits of using this method is that only L3 larvaeThe 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. can escape from the feces and be collected in the water.

After a week or two, pipette all the liquid from the bottom of the container and put this liquid into a conical-bottomed container such as a wine glass, silicone snow cone cup, or glass centrifuge tube. Let this sit for at least a few minutes so the larvaeThe 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. have time to sink and collect at the bottom. [8] [9] The majority of the larvaeThe 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. will have settled in 2 or 3 hours, [10] and all of them by 24 hours.

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Conical bottomed centrifuge tubes (they come to a point on the bottom) were a God send for me. I pre-harvest the worms and put them in these tubes for 24 hours. The worms settle to the point of the tube at the bottom, and are concentrated there. [11]

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Pipette the water from the bottom of the wine glass onto the middle of a glass slide. Pipetting from 2 to 4 mm above the bottom will minimise the number of dead ones that are collected and save time at the next stage. [12]

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Place the slide on the microscope's stage and, moving systematically up and down across the fluid on the slide, examine for larvaeThe 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..

Some people may find it easier if a few small drops of fluid are placed on the slide rather than a single large drop.

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I do small drops on the slide so easy to check. I put three small drops and its very quick. [13]

If you don’t get larvaeThe 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. or don’t get enough larvaeThe 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., add more room temperature distilled water to the bottom of the container. Repeat until the desired number of larvaeThe 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. are obtained.

I only use squirming larvaeThe 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.. I poke them with a mechanical pencil to see if they are alive (requires a steady hand and practice), but larvaeThe 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. that are not moving are not necessarily dead. They conserve energy until they find some skin, so motionless larvaeThe 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. that are intact may be viable. Scraping some skin cells from an arm and adding these to the water will often get the larvaeThe 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. moving, or the light from the microscope may waken them after a minute or two.

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All my non-wigglers have worked. [14] Just because they are lying still don't mean they are dead, even with poking and skin cells to bait them. In my experience, worms that are dead quickly begin to show signs of decay. If they look sleek and smooth, they are probably still alive. [15]
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I often have "dead beat" worms that just lie still and do not move. I will try to poke them with a fine diabetic syringe needle, but they often still won't budge. So today I took the needle and coaxed them to the edge of the drop of water, and finally out of the drop. This seemed to wake them up quickly! I did this on three different worms, and it worked every time. [16]
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I have deliberately chosen immobile larvaeThe 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. to inoculate with, and they've all been viable. I just make sure they look completely intact - once in a while I find one missing its internal structures, so those are almost certainly dead. [17]

Storage

If you are not ready to use the larvaeThe 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. immediately, they can be stored in the same water for several weeks if kept in the dark, or at least out of direct sunlight.

One user of this method has found that, if the larvaeThe 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. are stored at between 60°F (15°C) and 65°F (18°C), some can survive for as long as 4 months, [18] and occasional fluctuations between 54°F (12°C) and 70°F (21°C) are unlikely to be a problem. [19]

The larvaeThe 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. do need oxygen, which can become depleted at higher temperatures and by bacterial activity in the jar, although this is less likely to be a problem when using jars rather than test tubes, and it can be prevented by opening the lid every few days and gently shaking the jar’s contents. [20]

It has been suggested that a greater weight of water above the larvaeThe 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. in their storage vessel may shorten their lifespan, so it may be best to limit the volume of water to just enough to cover the larvaeThe 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. - perhaps just 3 mm. [21]

Even when kept in perfect storage conditions, the larvaeThe 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. will inevitably become progressively weaker as a result of the depletion of their fat stores.

Inoculation

Have a bandage ready that seals on 4 sides, or use a gauze pad with tape. Test bandages first to rule out allergic reactions.

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Use a pipette to gather larvaeThe 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. in a few drops of water on the slide, but I don’t do this while looking through the microscope. Once the liquid is on the slide, I put this under the microscope and count the larvaeThe 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., then add or remove liquid as necessary and recount. If there are a lot of larvaeThe 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., I suck the water up until I reduce the numbers. Then, when I get the amount I want, I let the water from the slide drip on to a prepared bandage, or gauze pad with tape, and finally wipe the slide onto the gauze to get every last drop. Then I immediately apply this to my skin. I repeat this until I get the number of larvaeThe 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. I want, but can often get the whole dose on a single gauze.

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I recommend 1-3 larvaeThe 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. at a time, depending on your goal dose. (See Hookworm dosing and response.) Place the bandage on a hairless, convex area of skin, such as your (shaved) leg. I don’t recommend the upper arm because the skin here is thin, and entry spots hard to observe, but a variety of sites have been used successfully. (See Body sites used for hookworm inoculation.)

Tingling should start after 10-20 minutes as larvaeThe 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. enter the skin, but let the bandage sit for at least 3 hours. (For more on this, see Inoculation with NA.)

Clean up supplies by freezing them in a ziplock bag overnight before disposal.

After a few hours or days, you should be able to observe the entry spots with the naked eye or a magnifying glass. If I didn't get a tingle and rash I would suspect failure.

Safety

This paper describes some methods you can use in your lab to kill hookwormA 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. larvaeThe 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..

However, freezing the larvaeThe 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. is one of the easiest ways to kill them off at the end of incubation. Simply throw your slides, beakers, gloves etc. into a plastic bag and put them all into the freezer overnight.

Notes

  • Research safety extensively before starting (including reading The laboratory and safe practice), and have a doctor supervise you.
  • Have a script for a course of an anthelminthic filled and ready before you start experimenting. Albendazole is more effective against hookwormsA 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. than mebendazole, but isn't available everywhere. (See Terminating a helminth infection.)
  • If using a donor, get them tested for HIV, Hepatitis B and C as a minimum. Strongyloides blood test is also recommended especially if you are on immune suppressing drugs.
  • If using donor stool, take a peek at this under the microscope to see what’s in it when you first get it. Do a fecal float and check for eggs. (See Stool testing (egg counting).) If you see larvaeThe 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. in less than 24 hours, do not use, since it may contain Strongyloides.
  • Get bloodwork done on yourself before inoculating (CBC, Hiv, Hep A+B) and afterwards (Hiv, Hep A+B, Stronglyoides).
  • Do not prepare or open the jars of feces outdoors. Bathroom is recommended, as flies can lay eggs in seconds and flies will ruin the feces. Feces are their favorite food.
  • If you put your container outside to incubate, make sure the temperature stays between 77ºF (25ºC) and 95ºF (35ºC).

Tips and adaptations

Several users of this method have made modifications, and suggested tips, as follows.

Using garden soil instead of vermiculite

Soil can be used on its own as the incubation medium, as described by Alana in her incubation protocol.

Others have also had success using soil alone.

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I just used soil from organic garden bed. So just a spot that I knew had not been sprayed with chemicals. I baked 2 litres of soil in oven at 120C for two hours then put in large tin. I have used this same soil for over a year. I just mix about 2/3 soil to 1/3 fecal. Add water till like lentil soup and apply thinly to paper towel on slide. I have always got NAthe human hookworm, Necator americanus using soil. [22] [23]

Using more feces

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Larger amount of poop (more than a dime, more like a big cherry size). I was going off the photo in Sarah’s method but there's no reason not to go big 😀 [24]
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Maximal ”sample” and minimal water makes for a good harvest. [25]

Mixing feces to a thinner consistency

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I use about half a teaspoon of 50/50 sterilised dirt/fecal per slide, mixed like runny oatmeal and spread out thin over the slide surface. The paper towel on slide soaks up a bit of moisture. This seems to give a better yield. I think a lot (of larvaeThe 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.) get stuck if it’s a sticky ball of goop. [26]

Using less water in the jar

This makes it easier to find the larvaeThe 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..

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I use much less water (a few pipettes' worth) and cover the top. [27]
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As little water in the bottom of your jar as possible: 1mm depth. [28]
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If the lids are on the jars, then all you need is a teaspoon or two of water in each. Less water, less searching (for larvaeThe 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.). [29]

Incubation temperature

The ideal temperature for incubation is said to be between 73°F (23°C) and 86°F (30°C), although one grower has found that a room temperature of 68-72°F (20-22°C) works well for him, [30] and another had great success at 64-65°F (18°C). [31]. At the lower end of the range, the larvaeThe 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. might be ready after 12-14 days and at the higher range after only 4-5 days. Larvae incubated at a lower temperature have greater longevity.

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Mine are at 72-75ºF (22-24ºC) and seems ok there. If it drops lower or goes higher it get wonky for me. [32]
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I do not use an incubator but store jars in a towel-lined box in a stable part of my house. Not particularly warm either... as long as you have a stable temperature between about 50-70ºF (10-21ºC). I have found that cooler and even temps, given a bit more time, produce weeks of long-lasting larvaeThe 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.. When I started with rapid and warm I frequently killed them. [33] [34]

Controlling the incubation temperature

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Egg incubators can run a bit hot, so get one with an adjustable temp if you can. [35]
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Get a thermometer that records high and low temp for the last 24 hours. Heaters (such as reptile warming mats) sometimes get just a bit too hot, so you need to check the temp range. I have used a seedling mat, which is a similar idea, and used a ‘dimmer switch’ (actually a fan speed controller that plugs into the wall, then the heater plugs into that) to get the perfect temp range. [36] Or you can put something like a wire baking rack between the heater and the samples to allow a bit of air circulation to drop temp a few degrees... [37]

Keeping the sample moist

The sample will remain moist if the container holding it is covered by a solid lid. However, if it is covered with a coffee filter or cloth, daily misting may be necessary to keep it moist.

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I misted my poo each day during the last incubation and it greatly improved my results. [38]

If you do mist the sample to keep it moist, only use a small amount of water. A garden or domestic sprayer may deliver too much.

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I use a very small sprayer that I bought at a health food store. The bottle only holds two ounces of water... about 60ml. [39] And I only spray one spritz per day. Maybe two if I think I didn't get it right. [40]

Concentrating the larvaeThe 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.

An alternative to using a conical-bottomed container to concentrate the larvaeThe 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. after incubation is to draw them up into a transfer pipette and then stand this up in a container.

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Suck up fluid with a transfer pipette. Let it sit 10-15 minutes upright in a paper cup. If you get a tiny bit of air at the bottom , it acts like a plug and there is no leakage. [41]

Isolating single worms

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My best simple idea for isolating single worms is to use multiple slides, i.e. if you draw up a batch, and have many hookwormsA 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.. Just keep siphoning off drops onto new slides until you get a number that suits you. Then put that drop from the slide onto a bandage and apply. [42]

Transferring larvaeThe 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. from slide to bandage

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This step vexed me for months. I found that a large wooden spoon really helped. You lay the bandage over the back of the spoon, so that the bandage is curved. Then when you transfer the hookwormsA 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. from the slide to the bandage, the slide doesn't get tangled up in the sticky part of the bandage, because it is curved over the back of the spoon. [43]

Possible reasons for failure

Novice hookwormA 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. growers may need a period of apprenticeship before succeeding at the art of hookwormA 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. incubation, even when using this simple method. For more details, see Some find incubation surprisingly difficult.

HookwormA 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. incubations can fail for a variety of reasons other than a lack of experience. Long-term hookwormA 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. growers, and even hookwormA 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. providers, can experience fallow periods when their incubations produce no larvaeThe 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.. One reason for this can be the egg donor’s diet (for more about this, see Promotion of egg viability by dietary manipulation) and certain brands of probioticMicroorganisms that are believed to provide health benefits when consumed. have been implicated by one grower, so diet should be considered if there is a continuing failure to grow larvaeThe 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..

Suggestions/observations

If anyone using this method has any questions, suggestions for improving this page, or any other observations, please post these to the Helminth incubation group.

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