Difference between revisions of "NA incubation: very simple Harada-Mori method by Sarah"

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{{Quote|indent}}I've had two containers, side by side, using the same poop preparation - one has some L3s, the other has nothing. [https://www.facebook.com/groups/678894952216125/permalink/1183357308436551/?comment_id=1183714441734171&reply_comment_id=1183803391725276&comment_tracking=%7B%22tn%22%3A%22R%22%7D]{{Quote|/indent}}
{{Quote|indent}}I've had two containers, side by side, using the same poop preparation - one has some L3s, the other has nothing. [https://www.facebook.com/groups/678894952216125/permalink/1183357308436551/?comment_id=1183714441734171&reply_comment_id=1183803391725276&comment_tracking=%7B%22tn%22%3A%22R%22%7D]{{Quote|/indent}}


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, place the container in an airing cupboard, [https://www.facebook.com/groups/678894952216125/permalink/1449292451843034/] use an egg or reptile incubator, [https://helminthictherapywiki.org/wiki/index.php/Helminth_incubation#Ready-made_incubators] a plant propagator, or a suitable container plus a heat source. [https://helminthictherapywiki.org/wiki/index.php/Helminth_incubation#Home-built_incubators] For more detail about incubation temperature, see [[#Tips and adaptations|'''Tips and adaptations''']], below.  
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, place the container in an airing cupboard, [https://www.facebook.com/groups/678894952216125/permalink/1449292451843034/] use an egg or reptile incubator, [https://helminthictherapywiki.org/wiki/index.php/Helminth_incubation#Ready-made_incubators] a plant propagator, or a suitable container plus a heat source. [https://helminthictherapywiki.org/wiki/index.php/Helminth_incubation#Home-built_incubators] For more detail about incubation temperature, see [[#Incubation temperature|'''Incubation temperature''']], 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 larvae get stuck to it when I’m trying to count them in the solution.
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 larvae get stuck to it when I’m trying to count them in the solution.

Revision as of 12:08, 15 October 2021

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


Introduction[edit]

This simple protocol provides the necessary basics for hookworm incubation. Anyone wanting more detail should read Alana's very detailed NA incubation method.

Video demonstrations of this method[edit]

The following brief video presentations show the main steps in the preparation, incubation and harvesting processes using the Harada-Mori culture method.

  • Hookworm incubation.

  • Harvesting the larvae.

Preparing the mixture[edit]

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 and resting place for contaminated instruments)
  • plastic knife
  • vermiculite (with no added material)
  • activated charcoal (capsules are fine)
  • distilled water (filtered or dechlorinated tap water and bottled water are also suitable)

Procedure

Take some feces from a hookworm host who has been infected for at least 6 weeks. (Eggs aren’t produced before this, and the quantity of eggs increases in the weeks following, up to a maximum at 20 weeks.) Place the feces in a covered container. A large amount of feces is not necessary, a few heaped teaspoons will be plenty.

Incubate1.jpg

Add vermiculite (one part vermiculite to one part stool) and activated charcoal. I use one charcoal capsule, or two if I’m doing a big batch. The charcoal kills the smell and balances the pH (healthy stool is acidic, but a neutral pH of 6.0 or 7.0 is optimal for hatching hookworm eggs) 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.

Incubate2.jpg

Incubation[edit]

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 placed in upturned egg cartons. In this case, the larvae 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 microscope slides
  • coffee filter paper (or flat, uncoloured paper towel)
  • plastic spoon
  • distilled water
  • pipette
  • conical-bottomed container such as a wine glass, silicone snow cone cup, or glass centrifuge tube
  • microscope (This needs to be capable of 100x magnification, preferably binocular compound and with a moveable stage. See: Microscope selection notes.)
  • gauze
  • tape/ bandage

Procedure

Cut a strip of filter paper to the size of the glass slide, leaving an area at one end free, so you can pick up the slide without touching the filter paper. If using centrifuge tubes, cut the paper slightly narrower than the slide to prevent it from touching the sides of the tube. (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 CD 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.

Incubate5.jpg

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

Incubate6.jpg

Add some water to the bottom of the jar. The mature larvae 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 larvae!)

Incubate7.jpg

If you have more prepared mixture, 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, place the container in an airing cupboard, [3] use an egg or reptile incubator, [4] a plant propagator, or a suitable container plus a heat source. [5] For more detail about incubation temperature, see Incubation temperature, 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 larvae 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. [6]
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I never understand why some batches are moldy and some aren’t. I’ve had different samples from the same batch incubated side by side - some with crazy mold and others with almost none. I’ve never noticed that it affected the outcome. [7]

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. NA larvae die in a matter of minutes once they become dry. [8]

Hookworm eggs do need air for development [9] but there is plenty of air for them in a jar, even when this is sealed with an airtight lid. [10] 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[edit]

One of the benefits of using this method is that only L3 larvae 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 larvae - which cannot swim - have time to sink and collect at the bottom. [11] [12] The majority of the larvae will have settled in 2 or 3 hours, [13] 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 godsend 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. [14]

An alternative to using a conical-bottomed container is to draw up the liquid 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. [15]

Incubate8.jpg

Pipette the water from the bottom of the wine glass, or alternative container, onto the middle of a glass slide. Squeezing the pipette bulb before it enters the liquid will prevent bubbles from stirring up the collected larvae. Some growers recommend pipetting from 2 to 4 mm above the bottom to minimise the number of dead larvae that are collected, and to save time at the next stage, [16] but others don't bother doing this, and just pipette right from the bottom. One grower swirls the larvae and then lets them settle for a few minutes, and recommends practicing this with fine dregs from tea leaves, or something similar, to watch how the swirling causes them to gather. [17]

Incubate9.jpg

Place the slide on the microscope's stage and set the magnification to 40x. Then, moving systematically up and down across the fluid on the slide, examine for larvae.

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. [18]

It's easier to get small drops if you use a fixed-volume micropipette/minipipette. e.g., a 100 microlitre mini pipet such as this model which takes removable tips.

A single drop from a 100 microlitre micropipette may contain 100 larvae, but 10 larvae per drop is easier to count. If there are too many larvae, add more water to each drop, or divide each drop on the slide using a pipette tip, and then add water to these daughter droplets.

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

I only use squirming larvae. I poke them with a mechanical pencil to see if they are alive (requires a steady hand and practice), but larvae that are not moving are not necessarily dead. They conserve energy until they find some skin, so motionless larvae that are intact may be viable. (I use 100x magnification for a closer look at individual larvae.) Scraping some skin cells from an arm and adding these to the water will often get the larvae moving, as will gently pushing them to the edge of the drop of water. [19] The light from the microscope may also waken them after a minute or two.

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All my non-wigglers have worked. [20]
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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. [21]
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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. [22]
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I have deliberately chosen immobile larvae 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. [23]

Storage[edit]

If you are not ready to use the larvae immediately, they can be stored in the same water for several weeks if kept in the dark, or at least out of direct sunlight. If you need to add extra water, use distilled water, filtered or dechlorinated tap water or bottled water. (When used to store larvae, the osmotic pressure of reverse osmosis water may be too low and put undue pressure on the larvae's excretory system. [24])

One user of this method has found that, if the larvae are stored at between 60°F (15°C) and 65°F (18°C), some can survive for as long as 4 months, [25] and occasional fluctuations between 54°F (12°C) and 70°F (21°C) are unlikely to be a problem. [26] However, out of one group of eight 4-month-old larvae, only one successfully penetrated the skin, [27] therefore 8-10 weeks might be a more realistic expectation for larva survival. Also see, Hookworm larvae storage and survival.

The larvae 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. [28]

It has been suggested that a greater weight of water above the larvae 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 larvae - perhaps just 3 mm. [29]

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

Inoculation[edit]

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

Incubate10.jpg

Use a pipette to gather larvae 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 larvae, then add or remove liquid as necessary and recount. If there are a lot of larvae, 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 larvae I want, but can often get the whole dose on a single gauze.

Incubate11.jpg

I recommend 1-3 larvae 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 larvae 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[edit]

This paper describes some methods you can use in your lab to kill hookworm larvae.

However, freezing the larvae 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[edit]

  • 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 anthelmintic filled and ready before you start experimenting. Albendazole is more effective against hookworms 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 larvae 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[edit]

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

Using garden soil instead of vermiculite[edit]

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 NA using soil. [30] [31]

Using more feces[edit]

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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 😀 [32]
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Maximal ”sample” and minimal water makes for a good harvest. [33]

Using only feces[edit]

One hookworm grower uses no soil, vermiculite or charcoal at all, and just adds enough water to produce a suitable consistency for the sample to stick to the slide without slipping off. He says there’s no smell and he gets lots of larvae. [34]

Mixing feces to a thinner consistency[edit]

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I got almost no worms with thick poop, and tons of NA with small amounts of runny poop. [35]
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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 larvae) get stuck if it’s a sticky ball of goop. [36]

If the poop mixture slides down whatever it's placed on and ends up in the water at the bottom of the container, try a wider container to allow the samples to sit at a flatter angle. [37] Also try leaving whatever the poop mixture is placed on completely flat for 30 minutes before putting it in the jar. This helps to firm up the mixture and prevent slippage. [38]

Using less water in the jar[edit]

This makes it easier to find the larvae.

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I use much less water (a few pipettes' worth) and cover the top. [39]
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As little water in the bottom of your jar as possible: 1mm depth. [40]
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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 larvae). [41]

Type of container used may affect outcome[edit]

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My incubations were successful using urine specimen cups, then someone gave me a bunch of old baby food jars. Using those, I kept finding all of the larvae dead. All I can think of is they washed the jars in something toxic to hw. I tried really scrubbing the jars before using and then found a few alive, but not many. [42]

Incubation temperature[edit]

The ideal temperature for incubation is said to be between 73°F (23°C) and 86°F (30°C), but there are growers having success with a wide variety of temperatures.

  • 90°F (32°C) [43] - "I incubate at 90F and 70-80% humidity." It takes 6-7 days at this temperature. [44] This may be close to the upper limit. One very experienced incubator has reported that she has completely failed to get any larvae at all when the temperature has even temporarily approached 95°F (35°C). [45].
  • 80°F (27°C) [46] - "Keep mine at 80 and it works fine."
  • 79°F (26°C) [47] - "I incubated for 5 days and a few hours. I had enough worms but I think one day more would have produced more."
  • 72-75°F (22-24°C) [48] - "If it drops lower or goes higher it gets wonky for me."
  • 68-72°F (20-22°C) [49]
  • 65-70°F (18-21°C) [50] "I keep them in a dark cabinet in a space that is between 65-70 depending on the season."
  • 62-65°F (17-18°C) [51] [52] - Without a heat source, just in a dark box in a basement. "Takes 12 days or so."
  • 50-70°F (10-21°C) [53] - Without an incubator, just in jars in a towel-lined box in a stable part of the house. "I have found that cooler and even temps, given a bit more time, produce weeks of long-lasting larvae. When I started with rapid and warm I frequently killed them." [54]

Larvae incubated at higher temperatures may be ready after only 4-5 days. Those grown at lower temperatures might not be ready until 12-14 days, or later, but they will survive for longer.

Fluctuations in temperature should not be a problem so long as the upper and lower extremes fall within the range above.

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Mine typically go through a 10 degree temp swing sitting in top of the water heater, depending on ambient temp & water use. And they’re used to being outside with natural temp changes. [55]

Controlling the incubation temperature[edit]

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Egg incubators can run a bit hot, so get one with an adjustable temp if you can. [56]
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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. [57] 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... [58]

Keeping the sample moist[edit]

NA larvae die in a matter of minutes if they are allowed to become dry. [59]

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. [60]

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. [61] And I only spray one spritz per day. Maybe two if I think I didn't get it right. [62]

One experienced hookworm grower likes to maintain his cultures at a humidity of 70-80%. [63]

Isolating single larvae[edit]

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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 hookworms. 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. [64]

Transferring larvae from slide to bandage[edit]

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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 hookworms 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. (Link expired)
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I like using a Q-tip to soak up the drop on the slide, and then tape or bandaid it to my skin. [65]

Cleaning larvae to be shared with others[edit]

Hookworm larvae do not need to be cleaned if they have been grown at home for use by the same individual who provided the stool sample for their incubation. However, if that individual wishes to share the larvae they have grown, these can be cleaned using the following method.

Add one drop of commercially available 5% Lugol’s iodine to 5 ml of water (distilled, filtered or dechlorinated tap water, or bottled water). Then add 1 ml of this 0.02% iodine solution to 1 ml of water containing the larvae. After the larvae have spent 20 minutes in this solution, the iodine should be neutralised by adding a pinch of ascorbic acid (vitamin C) powder to the water. Neutralisation of the iodine is confirmed when sufficient ascorbic acid has been sprinkled into the water to make this completely clear.

Note. Larvae that have been cleaned by any method will have a shorter shelf life than those that have not been cleaned.

Possible reasons for failure[edit]

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

Hookworm incubations can fail for a variety of reasons other than a lack of experience. Long-term hookworm growers, and even hookworm providers, can experience fallow periods when their incubations produce no larvae. 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 probiotic have been implicated by one grower, so diet should be considered if there is a continuing failure to grow larvae.

Suggestions / observations[edit]

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.