Helminth incubation
Introduction
This page is concerned with the incubation of the mutualistic helminths used in helminthic therapy.
Of the four species of helminth being used in therapy, three are suitable for home cultivation.
- Rat tapeworm, Hymenolepis diminuta (HDC)
- Human hookworm, Necator americanus (NA)
- Human whipworm, Trichuris trichiura (TT/TTO)
The only therapeutic helminth not suitable for cultivation at home is the pig whipworm, Trichuris suis. The incubation of TSO involves a complex process spanning many months, and requires the use of pigs - preferably sterile ones.
In terms of risk, HDC is arguably the least problematic, since only a relatively small number of cysticerci - typically somewhere between 0 and 80 - will be found in each beetle dissected, whereas a single batch of TTO or NA may contain thousands of infectious organisms, with a potential to cause serious illness if not very carefully managed during incubation and inoculation. For example, one of the risks with TTO is that, if too large a dose was ingested, this could cause rectal prolapse. TTO is also more challenging to grow than either HDC or NA.
There is additional risk attached to using organisms obtained from other private growers, due to the possibility that some, or all, of these may not be the species they are claimed to be. A donor may have unknowingly become infected with a pathogenic helminth, especially if they have engaged in foreign travel. It's also possible for a private donor to pass on other pathogenic microorganisms. It is therefore recommended that any incubation process is begun by obtaining a dose of the organism to be cultivated from a reputable commercial source that guarantees its product contains only the specified helminth species and that this is free from contamination by other organisms.
HDC and NA are both available for purchase as a single dose. See the list of helminth providers.
Before sourcing any products from a private grower, the would-be incubator should explore the legality of doing this in their particular locality. What regulations there are are often imprecisely defined, so are open to interpretation, making it difficult to predict exactly how a regulatory authority might react if someone in their jurisdiction were identified as having supplied or received helminths, or been involved in their cultivation.
While the precise legal position concerning helminths is still uncertain and untested in many countries, it is clearer in the US, where hookworms, whipworms (both human and porcine) and rat tapeworms are currently classified by the Food and Drug Administration as biological agents (i.e. drugs), as defined in Section 351 of the Public Health Service Act, and subject to an Import Alert.
Helminth incubation methods
The following incubation protocols were written by members of the helminthic therapy community.
Necator americanus
- NA incubation: very simple Harada-Mori method by Sarah (A well-developed method featuring many supplementary details that will also be useful to users of other methods.)
- NA incubation: very simple petri dish method by Steven (This method uses petri dishes and supplementary heat to produce very clear water.)
- NA incubation: simple method by Janice (This approach gathers larvae onto cork particles, providing a clear sample for microscoping.)
- NA incubation: simple method by Anon (This method gathers larvae onto cotton balls, which are then dropped into water, creating a clean sample.
- NA incubation: very detailed method by Alana. (This method provides information about every aspect of hookworm incubation. A valuable resource for users of other methods, as well as an effective method in itself.)
Hymenolepis diminuta
- HDC production protocol from Duke University, 2017 See Section 2: Materials and Methods.
- HDC production protocol from Duke University, 2018 (PDF) See sections 2, 3 and 4: Materials, Methods and Notes.
Trichuris trichiura
- TTO incubation: very detailed method by Alana. This comprehensive manual is the only TTO incubation protocol available for home growers.
Helminth incubation discussion groups
See the following page section.
DIY biology support
Expert help with incubation can be found in bio-hackers clubs (DIY biology, DIY bio) which are often located in university science departments and form part of a growing biotechnological social movement (‘Do-it-yourself biology’) in which individuals with extensive research training mentor and oversee DIY biologists with little or no formal training.
DIY bio clubs can be found on this list, through Meetup, or by googling “community science" groups, or “meetup" science groups.
Hookworm incubation
Hookworm incubation issues
Some find incubation surprisingly difficult
Many people find the cultivation of hookworm larvae simple and straightforward, especially those living in a favourable climate, but others have failed, sometimes repeatedly, even when they have had appropriate previous experience, and in spite of following good instructions. This has led to the suggestion that this process is as much an art as a science.
Sometimes, even very experienced incubators can have incubations that inexplicably fail or produce very low yields.
And a few people, including some with a high level of education, have found that they just can’t grow any larvae at all, whatever they try.
Infection risk
Some would-be hookworm hosts may consider traveling to an area where these worms are endemic, in the hope of acquiring their larvae in the same way that the locals do - by walking barefoot in open-air latrines. For a number of reasons, this is a not a good idea, as is explained here by the only Westerner who claims to have done this.
Anyone seeking hookworm larvae from someone who is already cultivating their own supply, or faeces containing hookworm ova, needs to consider several issues. Included amongst these is the risk of inadvertently acquiring a different type of helminth, such as the less desirable species of hookworm, Ancylostoma duodenale, which causes an estimated nine times more blood loss than NA, can infect a foetus by crossing the placenta, and is able to migrate via breast milk. [4]. Even more risky is the roundworm, Strongyloides stercoralis, which is autoinfective and potentially hyperinfective, with a risk of fatality, [5] [6] and which has already contributed to the death of one member of the helminthic therapy community. [7] The eggs of both these species are virtually identical to those of NA, and a DIY grower would be very unlikely to be able to distinguish between the larvae produced from them.
Accurate methods for identifying the species of larvae are likely to be beyond the resources found in the home setting. The old style approach was microscopy, but this requires the examination of a stained specimen using a more powerful microscope than is typically available to home growers. Microscopy also requires a lot more experience than is possessed by most amateur incubators. More modern methods of species identification are molecular, either polymerase chain reaction (PCR) or DNA sequencing.
In the context of the domestic incubation of NA, the major infection concern is the possibility of contamination by cat or dog helminths such as Toxocara cati and Toxocara canis, which can result from using unsterilised soil in incubations. This medium may also contain the larvae of free living nematodes which, while not at all harmful, can interfere with the counting of larvae for inclusion in therapeutic doses. To avoid these risks, any soil used in incubations must be fully sterilised, using, for example, the protocol explained here.
Another issue is that an untested donor may prove to be a source of any one of a range of pathological bacterial and viral infections. Commercial providers of hookworms ensure the safety of the products they supply by regularly testing their donors for Hepatitis A, B & C and for HIV, and by employing a method similar to the one detailed below, which has been used by researchers since the 1950s and in the original studies into the efficacy of the anthelmintic drug, albendazole.
- The sample, obtained from an individual that has been tested for Hepatitis and HIV, is cultured in a temperature- and humidity-controlled environment to facilitate development of the embryos to stage 2 larvae, which takes 8-10 days.
- The larvae are forced to migrate through a 3 inch layer of activated charcoal, taking advantage of the larvae's instinctive behavior to migrate up and through whatever they are covered by or contained in. This takes a further 1-2 days.
- Once sufficient numbers of the larvae have gathered on the surface of the charcoal, they are removed by placing two layers of fabric over the charcoal. Once they have migrated through this, removal of the top layer will bring the larvae along.
- The sample is washed in buffered saline solution by agitating it mechanically for twenty minutes.
- The larvae are extracted from the saline by pipetting them from the surface (they float in saline). They are then placed on the surface of a piece of filter paper.
- The larvae are submerged and flushed ten times using a solution of Chlorohexidine 0.2% by weight.
- The larvae are washed from the filter paper into a solution of sterile saline. After allowing them to recover, motile individuals are individually pipetted onto a dressing for application to the user's skin, or placed into a suitable container for dispatch.
In addition to these steps, current providers of NA also add an antibiotic to the solution in which doses of larvae are shipped.
Legal risks
Yet another often overlooked issue is the risk of legal action against a donor of hookworms or hookworm-infected materials.
The donation of larvae or infected faeces could expose the person supplying them to legal action by the recipient’s relatives in the event of that recipient dying after using the donated material. The grieving relatives might sue the donor in the belief, whether right or wrong, that the donation was somehow responsible for the death of their loved one. Someone contemplating the use of donated materials may think that their relatives would never react in this way, but death can spark unpredictable and irrational behaviour in family members, and the fact that the organisms had been given away rather than being sold would not necessarily free the donor of liability. The outcome of such a scenario would only be revealed after it had been tested in a court of law, and this could be a very costly and stressful process for all those involved.
The risk of attracting damaging publicity
There are some people who choose the DIY hookworm culture route because they object to having to pay for the treatment, even though the cost of a dose of hookworm larvae is now minimal. Unfortunately, the sometimes reckless attitude displayed by some in this group not only threatens their own safety and possibly that of those around them, but could also have much wider implications.
A number of reports have already surfaced about people who have successfully incubated hookworm larvae but, then, due to a failure to carry out the appropriate research, sloppy technique, undue haste, or just plain carelessness, have taken unbelievable risks with their health. One individual attempted to collect larvae for inoculation without a microscope, using only a magnifying glass. Others have chosen not to even bother counting out a number of larvae with which to inoculate, and, in one case, someone who had incubated larvae proceeded to smear faeces from the sample, along with liquid from its container, directly onto his skin, potentially giving himself a dose of hundreds, if not thousands of hookworms!
A medical doctor, who hosts therapeutic helminths himself, has reported seeing the arm of another individual who he estimated had probably inoculated with at least many hundreds of hookworm larvae on a single occasion, resulting in a rash covering the individual's entire upper arm. Since infection with more than 1,000 NA can be lethal [8] it was fortunate that this individual sought medical assistance. He was treated immediately with steroids, and followed the advice he was given to eliminate the worms.
This experience serves as an example to all who might consider DIY hookworm culture. Even though this individual is well educated and has a job requiring intelligence, his research, and the level of care he applied to the task, had obviously not been sufficient to ensure even his own safety, and this scenario is unfortunately repeated all too often.
What is perhaps of greatest concern about incidents like this, is that their ramifications could extend well beyond the people who are directly involved. If news of such a story were to be picked up by the media, it would undoubtedly be seized upon by news-hungry journalists and milked for profit by publishers. This publicity could result in regulatory crackdowns that could well impact the ability of others to access this therapy, and there are now thousands whose health depends on them being able to obtain helminths. There are also millions of others who are desperately ill but have not yet heard about this therapy, who, as a result of the actions of some thoughtless individual, might be deprived of the opportunity to try it.
So it is hoped that anyone intent on incubating hookworms themselves will take great care in sourcing their live materials, and then proceed with the utmost caution, conscious of their responsibility for the health and safety of others.
Resources for helminth incubation
Incubators
Ready-made incubators
Time and effort can be saved by buying an entire incubator with built-in temperature and humidity control Examples range from laboratory quality incubation cabinets to simple plant propagators. (Note that many people have found that they get better and more dependable results without heat. Temperature control is arguably more important in colder climates. Also, closed container methods maintain humidity automatically.)
Laboratory incubators
Laboratory supply outlets offer incubators that have all the bells and whistles, but come at a price. For example, this small cabinet from Interlab.
Reptile incubators
Devices designed for incubating reptile or poultry eggs can be very inexpensive and can work well for incubating hookworms. They may not control humidity, but humidity could be increased by incubating in a semi enclosed container within the incubator.
- A Terrarium set-up for growing NA (Scroll down thread for more details.)
Egg incubators
If purchasing one of these incubators, make sure you get one with the right ac plug / voltage for your country.
Plant propagators
This type of propagator can be found at garden centres.
The 'Propagator 43' is working well for one of our members who lives in a warmer climate. “So far all my incubations were successful."
It might also work in a cooler climate, after the addition of a source of heat such as an aquarium reptile heat rock.
This, or a standard incandescent light bulb, can be connected to a temperature switch, such as the Red 12V Heat Cool Thermostat Temperature High Low Alarm Control Switch -55-120C. [9] using a 5.0V DC power supply (e.g., an old cell phone charge adapter) and wired as described in the Temperature control switch circuit for HW incubator doc in the Files section of the Helminth Incubation discussion group on Facebook.
Home-built incubators
In most climates, a cooler, or other similar insulated box, should work just fine, and this type of DIY incubator can be built for well under $50.
One incubator built hers using a widely available, $15 temperature controller that she wired with a regular double socket outlet and a project box from Radioshack. She plugged this into the cool plug and used a light bulb as her heat source.
Below are the exact materials she used.
- A widely available STC 1000 controller, such as the Inkbird All-Purpose Digital Temperature Controller Fahrenheit &Centigrade Thermostat w Sensor 2 Relays. [10]
- A project box, such as the Radioshack Project Enclosure (6”x4”x2”). [11]
- A $5 ikea hanging lamp cord with a fluorescent bulb as a heat source.
She had never wired anything before, but just followed Youtube videos that described setting up a temperature controller. The video she followed is How to wire your STC-1000 temp. control unit, but there are many alternative ones to be found by searching YouTube for “STC 1000”.
Another home grower described his DIY incubator as follows.
Potential issues with temperature control relays that could cause incubation failure
One incubator identified a potential problem with a W1209 temperature control relay purchased from eBay.
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, [13] and another had great success at 64-65°F (18°C). [14]. At the lower end of the range, the larvae 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.
Styrofoam enclosure
A low cost incubator can be rigged from a Styrofoam cooler, or, with a little more work, constructed from Styrofoam insulation from a home improvement store, and glued together with silicon sealant.
List of materials
- 1 x Styrofoam Box with Lid
- 1 x Old Reading Light
- 1 x 15 Watt Light Globe, resistance heater with light-bulb base, or infrared bulb (available at pet shops or online)
- 1 x Combination Thermometer and Hygrometer
- 1 x Plastic Container
- 1 x Duct Tape
Assembly and operation
- Buy a combination thermometer/hygrometer from ebay. They're cheap!
- Acquire a styrofoam box or cooler from the local shopping center.
- Acquire an old reading lamp, ideally one with a dimmer switch. Inline dimmer switches are also easily added to the line cord of any lamp.
- Cut a hole for the lamp in the styrofoam box, then tape up the gap between the box and the light fitting. Hopefully by this stage, you will have a empty styrofoam box with a light sticking through the side.
- Tape up all the corners of the box, and generally make it sturdy. Minimize the amount that the lid needs to open to maintain temperature and humidity in the box. You just need enough access to pull thing in and out of the box.
- Punch a hold in the side of the box which is opposite the heat source. This will encourage a flow of air (not too much) through the incubator.
- Put in a low wattage light-bulb. This may vary somewhat according to the size of the incubator.
- Find a good spot for your incubator. If you have a garage, use this. Inside is a bad place for it, as incubated feces tends to get a bit smelly. Keeping the incubator outdoors and using a waterproof extension cord could also work.
- Plug in your incubator and place a container filled with water inside.
- Place the thermometer/hygrometer inside, and keep careful observations.
Troubleshooting
- If the incubator is too hot: put in a lower wattage bulb, or punch another hole in the side.
- If the incubator is too cold: Place a higher wattage bulb in the light, and consider covering the hole you made.
- If the incubator is too dry: Consider placing a container with a larger surface area in the incubator, or place two containers in there.
A beer 12-pack box
Alternatively, an effective incubator can be made from a beer 12-pack box and a cat heater.
Aquarium heater and two tubs
Homemade Incubator less than €20/$20.
Use two plastic tubs of the same size. Put a 5 watt submersible aquarium heater on the floor of the bottom tub (it has suction cups). Cover the heater well with water. You will be floating the other tub on this water, so put something (rocks for instance) in the bottom tub to prevent the top tub from touching the heater if the water goes too low. The top tub (covered) is where you put your samples. You may need to add weight in this tub to keep it nested in the water a bit. A cover on the tub helps keep the temperature steady.
I find the adjustable heaters take too much work to keep the temperature just right. Even the ones with thermostats need adjusting. So I like the ones without adjustments; they keep the temperature in a good range for worms, 78°F/26°C. However if you you want it warmer, you can buy an adjustable aquarium heater and be prepared to spend about a week to be sure it is the correct setting and it holds the temp well.
Incubator accessories
If a source of humidity is needed, this can be provided by placing a pan of water in the incubator. Or humidity can be controlled remotely by using a small aquarium pump and an airstone in a pan of water. The air bubbles carry humidity out of the pan much more rapidly than just evaporation.
Aquarium stores and pet shops have several items that will be useful in constructing incubators. Resistive heaters with standard light bulb sockets are available as are infrared bulbs. Hookworms don't like light but many people report success using standard light bulbs for heaters, so this appears to be fairly unimportant. Inexpensive remote thermometers for use in fish tanks and aquariums are very useful.
Controlling the heat source is an issue. It is possible to use an "open-loop" type of control over the heat source by controlling the heat, with a light dimmer say, and simply monitoring the temperature, while very slowly raising the power level. However there is an element of danger that should be carefully considered in putting a heat source in an enclosed place and we strongly recommend one of the inexpensive thermostatic controllers made for reptile terrariums (they have a wider range than aquarium heaters and also have useful remote sensors). Controllers with digital control and humidity reporting are available for a little more money. Other users have reported finding useful (and possible better-made) thermostatic controllers on eBay for about the same price, e.g., this one or this one.
Conservative design principles would still indicate that heaters should be sized as small as possible (similar to the open-loop method), in case of thermostat failure, so that peak temperatures remain as low as possible, with the heater on all of the time.
Inexpensive timers - that will control on-off cycles in as small an increment as 10 minutes are also available in aquarium or pet stores.
Stool testing
Checking one's stool for eggs will reveal whether or not one is hosting helminths.
Microscopes
Advice on selecting and using a microscope for helminth incubation.
Pipettes and pipetting technique
Buffer solution
A buffer solution suitable for washing hookworm larvae (e.g., from the gauze into a beaker before counting and extracting them from the solution) and for storing them.
- 4.1g Sodium Chloride (Table salt)
- 7.1g Disodium hydrogen phosphate
- 3.0g Potassium dihydrogen phosphate
Mix these ingredients individually into 1 Litre of distilled water. Store the buffer solution long term in a fridge as it contains no preservatives.
"Larvae were retrieved from the suspension by gravitational sedimentation, and stored in BU buffer (50 mM Na2HPO4, 22 mM KH2PO4, 70 mM NaCl) at 12oC to preserve their infectivity.[16]" [17]
Sterilisation of leftover live materials
Immersion in boiling water for one minute or a 10% bleach solution are affordable, fairly safe, and effective for sterilization.
A study reported in 2017 [18] shows that Virkon®S and Dettol® (Chloroxylenol) outperformed four other commercial disinfectants against Toxascaris leonina eggs and larvae, and the study authors highly recommend the use of these two agents against other potentially zoonotic helminths. This suggests that Virkon®S and Dettol® may be the most effective disinfectants for use in sterilisation by worm growers.
Another option is to place the leftover materials in a freezer for 24 hours, although this is not guaranteed to break the shell of the egg, depending on freezer conditions. "Freezing at −21 °C for 1–7 days generally inactivates parasites, but cannot be relied upon in home situations." [19], although the following link states that freezing in saline increases effectiveness. [20] and, for other suggestions, see the penultimate paragraph of this page section: Storage and clean-up.
Further incubation resources
NA
- Stool culture for Nematode larvae (PDF)
- An Agar Plate Method for Culturing Hookworm Larvae: Analysis of Growth Kinetics and Infectivity Compared With Standard Coproculture Techniques (PDF)
- An explanation of the Baermann apparatus (funnel) for isolating nematode larvae from media.
HDC
Shipping biological materials
There are several categories of biological materials regarding the manner in which they can be properly shipped. A human sample infected with nematode parasites would be a class B biological substance. Details about the correct way to package and label a specimen can be found at the following link.