NA incubation: simple method by Janice
Stage 1: Incubation
- Container -- I use a takeaway food container washed and sterilised using my old microwave baby bottle steriliser
- Disposable latex gloves -- pack of 50 £5.49 inc delivery ebay
- Disposable spoons -- for mixing - pack of 20 £1 local shop
- Purified water -- 5 litres £3.70 ordered from local pharmacy
- Vermiculite -- 10 litres £4.99 local garden supplier
- Activated Filter Carbon / Charcoal -- used in fish tank filters - 375g £10.49 local pet supplies
- Water spray bottle -- £1.99 local pharmacy
- Hydrometer -- to measure humidity - £2.70 inc delivery ebay
- Incubator -- I bought a Lucky Reptile Herp Nursery II second hand for £65
Always wear gloves.
Mix feces with equal amounts of vermiculite (to maintain hydration), a few spoons of activated filter carbon (to reduce smell) and a splash or two of purified water to create a wet sand consistency.
Tip: I prefer to make the mixture in a plastic mixing bowl then transfer it into the incubation container to avoid smearing the sides with feces that can contaminate the gauze used in the second stage.
Give the mixture a few sprays of purified water and place in the incubator at 30 degrees Celsius and humidity over 60%.
Put all equipment that could have come into contact with feces in a zip lock bag and freeze for 24 hours before disposal. This will kill all eggs and larvae.
Leave the mixture for 8 days, giving a daily spray of 4 or 5 pumps of purified water to maintain moisture.
Stage 2: Harvest
- Sand -- 20kg children’s play sand £4.99 local garden supplier
- Tea strainer -- £1.98 Ebay
- Gauze -- Non sterile first aid gauze swabs 10x10cm Pk of 100 £4.65 inc postage Ebay
- Disposable wine glass -- with hollow stem to create an “Imhoff cone” at the bottom for worms to collect - Pk of 24 £6.49 inc delivery Ebay
- Purified water
- Water spray bottle
- Disposable gloves
Put as much sand as you need in a pot, add water, bring to the boil and boil hard for 20 minutes to sterilise.
Strain through the tea strainer.
Tip: Use a large pot with a lid to avoid sandy water splashing on to your stove.
Always wear gloves – your incubation mixture should now have several thousand infective larvae and skin contact could result in a large overdose.
Spread about a 1cm layer of sterilised sand over the incubation mixture.
Dampen the gauze by spraying with purified water.
Place three layers of damp gauze on the sand and give it a good spray on top.
Return to the incubator at 30 degrees Celsius for a further 24 hours.
Put all materials that could have come into contact with the mixture in a ziplock bag and freeze.
Keep the gauze damp by spraying a couple of times over the next 24 hours and give a good spray about 2 hours before removing the top layer.
Wearing gloves, remove the top layer of gauze and put it in a wine glass filled with purified water. Give it a stir and leave for 24 hours at room temperature. The larvae will migrate from the gauze into the water and sink to the bottom ready for collection.
Put anything that could have come into contact with larvae in a ziplock bag and freeze for 24 hours.
Stage 3: Collection, Identification, Counting and Storage
- Microscope -- should be capable of at least 40x for counting, at least 200x for identification. Binocular with a mechanical stage would be preferable for ease of use but this vastly increases the price. I have a New Apex Learner Microscope £55 inc delivery from Amazon, and an eye patch!
- Slides -- 50 pk £4.09 inc delivery ebay
- Micropipettes -- micro transfer pipette fine tip 10 pk £1.93 inc delivery ebay
- Petri Dishes -- 70mm plastic petri dishes 10 pk £4.99 inc delivery ebay
- Storage vials -- 50 2ml plastic test tube vials £6.89 inc delivery ebay
- Purified water
- Disposable gloves
Always wear gloves.
Take a disposable micro pipette, squeeze the bulb before putting it in the water, and carefully suck up a small amount of liquid from the very bottom of the glass.
Squeeze a few big drops on to a petri dish and view at 40x magnification. Hopefully you will see plenty of L3 larvae.
This picture is from a large crop of necator americanus L3 seen at 40x magnification.
This picture is at 400x magnification. You can identify NA L3 by a pointed tail, oesophageal join just over a quarter of the way down the body, and a long mouth opening (buccal canal). These three points differentiate it from Strongaloides stercoralis – a parasitic worm that can be fatal in certain circumstances.   To differentiate NA from Ancylostoma duodenale (AD), look at the oesophageal join: in NA it is a straight line whereas in AD you can clearly see a bulb shape.
The L2 which are not infective are shorter, fatter and less motile.
For further reading and images on larvae identification here are some links:
- Strongyloidiasis identification
- Strongyloides and hookworm compared
- Comparison of filariform larvae of “hookworm-like” species
To count out a suitable dose, pipette up a small drop, squeeze it out on to a slide and count the larvae at 40x magnification by systematically moving over the drop in a grid-like manner. The number of larvae administered in a single dose can range from 5 to 25. See the Hookworm dosing and response page for more details about dosing.
If there are too few, pick up the slide and hold it over a vial so that the bottom corner is resting on the vial and the slide is almost vertical, then flush the fluid into the vial using a pipette with purified water. Put another drop under the microscope and repeat until you have the right amount in the vial.
If you have too many, flush them into a vial to dilute then repeat the counting process with the diluted water.
Larvae are susceptible to temperatures under 15 degrees Celsius and over 45 degrees Celsius and to direct sunlight. If they are stored in purified water at room temperature and away from sunlight, they should survive for several weeks. There have been accounts of survival up to 5 months.
For added purification you can store the larvae in bleach solution. Make it by adding one drop of bleach per litre of purified water. This may help to avoid contamination with unwanted bacteria over time.
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.)