Waiting for the Cure » incubation methods http://waitingforthecure.com/I ... a day in the life of Crohn's disease ... Fri, 16 Jul 2010 14:37:32 +0000 http://wordpress.org/?v=2.9.2 en hourly 1 We’re on our own in the US with worms http://waitingforthecure.com/I/2010/02/08/were-on-our-own-in-the-us-with-worms/ http://waitingforthecure.com/I/2010/02/08/were-on-our-own-in-the-us-with-worms/#comments Mon, 08 Feb 2010 16:37:09 +0000 admin http://waitingforthecure.com/I/?p=936 Looks like Jasper of AIT can’t ship hookworms or whipworms to the US because of legal reasons.  Wormtherapy is still offering hookworms, but you have to go to San Diego then Tijuana to get them.  I’d like to collect an aggregate of information on hookworm incubation methods here, so we can learn from one another’s mistakes and triumphs.  What I’m finding is most people need to dose way more often than once every 3-5 years.  I’m only dosing moderately (10 hookworms at a time) and I’ve had good effects for about 4 months, then things decline.  I’ve also heard personally from others that about the one year mark, things start to go downhill.  I’m sure it depends on dose and disease, environmental factors.  Of course, I’m not in touch with the vast majority of people trying worms, but the more we can learn and understand about this and share this information, the better.

I will be trying to incubate again this week.  I will share my methods of success or not.  Meanwhile, here is another place where someone is experimenting with incubating hookworms.  Let’s help one another, shall we?

Update:  someone suggested two links for incubating instructions.  The first:

http://www.mja.com.au/public/issues/178_02_200103/lan10157_fm.html#CACJFHGC

From this post (they were incubating dog hookworms, but I imagine it would be the same for necator americanus:)

”

1: Details of isolation of third-stage larvae from infected dog faeces and of preparation of faecal samples from the human volunteer for microscopy

Isolation of larvae: Equal volumes of faeces and vermiculite were mixed well in a one litre plastic container, moistened and incubated at 30oC, being stirred daily. After five days, the mixture was spread onto large glass Petri dishes, covered with a 5 mm layer of washed, coarse river sand and overlaid with two layers of damp surgical gauze. The top layer of gauze (into which infective L3 had migrated) was removed (and replaced) at 12-hour intervals, to be rinsed in distilled water. 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.”

And the second site, with a good second page on identifying the difference between hookworm larvae and stronglyoides:

https://openaccess.leidenuniv.nl/bitstream/1887/13930/12/Appendix+01.PDF

“Preparation of the culture:
1        Put a flat cylindrical disk (40mm x 4mm) in the middle of a 10cm petri-dish
2        Put a circular filter paper (80mm) on top of the disk
3        Add water up to just below the level of the disk
2g stool sample         Petri-dish
+ vermiculite
Filter paper Cylindrical disk Water
4        Mix 2g of faeces with equal volume of vermiculite/coarsely ground charcoal
5        Label the dish lid with the patient’s number and the date
6        Keep away from direct sunlight
7        Add water when needed
After 7 days incubation:
8        Remove the filter paper and the faeces
9        Pour the water from the petri-dish into a conical settling tube
10       Rinse the petri-dish with a little water, pouring it into the same settling tube
11       Label the tube with the patient’s number
Allow the larvae to settle at the bottom of the tube. 2 hours later:
12       Pipette 1 OOul of sediment from bottom of tube onto a microscope slide
13       Systematically examine the whole drop using X4 objective
14       Switch onto X10 to distinguish between hookworm and oesophagostomum
15       Count the larvae
16       Add a drop of dilute iodine in KI solution to kill the larvae if necessary
NB       If using tap water, boil to kill plant nematodes and to remove chlorine
Cultures checked daily, to remove maggots and break up fungal hyphae
Iodine solution: 0.3g KI and 0.2g I2 in 10ml water. 1-2 drops in 1 ml water”

Happy hookworm growing!  Scientists, we could use some help here…

]]> http://waitingforthecure.com/I/2010/02/08/were-on-our-own-in-the-us-with-worms/feed/ 20 incubation techniques for hookworm larvae http://waitingforthecure.com/I/2009/02/28/incubation-techniques-for-hookworm-larvae/ http://waitingforthecure.com/I/2009/02/28/incubation-techniques-for-hookworm-larvae/#comments Sat, 28 Feb 2009 15:50:36 +0000 I http://waitingforthecure.com/I/?p=555 I’m researching incubation methods, and hopefully in a few weeks I will embark on my first McMaster egg count. There seem to be 3 choices for incubating the hookworm larvae; the Baermann, Harada-Mori, and the AP method (agar plate.) Here’s a nice visual and thorough list of requirements for the first two methods:

http://books.google.com/books?id=8AWz0cS6e9kC&pg=PA115&lpg=PA115&dq#PPA115,M1

This article goes into great detail on the advantages of each method:

http://www.ajtmh.org/cgi/content/full/77/6/1087

Incubation temperature is important, and 30C (86 degrees farenheit) is optimal:

http://www.ncbi.nlm.nih.gov/pubmed/3569472

I’ll provide an equipment list and write about the process once I’ve ordered the supplies, and tried it out. Oh why didn’t I take a science class in college? An art major in oil painting is not helping me out here…perhaps I’ll start a larval triptych!

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