Especially interested in:
- Collecting methods
- Useful feces containers
- Preservatives
- Storing conditions for further molecular work with the helminth parasite eggs or larvae in stool samples
Please only share empirical knowledge! Many thanks!
I cannot give you empirical knowledge yet, but, what we are going to do this season with the salamanders is place them in plastic recipients with a "film" of water, what is supposed to stimulate them to defecate (maybe also urinate?). Then you just have to wait. In the case of salamanders the faeces are big and hard enough, so that you can take it or take part of it with forceps and place it in an eppendorf.
We are also going to work with larvae of salamanders, and in that case we will place the larvae in falcon tubes, obviously in water, and wait a couple of hours. After that the idea is to take the faeces / water at the bottom of the tube.
We will work with DNA, so, freezing the samples at -20°C is ok for us.
I hope it helps you somehow
Thank you Eugenia!
It sounds absolutely reasonable but I'm just wondering how effective will be? Of course If you guard the animal and wait for the defecation happens can raise the rate of succes to collect the whole parasite "pool" from feces. But when you have many host individuals it could be problematic. In my opinion when the frog defecate into the water it is starting to dissolve and parasites could dissapear in the "environment" and you may have lose important information about the parasite fauna of the infected frog. But anyway, I don't have better options than that at this point. :)
When you collect stool from plastic recipient and put them into eppendorf (??) tubes do you use preservatives like formalin, alcohol, etc. before freezing?
Thank you Toumas!
This way seems more reasonable for me but I will try both!
p.s. Most of the time 96% EtOH even better for molecular work than 70%.
I know a PhD student from Trent Garner (ZSL, UK, London) did this too. You may want to ask him
Dear Annemarieke,
Could you give me an email contact to that PhD student in private message? Many thanks!
Cheers,
David
keep them in separate containers overnight and feces can be collected.Take a plastic cover and insert the frog in to it and hold the frog and apply little pressure on the back side.They will urinate and the urine can be collected from the cover.
We have had good luck with the overnight method, leaving them in a plastic bag ('poofed' up with a small paper towel on the floor) and collecting the wet spot and poo in the morning. This works >90% for large frogs (50mm svl and bigger) but not as well for smaller frogs. We then just cut the paper towel with what we want - usually the poo, looking for parasites. And I also recommend 95% EtOH for preservation for all but nematodes, and then the lower concentration can be used. Good luck!
Thank you Marcy and David for sharing useful experience!
Actually after collecting stool we want to store samples in freezer for future molecular work with the parasites, but maybe it would be more reasonable to separate the parasites first from stool with flotation or other method and then put them into EtOH and store in -20°C freezer.
I can't find how to send you a dm, please email me at: a.spitzenATravon.nl ;-)
You can hold the specimen in a plastic bag for a little while to collect feces. Frogs will usually defecate in the bag after being collected.
Poo: small wet paper towel on the floor of a plastic box and wait.
Pee: as you hold the animal usually Pelophylax pee on you up to 3 ml, be quick with collection, Falcon tube ready before catching the frog.
As you are interested in Parasites, a washing of the cloaka with a physiological (0.6% NaCl, Holtfreter, general Amphibian-Ringer) could be practical to collect helminth eggs. Use a plastic pipet. I fixed in 80% EtOH. In my hands it woks also to collect sperms.
For urine samples, have a falcon tube ready when sampling. When you catch the frog they will usually urinate within a few seconds.
For feces, a wet towel on the bottom of the box works, with a bit of patience.
Good luck!
You could try get in touch with Pat McLaughlin (University of Drexel, USA). He's an excellent frog researcher (usually deals with skin metabolites) and has great knowledge of survey techniques.
Best wishes
Using a fire polished glass pipette to cannulate the cloaca for urine collection. Works well with frogs and toads with large bladder. If cannulation is not possible, place anuran in a 5/8 mesh hardware cloth container and suspend over an angled sheet of wax paper or aluminum foil. Voided feces can be collected from the inclined aluminum foil; the urine will separate from the feces and run down the incline where it can be collected in a capillary tube. Use wax or critioseal to close both ends of the capillary tube and freeze until analyzed. Protocol requires constant observation and has been used to collect feces and urine for metabolic and water/electrolyte studies on anurans and avian species.
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