Dear Jennifer,

I have used lactophenol for clearing sheep strongyles. It works just fine. 

I have attached an image of head end of Oesophagostomum columbianum to see how it was cleared. I assume that lactophenol would be useful for identifying equine small strongyles.

Best regards!

Stanislav

Thanks!

The person training me has been using Beechwood creosote for about 40 years and swears by it.  I appreciate the picture!  I am going to order some lactophenol and start practicing with it.

Dear Jennifer

I strongly agree with Stanislav, since I use lactophenol since the 80s for horse strongyles identification and it works fine.

Some tips for a good and safe use:

- Whenever possible, avoid a long direct contact of your finger tips with the liquid. Of course that does not happen much, but with dificult specimens, you must take a longer time to identify them and while manipulating the nematode(s), you must clean more often your fingers with lab tissues.

- Use a lab with good ventilation, because the scent at the beginning is nice, but can be annoying if you work continuouly for 3-4 h.

- For larger and thicker specimens, like Strongylus, Triodontophorus or even Poteriostomum, some times you must need to leave the strongyles some hours in a petri dish with lactophenol and only after that you are able to see the internal structures. On the other hand, don't leave tiny cyathostomins, like Cylicostephanus longibursatus or C. minutus, too much time in lactophenol (more than 24 h) , because otherwise they will be too much cleared and you will loose the necessary contrast.

- To save time, organize strongyles by size in a Petri dish and then mount 10-20 at same time, for a faster identification.

- Stored nematodes in ethanol 70% can be easily cleared with lactophenol. After identifying the specimen with lactophenol (that we also call lactophenol of Amman), the nematode can return again to ethanol 70%.

In conclusion, using lactophenol it's easy, namely for working  on horse strongyles identification. I am sending a PDF with the Top 10 Cyathostomins of a study performed with lactophenol, so that you can see how cleared they are.

Best regards

Luis M. Madeira de Carvalho

We use lactophenol for clearing nematodes

Lactophenol for smaller nematodes and phenol for thick nematodes

Hello Jennifer,

I'll probably not add much as many experienced parasitologists have already contributed.

Clearing and mounting media preferences will change according to purpose, personal experience/preference, and resource availability.

In my recent work, for temporary mounts, I have used either lactophenol or phenol-alcohol solution. I believe that you just want to identify specimens to genus/species level and not necessarily have them mounted long term.

Remember that after identification/examination you have to clean them in 70% ethanol for a few minutes to remove the clearing solution and stop the clearing process, before storing in ethanol (as Dr. Madeira de Carvalho mentioned). You may revisit the same specimens if necessary (mounting also in clearing solution but often you won't need to wait for too long as they have been previously cleared - this is my experience in examining specimens deposited at the US National Parasite Collection).

I have used creosote before, with very good results, but yes... it is (more) dangerous (using it in a fume hood is recommended). I recall it was a longer process and mostly for permanent slides (e.g. teaching purposes/collection), which would be later mounted in Canada Balsam.

Cheers,

Gui Verocai

After fixing the nematode hot acetic formalin, taking care to leave a small amount of saline solution in the petri dish (for strain of the parasite), places them in jars containing acetic cold formaldehyde.

To clean, put them in a Petri dish containing lactophenol for helminths, and follow the clarification with the aid of a stereoscopic microscope.

Hi Jennifer,

All methods mentioned by researchers work very well, but the effect will depend on the size of the specimen. I use these methods and other, it is a mix of 1 part of ethanol at 50% (in destilled water) with 2 parts of phenol p.a. (crystals). This solution work very well.

Good lucky! 

It seems that the responses above are quite comprehensive.  I'll just add that I also favor the lactophenol method, with mounting in glycerine jelly.  There is a problem with making such slides permanent for museum collections.  I have solved this by mounting the small nematode in glycerine jelly between two cover slips of two sizes, then mounting the entire coverslip sandwich, small coverslip down, on a glass slide with natural resin such as gum damar or Canada balsam.  The resin seals in the glycerine jelly so there is no evaporation and deterioration over the years.  I have nematodes mounted like this that are still in excellent shape after 35 years.  PLEASE TO MAKE PERMANENT MOUNTS AND DEPOSIT VOUCHER SPECIMENS IN A MUSEUM WHERE THEY CAN BE ACCESSED BY OTHERS FOR VERIFICATION!

Dear Jeniffer,

you can use various types of solutions for clearing nematodes but it depends on the following nematode utilization (morphological identification only, permanent or temporary mounts, morphology + molecular analysis etc.). The fixative medium is also important. After recovering nematodes I usually wash them in PBS buffer (pH 7.4) or saline for GI tract debris removing. Then I store nematodes in 70% ethanol for possible molecular identification. For clearing nematodes I use phenol-alcohol (80 parts melted phenol crystals and 20 parts absolute ethanol) and it works well. I work mainly with nematodes of ruminants but I am convinced that it will work well on horse nematodes also. See attached pictures.

Good luck