I am having trouble histologically sectioning whole nymph ticks. I am experiencing problems of keeping the ticks within the wax or resin block. I have tried the use of carnoys and bouins fixative solutions to soften the cuticle combined with embedding whole ticks in both historesin and paraffin wax but am having no luck.
Does anyone have any experience in this area or know of someone that can perform the sectioning for me? I will be using the sections for immunohistochemistry so the procedure would need to avoid any intereference with this.
Thank you in advance.
Dear Stacey,
We performed some histological sectioning of whole ticks with sucess few years ago. This work was not published but here is briefly the protocole that we followed:
-ticks were fixed in Carnoy for 15 min after having cut legs so that the fixative solution penetrates well,
- a deshydratation step in alcohol 70%-95%-100%
- A "softening" step of tissues in butanol for 2 days
- inclusion in paraffin wax
Hope it will help
All the best
Sarah
Stacey, what comes to my mind is the following:
i) choice of the right fixative (Carnoy’s as Sarah said is one of them),
ii) use of microwave oven (tests should be made e.g. with sufficient WL=waterload besides the specimen containing vial(s), the temperature rise in the fixative should be monitored, at least by measuring temperature after a while/cycle [initially say 350 -500 W, 30-45sec, then measuring with thermometer; Temperature of the fix-solution should not exceed 55-60°C). Repeat MW-fixation cycle (e.g.2-3 times).
iii) during/after fixation (guessing 15 mins fixation is TOO SHORT!, at least 2 hrs at RT, then perhaps over night at 4C) try (after 15-20 mins fix.) to divide the nymphs in an appropriate plane by using a fresh, cleaned razor blade (stress free = without applying pressure or traction, longitudinally, cross sections, etc.) thus decreasing also the size of the specimen, allowing further incubation in fixative for a while and aiding afterwards the processing fluids to penetrate more easily.
iv) another possibility eventually could be to perforate the tegument of the nymphs with fine needles
(cf.Exp Appl Acarol (2010,Springer) 50:151–161 DOI 10.1007/s10493-009-9286-7 by Veronez V, Botelho de Castro M, Bechara GH and Szabo MPJ:
“….Immediately after removing ticks from the host, tegument of each parasite was carefully perforated with fine needles and was then immersed in fixative solution (buffered formalin, pH 7.0) for 12 h. After fixation, ticks were embedded in paraffin and processed according to routine histological techniques. Each sample was serially sectioned longitudinally at a thickness of 4 µm. Sections were stained with either Hematoxilin–eosin or May-Grünwald-Giemsa. “
Softening by butanol (as propoesd by Sarah above) certainly will help. Thinking about the quality of your paraffin mixture perhaps could be helpful too: cf.:
LIM et al, Modified paraffin wax for improvement of histological analysis efficiency. (Microsc Res Tech. 2010 Aug;73(8):761-765)http://www.ncbi.nlm.nih.gov/pubmed/20063315, (If you can't localize a source for downloading that article as a pdf write again....).
If there is no increased quality of embedding/sectioning one has to look for other parameters’ optimization….Best of luck, Wolfgang
Dear Stacey
Maybe the problem is the calcification of the tegument. After removing thicks from fixative solution (bufferd formalin), parasits were decalcified in 10% EDTA in the same buffer formalin, for 2 days at 4°c, then dehydrated ...........
good luck
Hi All,
Thank you so much. It is definately the cuticle that is causing the problem. I had considered the microwave fixation however my ticks are infected with both CL2 and CL4 pathogens so this method is not feasible at the higher containment level. I will definately try the softening methods you have described. Thank you so much for all your help.
hello, I do a fast search in scopus (Key: tick and histology) and I found with more that 700 articles in this item and organism of interest to you. Maybe through of these articles, you can take direct contact with specialist in this Acari.
and directly I think your need descalcification to avoid float of organism in paraffin. You can uses comercial vinegar (acetic acid).
I send you the list of articles:
best regards,
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Dear Stacey,
Personally, for Coleoptera I have used the following protocol. If any of the chemicals or the technical steps involved does not interfere with the imunohisto techniques, you can try it.
1. fixation in Duboscq-Brasil: 1 to 3 days, RT (legs removed)
2. tert-Butanol: (3 baths - 6 to 8 hours each) - in the paraffin oven (56-58 degrees C)
3. tert-butanol - paraffin (same m.p.): 1:1 (1 bath, 0,5 to 3 hours)
4. pure paraffin (same m.p.): (3 baths, 3 hours each)
Concerning the problem of losing the specimen from the block - maybe your ticks have not been perfectly dehydrated before the embeding, or maybe they haven't lost the last trace of solvent before the embeding - or they are simply underfixed.
In the mentioned protocol, the use of the alcoholic Bouin (a.k.a. Duboscq-Brasil) and of the tert-Butanol elliminates the need to dehydrate the specimens by passing it through the 70, 96 and absolute ethanols.
You have to try which period of time per tert-butanol and paraffin bath is the best for your material. The tert-Butanol does some decalcification of the specimens, but this propriety is greatly enhanced by performing it in the oven (56-58 deg. C).
You should do the tert-butanol baths only in wery well stoppered (and the caps provided with two or three tours of Parafilm) bottles, because the tert-Butanol is miscible with the paraffin, and, if any trace of vapours reaches the melted paraffin used in the baths or for the embedding, the specimens will not get perfectly included.
To improve the sectioning, you can try to cut some sections from the block until you just reach the specimen. After that, the whole paraffin block in immerged in distilled water, at RT, left in it, and sectioned the next day.
Hope that will help you. Good luck !
Greetings all,
It is good to see there are expert there ! I enjoyed all the answers really much. Unfortunately I can't do such experiments/specimen processings any more (soon retiring) but have learnt a lot (especially from the recipe-proposals for processing "hard" materials like "carapax" and similar tegumental /"cuticular" elements in insects and other species).
@Omar Hernando: NOT to teach you but just as a kind PROPOSAL for another Scopus-search results (other colleagues for sure are thankful for your literature collection!): just for better readability of the thread it would be better to include the whole list of results into a word.doc, save and attach to the reply either as a word.doc file (for ease of work-up and perhaps also to be able to "search for authors, titles etc IN the file and / or add new references) OR convert to a pdf and attach the file to your post.... Just to keep your important collection of relevant literature regarding the matter [ I haven't done a scopus-search forever myself, shame on me (;_() ] I have done this and enclose that pdf file as an example to my response. Best wishes, good day everywhere and good luck, dear Stacey, Wolfgang
Hi! We use the next protocole: The tickswere fixed in 4% paraformaldehyde, processed according to conventional histological techniques and stained with hematoxylin and eosin. Softening by estiren monomer.
The action of two ethyl carbamates on acetylcholinesteraseand reproductive organs of Rhipicephalus microplusM.G. Prado-Ochoaa, P. Ramírez-Noguerab, R. Díaz-Torresc,G.I. Garrido-Fari˜nab, V.H. Vázquez-Valadezd, A.M. Velázquez-Sánchezd,M.A. Mu˜noz-Guzmánb, E. Angelesd, F. Alba-Hurtadob. God luck
Hi All,
Thank you Omar for the comprehensive reference list, it is much appreciated! I have ended up cheating and have found a company in New York that has 20 years of experience histologically sectioning ticks so i am going to use them as i am on a tight shedule. However, i am going to use non infected ticks to work up a method to use in house and will try the removal of the cuticle versus softening agents. I shall let you know how it goes!
Dear madam,
I have some research articles regarding tick histology. I am sending references as below, I hope these articles may be useful.
The Use of Vacuum in Processing Intact Ticks for Histology, G. A. Carranza, L. M. Martinez, R. M. Rios and G. G. Wagner, J of parasitology.
Med Entomol. 1990 Nov;27(6):1067-70.
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Thank you
Hi
I think you should use EDTA after fixation to prepare the sample and next, perform the other tissue processing method
Hi Stacey,
I know this problem very well, I had the same experience with embedding mites in epoxy resin. The cuticle of oribatids, too, is very smooth and somewhat flexible, which results in them braking off the blockface frequently. I tried to roughen the cuticle with bleach and even potassium hydroxide, but that did not work very well, plus it destroyed a lot of the samples, you have to be very careful. In the end, I just embedded the samples in a way that the legs were oriented away from the blockface, and could thus work as an anchor. I would loose a couple of section in the beginning, when the smooth cuticle had to be cut in a flat angle, but that was no problem to me.
Also, I started to puncture the specimen and cut off some legs during fixation, initially for better penetration of the fixatives, but I also got better support of the samples in the block that way. Unfortunately, I have not found the perfect solution, except trying to match the embedding materials mechanical properties as closely as possible to the cuticle ( Araldite Cy212 and Epon 812 worked best for me), providing as much structural support as possible (e.g., by specimen orientation), and cutting as slowly as possible. But still, it is difficult, and I always lost some sections. You might also want to play around with other settings on the microtome, e.g. free angle.
regards
Paavo
Hi Stacey!
Me again. I just thought you might be interested in the diploma thesis of Petra Föttinger from Graz, 2007: " Comparison of histological techniques in heavily sclerotised soil-dwelling arthropods". Unfortunately, I can seem to locate a download source, but in case you are interested, you can send me a message and I can provide a copy.
regards, Paavo
Hello!
I have got some experiences with the fixation, paraffin embedding and sectioning of the whole adult ticks (unfed and partially fed). Used was a Bouin's fixative slowly injected (nanoinjector) into the tick until swollen and fixed for several hours. Since Bouin's causes hardens a tissue a little, when tick seems hardened a small portion of rear of tick is transversely cut to facilitate further dehydration and paraffin infiltration steps. Approach here is the longer processing the better (as many TEM guys'd agree). As long as the tick specimen sinks down easily during dehydration steps it signals you are very likely on the right track. If you see the same during paraffin infiltration microtome sectioning should not be a problem although to section the whole tick may not be a snap sometimes.
I hope I will help a little.
Regards,
Juraj
dear Bergman,can u send me the diploma thesis of Grez .in.it will be nice of you if you can do that.
thanks
The diploma thesis of Maga. Petra F. ÖTTINGER, Graz,Austria has an abstract (in German) on http://www.landesmuseum.at/pdf_frei_remote/ENTAU_0014_0273-0288.pdf, pp 277-278 (2006/CORRECTED 2014-10-09 to: 2007 by W.M.) (free access), entitled:
(Engl.translation: Comparison of histological techniques in heavily sclerotised soil-dwelling arthropods)
With a short Google search I have not found either a German or an English full paper which can be downloaded (for free).
Mag.a Föttinger explicitely stated (translated from German): "....animals were either punctured or left unpunctured..., this was followed by infiltration with rapid penetrating fixatives for arthropods (not specified, but: ).... ... Fixation was most efficient with Picric-acid-, as well as sublimate-based fixatives. .... ....special treatment using ultrasonic bath or vacuum (in an exsiccator) did NOT yield significantly better results. Paraffin embedding (paraplast) yielded constantly and generally the poorest results, section thickness below 5 µm could not be achieved. Additional drwabacks with paraffin embedding: the long lasting process, use of Benzene as intermediate fluid which hardened the objects/specimens even more.....
..... embedding into LR White soft grade (without intermediate fluid, NB: i.e. either from 70% or at least from 100% EtOH) yielded good results, and, last but not least, "constantly good results" concerning preservation of morphology and section quality (ease of) could be seen with Araldite embedding.
I am trying to come into contact with her, perhaps she has some archived info which would be helpful....
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