Dear Raja Kulanthaivel,

The below link introduces a very similar procedure to the one which I performed successfully (Gold Chloride can be left out!)

Proper Bouin fixation is ok against 'burst'.

http://ameba.i.hosei.ac.jp/BIDP/Ciliate/QPS/index.html

Julia Torok

hi dear u will be check this link (http://www.stainsfile.info/StainsFile/bdl.htm)

Fix: Bouin, saturated HgCl

Unfortunately chemical company stop producing silver protein (protargol). If you are using dark brown silver protein, it will not work.

A suitable silver protein is available from Polysciences

http://www.polysciences.com/Catalog/Department/Product/98/search__silver+protein/productId__3174/categoryId__200/

If you do not have access let me know.

Mr. Snyder, I couldn't confirm that the silver protein working well. Did you use that for ciliate staining ? Because dark brown powder is not suitable ciliate staining. Additionally, protargol from Polyscience stop producing.

I have a batch from years ago, works, Polysciences still carries it, but If the newer supply does not then we may be out of luck... Silver nitrate?

oof I am also having same problem in making permanent slides in ciliates

do anyone have Foissner methodology

1).Air dried smear put in 2 % silver nitrate(5-10 min)in dark.wash in water and expose to the sun or UV for 10 min. 2)Smears with ciliates will be dried in air,then fixed in Bouin solution -20 minutes,decolored in70 %ethanol,rinse in water and 15 minutes in haemotoxylin.

Hi

The following literature may be of some help to you, in case you are successful in procuring protargol.

1. Foissner, W. 1992. Protargol Methods. In Protocols in Protozoology. J.J. Lee and A.T. Soldo, editors. Allen Press, Kansas. C-6.1 - C-6.8.

2. Lynn, D.H. 1992. Protargol Staining. In Protocols in Protozoology. J.J. Lee and A.T. Soldo, editors. Allen Press, Kansas. C-4.1 - C-4.8.

3. Montagnes, D., and D. Lynn. 1987. A quantitative protargol stain (QPS) for ciliates: method description and test of its quantitative nature. Mrine Microbial Food Webs. 2:83-93.

4. Wicklow, B., and B. Hill. 1992. A short procedure for protargol staining. In Protocols in Protozoology. J.J. Lee and A.T. Soldo, editors. Allen Press, Kansas. C-5.1 - C-5.4.

Best

Dola

Hi, there is a new paper about the methods of ciliates taxonomic studies:

Wilhelm Foissner. An update of ‘basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa’. International Journal of Systematic and Evolutionary Microbiology (2014), 64, 271–292

But as Mr. Jae-Ho Jung said, the company (SIGMA, I think) have stopped producing protargol powder with light brown.

Hi,

The Polysciences protargol is now the dark variety that will not work for ciliate studies. It's a shame that they continue selling it. Their "old' product was discontinued years ago. As far as I am aware there are no commercial sources for effective protargol currently. It can be synthesized without too much difficulty: Pan X, Bourland WA, Song W 2013 Protargol Synthesis: An In-house Protocol. J Eukaryot Microbiol 60:609–614 Available at: http://www.ncbi.nlm.nih.gov/pubmed/23870015. Silver nitrate methods with marine material can be problematic due to precipitation of the silver by salts in the medium. However there are several modifications that provide adequate results: Ma H, Choi J-K, Song W 2003 An improved silver carbonate impregnation for marine ciliated protozoa. Acta Protozool 42:161–164; Bassham RD, Browning JS, Landers SC 2020 The complete life cycle of the unusual apostome Hyalophysa clampi (Ciliophora, Apostomatida), a symbiont of crayfish in Alabama (USA). Eur J Protistol 72. Good luck!

We've been working with a lot of researchers and have finally concluded that the product below seems like an almost complete replacement of the Protargol product.

https://www.srlchem.com/products/product_details/productId/142168/Silver-Proteinate-extrapure--8--Ag

Dear Akash Agarwal,

I have tested and the protargol from SRL did not work for ciliate staining (e.g., 'protargol impregnation using acetone developer' by Foissner (2014)). If you can show us more about 'almost complete replacement of the protargol product', it should be better for researchers.

Hi Jae

Could you elaborate your procedures on the mail [email protected]. We would like to involve our RnD on this to assist where we can course-correct if possible.

I was unable to follow the link provided by Akash

https://www.srlchem.com/products/product_details/productId/142168/Silver-Proteinate-extrapure--8--Ag

If you are unable to synthesize a usable protargol (Pan X, Bourland WA, Song W (2013) Protargol Synthesis: An In-house Protocol. J Eukaryot Microbiol 60:609–614) you could also try so called "in-situ" approach (Kurilov A V (2017) Improvement of Silver Impregnation Technique Using in Situ Synthesized Protargol. Acta Protozool 56:109–118).

Alternative silver impregnation methods such as silver carbonate and silver nitrate ("wet" and "dry") are complementary to protargol impregnation rather than being substitutes. Although there is much overlap, no single method demonstrates all the pertinent features in many cases. In the case of hypotrichs, some variant of protargol impregnation is indispensable. Cell preservation of hypotrichs with the silver carbonate method is inferior at best but parts of cells may be well-impregnated. Occasionally adequate results are obtained (Olmo JL, Téllez C (1997) The ammoniacal silver carbonate method applied to oxytrich ciliates. Quekett J Microsc 38:3–5).

Unfortunately, some companies are still advertising and selling "Strong Protargol".

I've only tried a small number of these but none of them were effective. Brownish-black crystalline products are all ineffective in our experience. If anyone finds a reliable commercial source for protargol I would appreciate the details.

Good luck in your efforts!

The following is an excerpt from the publicly available website of the Biological Stain Commission: https://biologicalstaincommission.org/new-dyes/. Some of the historical info is not completely accurate (See Pan et al. 2013) but the bottom line is that the BSC does not currently certify any commercially available protargol. I noticed Polysciences now sells a "protargol" designated as "uncertified" (this product is ineffective for ciliate impregnation in my experience) at a substantially higher price than the their now unavailable product that was certified by the BSC. Note that the "CAS number" has nothing to do with testing and certification.

"Protargol-S. Protargol was originally a trade-name of the Winthrop Chemical Company for its brand of strong silver protein, an antiseptic made from a silver salt and peptone, and containing about 8% Ag. It is a buff to cocoa-brown powder and must not be confused with mild silver protein, a black crystalline solid made from silver oxide and gelatin or albumin, containing 20% Ag. (The term “mild” indicates a lower concentration of free, as distinct from complexed Ag+ ions.) Black products sold as protargol or silver proteinate are not suitable either for staining axons in nervous tissue by the method of Bodian (1933) or for staining ciliated protozoa. The latter application is considered essential for identification, description and classification of these organisms (Foissner 1991, 2014).

The BSC tests protargol in Bodian’s method and gives the designation protargol-S to batches that pass the test. For several years, protargol-S has not been commercially available, and protozoologists have had to rely on old stocks of the brown powder, which fortunately does not deteriorate with storage. Methods for making strong silver protein in the laboratory, suitable for staining ciliated protozoa, have been described by Pan et al. (2013), Kim & Jung (2017) and Kurilov (2017).

It is possible that brown protargol powder may again become an article of commerce. Vendors of any such products are advised to submit samples for testing and certification by the BSC.

References.

Bodian D (1936) A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat. Rec. 65: 89-97.

Foissner W (1991) Basic light and scanning electron microscopic methods for taxonomic studies of ciliated protozoa. Eur. J. Protistol. 27: 313-330.

Kim JH, Jung JH (2017) Cytological staining of protozoa: a case study on the impregnation of hypotrichs (Ciliophora: spirotrichea) using laboratory-synthesized protargol. Animal Cells Syst. 21: 422-418.

Pan X, Bourland WA, Song W (2013) Protargol synthesis: an in-house protocol. Journal of Eukaryotic Microbiology 60: 609-614.

Kurilov AV (2017) Improvement of silver impregnation technique using in situ synthesized protargol. Acta Protozool. 56: 109-118."

Try Feulgen Nuclear techniques. It will works.