A permanent preparation of any found new species, also known as holotype, is typically left at a museum and as such well known to the visitors of the museum. Holotypes play an important role to describe new species and are crucial for taxonomists as the anchorpoint of the first species description. In ciliate1) research and ciliate species description two things are important to determine species from morphological characters: The pattern of the basal bodies of the cilia, how they are arranged along the body surface, and the shape and number of their micronuclei and macronuclei2) which carry genetic chromosomal3) material  and macronuclei are important characters to determine species. The ensemble of cilia follows typical patterns along the body surface. Single lines of basal bodies (or kinetosomes) form characteristic patterns for each individual species. These lines are also referred as kineties

Until today, protargol impregnation is the gold standard to obtain holotype preparations of ciliate species. Initially, protargol was a brand name of specific antibiotics at the times when penicillin was not yet discovered. Chemically protargol is a compound of silver ions and a proteinate. From experiments with microscopy staining techniques protargol was found to stain certain structures in nerve cells. Later, scientists discovered it also stains important characters of unicellular ciliate species. Apart from medical therapy, protargol became an important microscopic stain. Protargol impregnation stains important morphological characters that are the basal bodies that form the kineties. Also fibre structures arising from the basal bodies will be stained that demonstrate some of the "mechanical parts" of ciliar motion. Protargol also stains nuclei of the ciliates. Thus, protargol impregnation is a perfect stain to demonstrate these important morphological characters. While the technique of protargol impregnation is more or less reproducible it is still the de-facto method to create holotype preparations. The binding sites and the way how protargol impregnation works are yet unclear. This is still a topic of research. 

In recent literature commercially available protargol sometimes is claimed to no longer work as expected. People claim old charges of protargol that were used over decades in certain institutions are guarded like life itself. Pan et al. (2013) provided recent work about how to conduct in-house synthesis of protargol following experiments of Davenport (1952). Such research is obviously important to achive and perserve reproducibility of the protargol impregnation. Following the protocol of Pan et al. (2013) I recently tried to create protargol by myself.

The trick to obtain a working protargol seems to be the selection of an appropriate proteinate. I own an amount of peptone bought from Carl Roth (Germany) that already gave satisfactory results from wet silver impregnation. Wet silver impregnation, however, does not yield satisfactory permanent slides. Apart from silver nitrate and a peptone the other chemicals required for synthesis are still available and can be bought from building supply stores, for instance. Thus, I also tested the possibility not to use pure supporting chemicals for synthesis, that are aqueous ammoniac solution, ethanol and acetone. The question, if it is possible to do the synthesis "at the kitchen desk", can be be answered with yes. However, care needs to be taken about evaporation of the chemical fluids that may cause harm or fire! Please read and follow suggestions from manufacturers and also provided by the Pan et al. (2013). As such, protargol synthesis leaves room for variations of the technique. Unfortunately, availability of chemicals to the public has been limited drastically by regulations of the European Union within the past years. Thus, hobbyists are impacted heavily and are no longer able to obtain these materials from any commercial distributor or e-commerce platform world wide. This is some sort of impact discussed at the end of my article. See my last section "Talking about education and amateur works".

Another success factor to obtain good results lies in the fixation of the living cells. Fixation id the process to obtain a dead cell, that shall be preserved as good as possible to preserve the natural cell structure. Unicellular species, like ciliates, are often hard to be preserved and kept in a good shape. Only a few fixative mixtures are reported to match high expectations about cell perservation. These are Bouin's fixative, Steve's sublimate fixative. Techniques used to other silver impregnation techniques, like Champy and da Fano's fixatives are also very toxic and thus critical in handling. Disposal of the fixatives needs to follow respective regulations: Collect separately and carry to disposal facilities. There are new fixatives at the horizon that are sporadically found to be tested in recent scientific literature. It shall be an important task to figure out in the wild, how well these less harmful fixatives will work with many different ciliate species of which certain genera are very fragile when fixed. I will certainly report about this special topic at a later point in time. 

The hand-made protargol synthesized works well and comparable to the results found in literature. Some special topics still are under investigation. Certain contradictory results are found in the broad literature about this topic, that stell shall be investigated to get more clarification about the unpublished secrets from literature. Overall, I yielded satisfactory results from the beginning, which made me happy. Kineties, basal bodies and also fibre structures are well stained looking at the details of my first slides.

Finally, I should add: Protargol synthesis is nothing for the impatient. A weekend shall be planned for the synthesis. A few days more shall be taken into account to collect experience to achieve perfection in results. The many factors that may form erroneous results are hard to discover. 


Figure 1: Protargol impregnation of an anonymous ciliate species. A moniliform macronucleus is easy demonstrated, as well as the kineties, ie. the dark impregnated basal bodies along the peristome of the ciliate.


Figure 2: Details of the peristome of another individual of the same species.


Figure 3: Chemically similar to protargol impregnation is wet silver impregnation following Fernandez-Galiano (1976). With this method silver proteinate will not be synthesized before the staining. Instead the solutions of silver nitrate and a peptone are mixed with a solvent, then reacts chemically in the fixed cell.

Talking about education and amateur works

I started amateur microscopy in the 1970ies of the last century, when I was 12 years old. My first microscope was a Christmas gift provided by my parents. When I was between 15 to 18 years old, I started digging deeper into microscopy staining and preparation techniques. At this time chemicals were available at the local drugstore. The owner of the drugstore close to where I lived, asked my parents about what I want to do with the chemicals I was interested to order from my sparse pocket money. So I got permission to buy any stuff like color staining powders, alcohol or mounting media, like canada balsam, as my parents confirmed: I'm doing amateur microscopy and biology studies at home. In recent years I restarted my microscopy hobby, but things changed in the years from 2013 until now. From my observation, even harmless chemicals like color stains became unavailable from e-commerce platforms. Pharmacists started to no longer sell pure chemicals to people. The main reason behind all the change: New regulations within the European Union limit the ways how chemicals are sold. This is limited now to professionals, only. Stupid enough when talking about education (see below).

The main goal of the heavy restrictions in chemical trading lies in prevention of terrorism. Funny enough to see, the exact same regulation explicitly state that authors of the paper knew that these limitations are not effective to prevent terrorism. Nevertheless, regulations are in place and prohibit people from doing amateur life science studies. This is a big impact and most likely will change worlds in many ways. Certain important supporters in science were and still are amateurs. Alfred Kahl, a teacher doing ciliate research in his private life in years 1926-1943 is a good example. His work took a huge impact on modern ciliate research until today. He discovered lots of new species and provided initial descriptions. Many of these species are still to be re-described by present life-science work.

Another impact from European government regulations prevents free trading of goods between European countries in funny ways. This is not limited to chemicals, but all goods in general. What did change? In 2015 European government decided that value added tax no longer will be declared in the country of origin, but needs to be declared in the country of delivery. The added amount of tax declaration rises with complexity of the countries these goods are delivered into. Funny enough to see this does no apply to Non-European countries. Guess what? This is like having re-invented and implemented complex customs regulation again. Simply speaking additional resources and also human resources are required for tax declaration, which makes trading more expensive to the origins. Small companies are simply not willing to accept the additional costs and effort. As a consequence trading companies started to no longer deliver goods into other European companies, because of effort and cost for the tax declaration. This also applies to well-known distributors of chemicals used in (amateur) microscopy. This again impacts international collaboration of the large amateur microscopy community in Europe.

My personal self-studies during the years of scholarly education opened a wide field of playing around with natural sciences. I was an enthusiastic amateur scientist, when I was a young child. Luckily my parents didn't stop me here. In contrast, they supported my personal development. This again influenced my choice of occupation. I'm still a private researche and scientist and also work in industrial science fields. However, I have strong doubts that current young people generation may follow my path, simply because they are not allowed.

Teachers talking to me tell that they no longer conduct chemical or microscopic experiments at school. Simply, because more and more effort to documentation of chemical experiments is required on top of educational work in order to prepare teaching in MINT fields. In addition, more and more chemical substances are forbidden at school, like color stains used in microscopy, either because they are suspected to be carcinogenic or simply "thought to be dangerous". I mean, there is not even a single microscopist known or confirmed who died from using congo red in microscopy. For the young people this means, certain aspects of scholarly education and self-study are more or more limited in many ways. Various educational tasks stopped at school, as I heard. This should be taken as a bright warning light to the public.

Regulations suppose, I'm a suspected terrorist. That same regulations also tell me, this is the reason, why I will no longer be able to get access to pure chemicals used to apply scientist techniques. Reading such work is stupid enough reading it. Honestly, as the owner of a small trading company being a citizen of European Union and also being an enthusiastic amateur microscopist doing scientific work as a private researcher, such limitations are absolutely unacceptable. 

This is not a direction a harmonized Europe should evolve into, simply because it is stupid. While claiming the freedom of choice of occupation, current European regulations takes away exactly this freedom due to the growing number of limitations to the people. This will probably limit science education and science outcome and excellence for future generations. Just thinking loud about the future and science within Europe.



  • Davenport, H. A., Porter, R. W., & Myhre, B. A. (1952). Preparation and testing of silver-protein compounds. Stain Technology, 27(5), 243-248.
  • Pan, X., Bourland, W. A., & Song, W. (2013). Protargol synthesis: an in‐house protocol. Journal of Eukaryotic Microbiology, 60(6), 609-614.


  1. The term ciliate historically is still in use in recent literature. It is hard to imagine that the name ciliate will ever be replaced in this special field of research. A genus of ciliates was already implemented for certain fish species at that time, when Perthy (1852) suggested this name. Thus ciliate (Perthy, 1852) taxonomically is an invalid name of the zoo of these unicellular species referred here. Nowadays, the correct name of the taxonomical group of unicellular species is ciliophora.

  2. Macronucleus and micronucleus are the two types that are typical and very special in this family of genera. Micronuclei carry genetic information for reproduction (cell division and conjugation), while the macronucleus carries genetic material important to somatic processes and process control of the unicellular ciliate species. Division of these two types of nuclei during life cycle (cell division, sexual conjugation) is manifold and also very typical within the different genera.

  3. The term chromosome is not well defined in ciliate research. Görtz (1988) for instance claimed that Paramecium species, the well-known slipper animalcule, doesn't carry chromosomes. Fokin (2019, however reported certain chromosomal type micronuclei for certain Paramecium species, well visible from light microscopy. For Tetrahymena species authors report to have found well separated DNA strangs from genomic sequences. Indeed, chromosomal-like division of the micronuclei are found with several species using special fluorescence microscopy techniques. This, however such nuclei reproduction during cell division doesn't seem to apply to all species.



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