Oct 09, 2024
Permanent specimen preparation by protargol staining
- Marek Valt1,
- Michael Kotyk1
- 1Charles University, Faculty of Science, Department of Zoology, Prague, Czechia
- SUM-K
Protocol Citation: Marek Valt, Michael Kotyk 2024. Permanent specimen preparation by protargol staining. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71or9gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 09, 2024
Last Modified: October 09, 2024
Protocol Integer ID: 109479
Abstract
Protargol staining according to the protocol by Bodian (Bodian 1936) and later modified by Nie (Nie 1950), further optimized for staining of small flagellates.
Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
Protargol staining optimized for a small flagellates, used for staining of Solarion arienae.
2d 1h 15m
2d 1h 15m
Mix 1 μm of culture sediment with a drop of sterile egg albumin, on a coverslip.
For Solarion arienae, egg albumin was not diluted to achieve better adherence to the cells to coverslip.
Fix in Bouin-Hollande’s solution overnight. (0,175 mol/l picric acid, 0,138 mol/l Cu+2 acetate, 4% formaldehyde, and 5% acetic acid in water solution)
Next day, transfer coverslips throught a graded ethanol series (70%, 50%).
Wash in distilled water.
Bleach in 0,5% KMnO4 solution for 5 minutes.
5m
Wash in distilled water (5x).
Treat coverslips with 5% oxalic acid for 5 minutes.
5m
Wash again in distilled water (5x).
Stain in 1% protargol solution, for 48 hours at 37°C in a beaker with copper wire pieces placed between the coverslips.
For Solarion, protargol produced by Bayer, I. G. Farbenindustrie Actinengesellschaft (out of business since 1952) was used.
2d
Wash in distilled water (2x).
Treat with freshly prepared reducing solution (1% hydroquinone and 5% Na2SO3), for 10 minutes.
10m
Wash in distilled water (5x).
Tone with 1% AuCl3, for 5 minutes.
5m
Wash in distilled water (2x).
Treat with 2% oxalic acid, for 5 minutes.
5m
Wash in distilled water (5x).
Treat with 5% Na2S2O3 for 10 minutes
10m
The final wash is done under a constant stream of tap water for 20 minutes.
20m
Dehydrate the coverslips in an ethanol series (50%, 70%, 80%, 96%, 100%)
and 3 times in xylene (5 minutes each).
Dehydrate in xylene, 3 times (5 minutes each).
15m
Finally, the stained, dehydrated coverslips are mounted on glass slides with DPX mounting
medium (Sigma–Aldrich).