Have you ever tried with 30 % of concentrated hydrogen peroxide (H2O2) to dissolve organic matter? It works for me.

You can try with conc. HCL (34%) first then wash with water after that add concentrated hydrogen peroxide (H2O2) to dissolve organic matter. Try if it improves as I work in that way to photograph out..

Maybe you can use the same protocol that are used to clean spicules of sponges.

http://species-identification.org/species.php?species_group=sponges&menuentry=inleiding&id=18&tab=foto

I have used 1% papain on bones to remove soft tissue with great success. I have also used sodium hypochlorite (bleach) effectively to clean bryozoans.

In many cases, using seawater samples, removing organic materials were non-essential process when identifying diatom in species level. I think that the frustule of many delicate species may dissolve at higher temperature during acid treatment, due not to the effect of acidification or Lugol preservation.

You could try this...

NITRIC ACID METHOD (Modification of Boyle et al. 1984).

Concentrated nitric acid (70% HNO3) is added to the sample pellet (1:1) and heated in a water bath at 80 ºC for 30 min. (the time varies depending on the amount of organic material in the sample). A marble is placed on top of each test tube to prevent loss of sample. The solution is vortexed gently every 10 min. to resuspend the sample.

Hi, have you seen this paper by Watanabe et al 2010: http://www.u-gakugei.ac.jp/~mayama/PDF/Watanabe_et_al_2010.pdf. I hope it may help.

I think, when the samples stand longer than a year in Lugols you will not get frustules out of it anymore. Not because of the acid you are using but of the bacteria working in your samples. Lugol is killing cells fast but is not good for long preservation of the cells. I would recommend hexamine buffered formaline.

Cooking the cells in 1 % SDS, 50 mM EDTA pH8.0 may be an option for you. The detergent will solubilize most of the organic material attached to the wall.

I prefer Patrick and Reimer method

Dear maxine,

I think that a protocol to clean diatom frustules gently has been described in: Prisholm, K., Moestrup, O. & Lundholm, N. (2002). Taxonomic notes on the marine diatom genus Pseudo-nitzschia in the Andaman Sea near the island of Phuket, Thailand, with a a description of Pseusdo-nitzschia micropora sp. nov.. Diatom Research 17(1): 153-175.

It is much more time consuming and as far as I know it has not been used widely, but I think that you could give it a try.

Best wishes,

Kimon

While there are many techniques for removing organic matter, a digestion with H2O2 is widely used. Thus, after removing the fixative from a given sample by successive washings by centrifugation, H2O2 100 vols is added at a ratio of one part H2O2 to two parts sample. Afterwards the sample is put in a stove at 60° C for 12 h. Then the samples are washed by centrifugation to remove the peroxide. This methodology has the advantage of reducing breakage of the material and is as well relatively easy to implement. In calcium carbonate-rich waters, its suggested that the carbonates are eliminated by the use or diluted hydrochloric acid.

Best wishes

Magdalena

Two alternatives:

1. Wash the sample with distilled water to remove salt. Then add hydrogen peroxide 20-30vv. Leave about 50min at 50C.

Then wash with distilled water.

2. Wash the sample with distilled water to remove salt. Add saturated potassium permanganate to the sample until It turns purple, that is, the same colour of KMnO4. Let stand for 24hs. Turn the solution transparent by adding oxalic acid. Then, wash with distilled water using a centrifuge as usual. Do not use any acid

Hi Towe

Are EDTA active to clean silicate in diatom frustule!

Dear Maxine

In your case, it is not the problems of acid treatment; the frustule degradation is related to bacterial activity. I had same problem with some samples preserved by Lugol preserved for several months. Before moving to acid treatment, we must verify the presence of the intact cells under a light microscope. If not!

Dear Towe,

Thank you, but the question was how to clean frustule, which is composed almost purely of silica!

i treat diatom samples by boiling in concentrated nitric acid and potassium permanganate (St. Clair & Rushforth, 1976), diluting, settling in distilled water and decanting until clear. it worked well, but the frustules of some delicate cells destroyed during the process of preparing samples for SEM.

Ok Maxine

thank you all for this methods

let's try the gentle "lundholm" method and another one with H2O2, perhaps 20v 50°50mn

Hello I think the best method is to use the following protocol

Use H2SO4 (Merck, 95-97) to fill about half the tube

Add 500 m L permanganate (solution saturated), mix well, leave overnight

If the sample is very clear add more permanganate.

Centrifuge 10 ', 2500rpm (25 ° C)

Remove supernatant

Add drops oxalic ac (saturated)

Shake your finger pellet; wait until transparent. Pour milliQ H2O (try to not make 2 phases)

Centrifuge 10 ', 2500rpm (25 ° C) 4 time

Hello Maxime

I think I have told you the method, maybe I forgot about the first step, concentrating the sample in a glass tube, you need approximately 50 ml of culture. If you have any specific question please let me know

sonia