[Nhcoll-l] fluid specimens and iodine staining

Tue Jul 4 07:38:28 EDT 2017

Hi Chris

A few brief thoughts building on Dirks reply a little;

I2KI - returning the specimens to a water based solution will introduce osmotic changes which you'll need to consider. Also just because something was chemically fixed in formaldehyde doesn't mean that crosslinking is necessarily permanent - some of the bonding formed is labile and will be open to degradation. Finally there is the potential effect of surviving molecular information such as DNA, via hydrolytic damage etc.

I2E may be the more preferential treatment for an ethanol preserved specimen as it is a simple step up, and you could then step it back to the original preservative. There will be some additional extraction of lipids and other mobile cellular components. I haven't looked into the literature so don't know the differences in optimisation the ethanol strength makes but if something like 90 or even 95% gives potentially acceptable results then I'd go for the options that results in the least change.

As ever a balancing act between access and preservation decisions!

Best wishes

Jules

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From: nhcoll-l-bounces at mailman.yale.edu [mailto:nhcoll-l-bounces at mailman.yale.edu] On Behalf Of Dirk Neumann
Sent: 01 July 2017 09:45
To: nhcoll-l at mailman.yale.edu
Subject: Re: [Nhcoll-l] fluid specimens and iodine staining

Hi Christopher,

this is a rather theoretical reply after looking into available literature which you surely also consulted (e.g. Butters et al 2014 & Balint et al 2016 on Plos One).

Both papers basically say that the staining results are hard to predict and largely dependent on perfusion rates and tissue thickness (which of course is no surprise but pure chemistry and physics). Obvious issues effecting the scan results are over-staining or under-staining, and the optimal incubation time is hard to predict and depends on specimens size, tissue composition and - surely too - deposits in and on the specimens (the cholesterol you already noticed surely is one of the most obvious ones).

Other influencing factor are is the x-ray source and data recovery.

Personally (gut feeling rather then proofed experience), exposure to water based solutions for weeks or even months surely damages the specimens and weakens the tissues, and my next question would be what happens will all those (dissolved) fats in the specimens if they are immersed in water for such a long time. Also, Balint et al (2016) report that over-staining can lead to deformation and tissue shrinkage .

The other method you mention, exposure to nearly 100% EtOH has just the effects that you describe, and the question for me is what would happen especially to those tissues near the body surface (e.g. in fish increasing the EtOH strength may cause cell rupture in the scale pockets and consequently may loosen the fish scales).

So it might be worth to explore two alternatives:

1. It might be worth to compare the imaging capacities of different ct-scanners; colleagues at the MNHN Paris for example used the synchroton in Geneva for imaging of or Lungfish larvae with amazing results.

2. You mentioned that targeted specimens in your collection were preserved +100 yrs, some probably used pre-formalin fixatives. If this is the case, it might be worth to test if any mercury salts have been added. Balint et al (2016) mention, that "Mercury (II) chloride provide the best contrast between different soft tissue types".

Around 1900, Mercury salts were not rarely added to enhance the "fixing" properties of the ethanol. Perhaps some of your specimens have already been treated with a contrast enhancing agent?

Hope this helps, even though it is not a direct answer to your question.
All the best
Dirk

Am 30.06.2017 um 19:59 schrieb Milensky, Christopher:
Hello,
I am hoping that some folks in the NHCOLL community have experience with iodine staining and would be willing to share their knowledge.  We are receiving more and more requests to use our fluid specimens (birds) for CT scanning projects, some of which require staining.  In the recent past, we have been asked to approve two different staining techniques.  One is a 'water' based staining method and the other an 'ethanol' based method.  We would like to hear opinions about which method is best for the short and long term care of the specimens.  As with any old collection, the exact methods of fixation used over the last 100+ years have been variable, but typically our birds were fixed in formaldehyde and then moved to storage in 70% ETOH.  I'm sure some of our oldest specimens were never properly 'fixed' since formaldehyde did not come into use until the early 1900's.

The two methods:

I2KI (potassium iodine and iodide) is dissolved in water and makes a naturally antimicrobial solution in which the specimen is removed from ETOH storage and soaked for weeks to months, depending on the size of the animal, in this water solution.  Pickled tissues (fixed in formalin and preserved in 70% ethanol) should remain pickled even after being removed from preservative due to the chemical changes that occur during fixation, and iodine prevents bacterial growth, but it still involves putting a pickled specimen in water for an extended period.

I2E is elemental iodide dissolved in 100% ethanol (200 proof), which raises another set of issues for specimens stored in lower concentrations of ethanol. Increasing the concentration of ethanol further dehydrates and shrinks the specimen, but it has been found to be the most effective concentration of ethanol for staining soft tissues.

So, the question is:  Would you rather have a specimen removed from 70% and stored in a water/iodine solution for weeks/months or stored in 100% ETOH for a similar period or neither?  What are the pro/cons of each?  Would you allow a specimen collected pre-1910, and presumably not fixed, go into water/iodine for a month or more?  Are you aware of other methods?

I look forward to hearing your thoughts!

Chris

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