Thursday, May 17, 2007

More on tissue preservation

A few days ago a book came for me that I'd ordered through the university's interlibrary loans system. Field Preparation of Marine Specimens, by GJ Meuller, published in 1972, is a very useful little book that was published by the university of Alaska press and distributed to a very limited number of libraries - my copy came from the University of Idaho; I found other copies at the Smithsonian Institution and the various branch campuses of the university of Alaska. Nobody in Canada carries it that I could find.

This is an interesting book for at least two reasons. First, from a practical standpoint, it contains a high density of useful advice for collecting, identifying and preserving marine invertebrates - it's essentially a handbook for practical marine biology. Second, it's a fascinating look at laboratory and field practices in biology from 35 years ago.

Most of the recommended fixative and preservative solutions, generally named after their inventors (Gray's mixture, Bouin's fluid, etc.), were invented some time between 1930 and 1960, with lots of work apparently happening in the 1950s. Did the people of that era, extending up to 1972, have no concept of laboratory chemical safety? All of the solutions, for which recipes are helpfully provided, contain at least one horribly toxic or reactive ingredient. Many contain Mercuric chloride, one of the nastiest chemicals one can buy! That shit passes right through gloves made of latex or lots of other materials, absorbs through skin, and then fucking kills you! Boo-ya! You're dead! Madness! There's a note that Mercuric chloride is "extremely poisonous" in one description for one solution (Gilson's fluid), but that's it. The explosive properties of Picric acid are not discussed at all, and no mention is made of the toxic effects of chronic exposure to formalin. Several of the mixtures contain controlled substances such as Chloral hydrate or the insecticide Sevin, without any mention of how to obtain such materials - were these chemicals only controlled very recently?

All of the information not directly related to poisoning oneself in this book is great, though. I'm happy I found it, but I'm still stuck trying to find a method for working with invertebrates that involves neither my own death nor destruction of cell nuclei. Oh well. A recipe for a graded Ethanol series is included, which may be useful as I've seen some mention of a gradual increase in Ethanol not dehydrating tissues. Experiments ahoy!


Laelaps said...

Well, at least if you die from some sort of poisoning described in your post, you'll be well-preserved...

Scary stuff though; sometimes I look at the slowly-decaying vertebrates lining various biology classroom shelves here at Rutgers and I can't help but wonder what horrible chemical cocktails are contained in those jars.

TheBrummell said...

You are right to wonder... especially for the specimens that appear not to be decomposing at all. What horrid chemical could completely shut down spontaneous sterile dead cell decomposition? What could penetrate cells so quickly and completely and inactivate all intracellular proteins?

I talked with my advisor; both of us are in full agreement that we don't want any Mercuric chloride or anything like that in the lab. We've already got Formalin, Ethanol, Methanol, Xylene, and Hydrochloric acid kicking around, none of which are particularly nasty (even highly concentrated HCl is pretty harmless as long as you keep it out of your eyes). We've also got completely harmless stuff like Propylene glycol and Menthol, both of which are food additives.

I'll be trying out a stepwise Ethanol series tomorrow on some hapless Psuedosuccinea columella snails, to see if the tissue and cell nuclei will be trashed if we gradually replace the intracellular water with Ethanol.

Laelaps said...

Good luck with your Ethanol series, and in case you weren't content with just borrowing Meuller's book, amazon has one used copy for about 37 bucks (a little pricely, but I thought I'd let you know anyway). It's yet another book that's on my list now because I know almost nothing about specimen prep outside of fossils, so thanks for blogging about it.

TheBrummell said...

Mueller's book is only 44 pages long, so I think you should wait a while until the price drops.

It's a little out of date in some respects, besides its cavalier disregard for laboratory hazards, for example the Phylum-level taxonomy used at the end of the book contains several taxon names that are no longer used.

There are almost certainly newer and safer field preparation guides available that I haven't found yet.

Laelaps said...

Thanks for the update; I wasn't suggesting that it was the most up to date or best buy, but (like you suggested in the post) I find how science has changed over the years very interesting. That's why I spent so much time reading G.G. Simpson's books from the 50's as well, but I have many more books higher up on my list I want to get to first. Thanks for the information!

Necator said...

Oh Picric my last lab, we had an old crystallized bottle of the stuff! We had to get the HazMat guys in there to remove it.

As an aside, acrylamide is pretty dangerous too. It’s supposed to be a neurotoxin, of course the first time as an undergrad I was totally careless weighing the stuff out until our tech told me that I was endangering the lab. I’m not sure if it was psychosomatic but I recall having tingling sensations in my fingertips afterwards.

SDS is pretty nasty in its powder form. I have yet to wear a mask that seals properly on the sides and doesn’t allow the noxious white substance in.

TheBrummell said...

As an aside, acrylamide is pretty dangerous too.

I spent about 18 months of my Master's running PAGE - trust me, I know about the toxicity of acyrlamide.

My favourite thing about acylamide is the way it's a suspected carcinogen, but a confirmed neurotoxin - in other words, the rats all died of brain lesions before the cancer had a chance to metastasize. On the plus side, the neurotoxic effects take so long to manifest you usually have time to publish before you develop Alzheimer's-like symptoms. There's a professor in the department here whose inner-ear damage (on one side) is probably the result of his habit when a grad student of plunging his hands into tanks of liquid acrylamide. See my point above about ignorance of lab hazards 30+ years ago.

In addition to the lovely acrylamide in my gels, I used Formamide and Urea to keep the DNA denatured, and used radioactive 32P to visualize the bands. Also, typical power levels for my gels when running were 50 to 75 W, at upwards of 1800 V. Sparky! So I had four of the five common laboratory nasties in one place - toxic, flammable, radioactive and high-voltage electricity. I was only missing biohazardous - that was upstairs in the HIV lab I would occassionally steal supplies from.

Note that I was also using two of the five definitions of "hot" - radioactive and live electrical current, but not sexy, high temperature, or spicy flavoured. I never tried tasting the 32P though...