Posted: Mar 24 2006

The following is a copy of my article published in Of Sea & Shore.

                                              COMMENTS ON DESSICATION AND PRESERVATION OF SHELLS

On March 12, 1998 I asked the expert Conch-L readership the question as to the best desiccation technique for preserving microshells (up to 10 mm). I received many answers that addressed desiccation and preservation for dissection or DNA analysis. I then integrated the responses and issued the following summary. Here it is again to serve as a refresher.

But first, a few comments about why to desiccate at all. Desiccation is best used when the amount of animal matter is not excessive. The basic standard for “excessive” is that the shell is too small or too small and delicate (especially the lip) to be cleaned by mechanical means, the use of chemicals will damage the shell as well as dispose of the animal, or the “rotting/flushing” technique will damage the shell. The other way around to say it would be, “if you can readily get the animal (or most of the animal) out via other means, then getting it out is preferable to desiccation. If not, then desiccation is a viable second choice. A few cautions about using the desiccation technique. For univalves, make sure the animal is withdrawn into the shell before placing it in the desiccation fluid. Although there will be shrinkage, there will not be enough to draw the animal into the shell. The portion of the animal outside the “mouth” will adhere to the shell blocking your view of the shell and once dried will be almost impossible to remove. For some shells (i.e., Bubble shells) where the animal is often larger than the shell, it will be very difficult to achieve desiccation without some portion of the animal clinging to the outside of the shell. Try to remove at least a portion of the animal. This is a bit of a Catch 22 since the small Bubble shells are exactly the most difficult category of “small and delicate” shells where mechanical removal of the animal is likely to cause damage to the shell. If it can be done without harm to the shell, it is always best to remove as much of the animal as possible before desiccating. Use of desiccation is not recommended for bivalves. It will be impossible to open the valves. No matter how small, the best method for bivalves is to just soak in fresh water until the shell opens, then clean the animal out.

Oh yes, there’s another reason to use desiccation on micros. They are usually collected in large quantities. Desiccation is fast and easy.

A. Definitions
1. Desiccation - to dry up; dehydrate.
2. Isopropyl alcohol (isopropanol) - C3H8O, usually used as rubbing alcohol.
3. Ethyl alcohol (ethanol) (alcohol) - C2H6O.
4. Hydrogen peroxide - H2O2, an oxidizing and bleaching agent.
5. Oxidize - to dehydrogenate; to remove hydrogen from. Good at deodorizing, but does not desiccate.
6. Denature - to modify the molecular structure of (as in proteins or DNA) or diminish some of the original properties.
7. Methanol - CH3OH

B. Desiccation
Isopropyl alcohol or ethanol can be used with equal success. Since isopropyl alcohol is far more readily available (any drug store) and far cheaper than Ethanol, it would be the best bet if the objective is simply to prepare specimens for storage in your collection. However, if the objective is to preserve the animal for dissection or DNA studies, isopropyl alcohol has certain disadvantages and ethanol is preferred. For this reason ethanol is most commonly used in labs. Ethanol has a greater affinity for water, and because of its smaller molecular size, theoretically it may penetrate and dehydrate tissues somewhat faster. Whichever is used, the concentration of alcohol is the most important factor in determining the degree of desiccation that will be achieved before the final step of air drying. The most commonly available 70% rubbing alcohol will do the job, but the higher the alcohol content, the better. The higher the alcohol concentration the more water dehydrated from the animal, desiccation is more thorough, and the faster the final drying process.

Alcoholic beverages can be used. However, note that the "Proof" of the beverage is twice the percentage of the alcohol content. Therefore, 140-proof rum has an alcohol concentration of 70% and would work the same as 70% rubbing alcohol.

The ideal method is to use 10 times the volume of 100% alcohol as the volume of the animal. This approach compensates for the dilution that occurs as water is drawn out of the animal. Therefore, if you use a 70% concentration rubbing alcohol the quantity should be increased by approximately 1/3. A good approach to assuring as thorough desiccation as possible would be to replace the alcohol after a day or two; especially if the alcohol becomes discolored (yellowish). Because, remember that as water is drawn out of the animal the concentration of alcohol is being diluted. Since the percentage desiccation cannot exceed the percentage concentration of the alcohol, in order to achieve 70% desiccation with a 70% alcohol you will have to replace the alcohol several times until the dilution effect is eliminated (until the alcohol no longer becomes discolored - yellowish). The remaining 30% of water is eliminated via air-drying.

Denatured alcohols have denaturants added that will damage the animal and which can also eat up the calcium carbonate in the shell. Do not use denatured alcohol. Potable alcohols do not have denaturants added.

It must be remembered that desiccation is no more than a dehydration technique. The purpose for drying is to prevent the animal from rotting. But, what has been dehydrated can also be hydrated. That is the down side of this technique. You must be careful that desiccated shells are stored in dry (low humidity) conditions. There is an alternative. But it also poses certain risks and involves a somewhat more hazardous chemical with which to work. Conch-Ler Andrew Grebneff of New Zealand treats his micros with 4% formalin. Formalin is an aqueous solution of formaldehyde (CH2O) containing a small amount of methanol. Formalin is more difficult to obtain than rubbing alcohol and is rather displeasing with which to work. Formalin will damage shells if they are soaked too long. Andrew uses 40% commercial formalin diluted down to a 4% solution. He soaks the entire shell for about 12 hours, rinses thoroughly in fresh water, and sets out to dry. This method of embalming (or mummification) fixes the tissues in a manner to better resist rotting in damp conditions.

C. Preservation for Dissection
Preservation for dissection is best accomplished by storing in 70% ethanol. Isopropanol will harden the animal to the point that it can't be dissected. Ethanol in concentrations over 80% will also harden the animal. (Note: I've gotten differing opinions on this from different researchers doing dissection work. Personal preference appears to be involved.)

D. Preservation for DNA Study
Preservation for DNA study is aimed at stopping enzymes that destroy DNA. Alcohol denatures enzymes that destroy DNA, but does not similarly denature the DNA. The faster and better the alcohol does its job the less harm to the DNA. Therefore, the optimal fluid to use is 90-100% ethanol (and it wouldn't hurt to change the fluid once). However, in a pinch use the best you can get and change the fluid a few times. Dr. Gary Rosenberg, Associate Curator of Malacology at the Academy of Natural Sciences of Philadelphia, indicates that methanol can also be used to preserve for DNA studies.
Note: Be careful. Ethanol sold in drug stores is often denatured. Do not use denatured alcohols.

E. Preservation for Later Cleaning

Soaking shells in alcohol will stop the animal from rotting. Therefore, when a shell cannot be cleaned promptly after collection many collectors will store the shell in alcohol until returning home and time permits cleaning. This technique is particularly effective for such sensitive shells as Cowries, Olives and others where the living animal normally covers the shell. The best (cheapest) product for this purpose is the highest concentration rubbing alcohol you can find. A 91% concentration is available at many drug stores. However, remember the “10 times volume rule” (see section B, above). Don’t think that just because you put a shell in alcohol it’s safe from damage, especially if it is a large shell with a large animal in a container not much larger than the shell(s). Check the storage container every few hours (the alcohol works very rapidly) and if the fluid has turned yellow, empty and replace with fresh alcohol. You went to a lot of trouble and expense to collect your shells, take the time and care to make sure they get home in optimal condition and that you have not killed them unnecessarily. And, when you get home, if you cannot remove the animal immediately, keep checking the fluid and changing it until it no longer turns yellow. Also, note that it may be more difficult than normal to remove the animal from a shell stored this way since desiccation will toughen the animal tissues. Be careful and patient. Don’t damage your prizes at the last.

F. Some Comments On Use of Hydrogen Peroxide
Hydrogen peroxide does have useful applications. However, it is not a desiccant and should not be used for this purpose. It is an oxidant and can do a good job of deodorizing by destroying odoriferous compounds. Since it can also dissolve some of the soft tissues of mollusks, it may make it easier to flush out micro shells after a short soak. Hydrogen peroxide is an oxidizing agent, and can harm the pigments in shells. If it is used, soaking should be restricted to no more than a few hours and the shell should be thoroughly rinsed afterwards. Note that hydrogen peroxide can also destroy radulae and opercula. Mr. Grebneff also reports that even if confined to only an “over night” soaking hydrogen peroxide will ruin the color of certain species. Unlike ordinary laundry bleach (sodium hypochloride - aqueous NaOCl), hydrogen peroxide will dissolve the organic matrix of the shell.