Write things. Estivate. Acclimate. Repeat.

The musings, brain droppings, and research interests of Nick D. Waters

Slide mounted reproductive systems of Sonorella species made by Lance Gilbertson, above, and Walt Miller, below. Above specimens collected by the author. Page header is the Walnut Creek talussnail, Sonorella coltoniana.

The Method

October 31, 2026.

Much of what we infer about the relatedness of snails comes from inspecting the secondary characteristics of the reproductive system. A fully phenotypically expressed structure is complex. In desert species, significant reduction and elimination of structures has occurred, presumably due to reduced potential for mate confusion in environs largely devoid of other terrestrial mollusks, restricted and specialized habitats, the necessity for size reduction to persist within the habitat during grueling estivation periods, and after all this, still produce viable eggs with hatchlings capable of surviving the first summer. From what remains of their secondary reproductive structures, their phylogeny has been inferred.

Helminthoglyptids are obligate hermaphrodites. The gastropod reproductive system employs a lock and key mechanism for mate recognition and serves to ensure simultaneous mating success. The penial sheath and its structures are expected to match the dimensions of the vagina, and those pairings occur simultaneously for the participants. Theoretically if differing species were engaged, a mismatch would occur and mating would halt. Theoretically, being the key word. As we know, sometimes, nature ... finds a way. In hermaphroditic species, a pairing is optional and the species may persist for a time in those dire circumstances. Nature finds a way.

Miller and Lance Gilbertson studied Sonorella hybridization. I have not investigated their results. I am unaware of field observations that document suspected occurrences. Hybridization is suspected to occur because traits that seem particular to one species also appear outside the geographic area expected within neighboring species.

The basic tissue-mounting protocol was developed by Wendell Gregg and published in 1958. It was later employed by Walt Miller's 1967 dissertation, and his students who continue to use it to this day. Edna Naranjo-Garcia matured it further in 1989, and at this point another opportunity presents itself to update it further to clarify euthanization, tissue preservation, and testing of alternative processess for tissue clearing. I have extracted these sources and tidied for readability, followed by my comments and materials list gleaned from the methods.

After review, the entire process, including relaxation of the specimen, extraction, and preservation, along with an updated slide mounting method and materials will be presented as a complete treatment for subsequent laboratory usage.

Tissue Clearing History

Werner Spalteholz pioneered techniques using Benzyl alcohol, aka toluene.

https://health.uconn.edu/cell-analysis-modeling/wp-content/uploads/sites/149/2022/03/wiley_zeiss-microscopy_the-art-of-tissue-clearing.pdf

The technique was apparently adopted by Gregg who then applied it to malacological specimens without the source being credited. S.S. Berry and subsequent authors were apparently unaware of their origin.

Tissue Clearing Resources

BaBB is a 2:1 mixture of benzyl alcohol (toluene) and benzyl benzoate. Benzyl benzoate is a tissue penetrating ester of benzyl alcohol and benzoate. The mixture is superior to toluene alone due to the penetrating ability of the benzoate additive. It can be prepared using hobby store or cosmetic quality material.

https://pubs.acs.org/cbihbp/article/doi/10.1021/cbmi.6c00155/5455973/On-the-Molecular-Basis-of-Shrinkage-in-Hydrophobic

Steps Forward

Tissue clearing and fixation using toluene chemistry is problematic due to tissue shrinkage. Further, it is a lengthy process. A hybrid approach may help improve the time for slide preparation. Dissection, isolation, staining, fixing tissues with formalin, clearing with BaBB rather than toluene alone, followed by mounting. If a live mouse brain can be viewed under a microcope in an hour, then dammit, a snail or slug can have its private parts on a slide mount, too.

Naranjo-Garcia, Edna. 1989. "Four Additional Species of Sonorella (Gastropoda, Pulmonata, Helminthoglyptidae) From Sonora, Mexico." The Veliger 32(1):84-90.

The reproductive systems were dissected out and preserved as whole mounts in accordance with the method of Gregg (1959; Miller, 1967a, and Miller, personal communication) as follows. [NDW: My numbering, and my materials breakdown afterwards]

Method

The specimen to be mounted consists of the dissected reproductive system of the snail, which is attached to the base of a cork by means of a short, bent insect pin through a flap of tissue at the genital orifice.
The specimen is immersed in a vial filled with haematoxylin (saturated solution), suspended from the cork, which is used to stopper the vial, for a period of about 9 min.
It is then removed and immersed in a similar-sized vial filled with destaining solution (2% HC1 in 70% ethanol) for 2- 5 min, until the specimen has changed color from deep black to light purple.
It is removed and placed in 70% ethanol for a period of 3-5 min, sufficient to dilute most of the absorbed acid.
Finally it is placed in an eosin stain (saturated solution of eosin-Y in 70% ethanol), where it may remain indefinitely, until it can be mounted. Protracted immersion in the eosin solution, however, will cause hardening of the tissues prior to mounting. Normally, 1- 2 min of immersion are adequate for eosin staining.
The specimen is removed from the eosin solution and placed on a slide (25 x 75 mm or 50 x 75 mm for large specimens).
At this time, the structures can be cleaned of bits of attached connective tissue, and the organs separated and straightened (for subsequent measurements and comparative examination).
A cover slip, or a similar slide, to which a very fine film of vaseline has been applied on the underside, is then placed on top of the specimen, and the slide, mount, and cover slip are tied firmly together with a piece of thread near each end to prevent the specimen from falling out during the dehydration and clearing processes. The vaseline is used to prevent sticking of the cover slip to the specimen when it is removed for embedding in Permount® (histological mounting medium from Fisher Scientific).
The whole mount is dehydrated in a series of three jars of 100% ethanol. Since the dissection is usually done under 70% ethanol, the mount can be placed directly into 100% ethanol. The whole mount is immersed in each jar for a minimum of one day. This assures maximum removal of water from the tissues.
After the third immersion in 100% ethanol the whole mount is transferred to a jar filled with 50% toluene and 50% ethanol, where it is kept for a minimum of one day.
It is then immersed in a series of four toluene-filled jars, a minimum of one day per jar, in order to assure that all of the ethanol has been removed from the tissues. This process also serves to clear the lipids from the tissues and allows the transparent viewing of internal structures.
After the fourth toluene immersion, the whole mount is placed on a flat surface, the cover slip is removed, and a layer of Permount is poured over the preparation.
A clean cover slip, or slide, is placed on top, and care is taken to eliminate bubbles. The whole mount is then set aside to dry. The drying process may take several days and additional Permount may need to be added, with a pipette, to compensate for evaporation during the drying process.
Permanent mounts are stored horizontally in covered, dust-free containers.

NDW Comments

It is unclear to me whether the method pertains to only one specimen in its entirety, or whether specimens can be processed synchronously through the steps and whether fluids can be topped off as needed, or have to be replaced after a single specimen or a certain number are processed.
"...through a flap of tissue at the genital orifice." The wording may imply that there is a flap of tissue present at the genital orifice, or, is it just that there is a flap of tissue as a result of the dissection? Is the placement intended to allow fluid to enter the orifice? I am thinking a specific flap of tissue that covers the orifice itself?
Presumably all fluids are maintained, and soakings performed at room temperature ~76 F?
For steps 2-5, can the same fluids be used for subsequent specimens, or just for the one specimen and immediately replaced? If re-usable, for how many specimens. Can one top off the fluids or is total replacement necessary?
For steps 9-11, the implication is the fluids may only be used once? If one wanted to stretch their dollar, how many specimens are reasonable, and can they be partially replaced, or topped off?
I believe Lance told me that one may prepare slides in advance by building a permount dam around the edge of the bottom slide and allow it to dry, such that when the specimens are placed they are more secure when final permount is applied. This would be unnecessary if the slide was compartmentalized or had an inert silicone gasket.
Toluene and ethanol mixed presents a high combustion risk. Mix only the amount needed. Dispose of in a container diluted with water in a well ventilated and cool room.

Mounting Materials

Four stoppered glass vials
Cork stopper(s) that fits the vials. Specifically cork such that a pin can be inserted. Only one cork needed as specimen will be transfered to different vials which presumably are either semi-permanently sealed with rubber, or the fluids are replaced into an appropriate sealed container.
Insect Pin(s), bent slightly to secure mounting of the specimen to the cork such that it may enter the vial, probably bent near the bottom third about 45 degrees
Haematoxylin (saturated solution) in vial
Destaining solution (2% HC1 in 70% ethanol) in vial
70% ethanol in vial
Eosin stain (saturated solution of eosin-Y in 70% ethanol) in vial
Two glass slides (25 x 75 mm or 50 x 75 mm for large specimens), one for base, second for cover.
Vaseline
Thread
Eight 12 oz lab jars, 2" diameter, 4" height approx.
One liter of 100% toluene
One liter of 100% ethanol
Three jars with 100% ethanol
50% toluene and 50% ethanol in one jar
100% toluene in four jars
Permount (Fisher Scientific)

Other Materials

Dissecting scope and lighting
Petri dishes
Squeeze bottles
Distilled Water
Dilute Hydrogen peroxide
Small pippettes / droppers
Dissecting tools and needle probes - fine dissecting scissors
Lab gloves
Well vented room with a fan
Disposal container for ethanol
Disposal container for toluene
Measuring flasks
Container to prepare 50% ethanol / 50% toluene, presumably by volume
Container with 70% ethanol 30% distilled H2O.
Timer

Miller, Walter B. 1967. Anatomical revision of the genus Sonorella (Pulmonata: Helminthoglyptidae). Doctoral Thesis, Univ. Arizona, Tucson, Arizona. 293 pp.

[NDW: My numbering and comments added]

Dissections

Snails are first prepared for dissection by drowning them in water for about twelve hours, to insure permanent expansion and relaxation.
They are then heated gradually to a temperature of about 56°0, the temperature of denaturation of collagen, at which time the dead animal can be pulled easily from the shell without harm either to the shell or to the animal. Care must be taken not to allow the animal to remain too long at this temperature or at higher temperatures, because the apical viscera may then harden and break off in the pulling process.
The snail is laid on its left side in a dissecting dish, under 50 per cent ethanol, and the dissection of the genitalia is performed under a dissecting microscope.

Whole Mounts

The technique used is essentially the technique of Gregg (1959).

The genitalia are partially fixed by letting them hang loosely in 50 per cent ethanol in a shell vial for 12 to 24 hours. This is accomplished by leaving a flap of body wall attached to the genital orifice and pinning this flap to the shell vial cork.
After this initial fixation, which predisposes the tissues to absorb stain, the specimens are stained in 1:6 hematoxylin/50 per cent ethanol solution for 75 to 120 minutes, depending on the thickness of the tissues.
They are then destained for about 30 minutes in acid ethanol and
... counterstained for 5 minutes in eosin.
They are then placed between two 1x3 inch slides, the top slide having been rubbed lightly with vaseline, to assure easy separation, and
... the slides are wrapped together with strong thread.
The mounts are then dehydrated in successive solutions of ethanol at 70, 80, 90, 95, 100 per cent, and absolute
... followed by immersion In 1:1 absolute ethanol and toluene,
... followed by clearing In four separate baths of toluene. Each Immersion lasts 3 days.
After the final toluene Immersion, the top slide Is removed,
... permount Is poured over the specimen,
... the top slide Is replaced, and the mount Is now ready to dry.
If voids develop during the drying process, permount Is simply added by means of a small pipette.

NDW Comments

Adding 4-5% ethanol, or beer, to water hastens the relaxation process. One can narcotize the snail for 75-90 minutes, then place directly into appropriate strength ethanol for dissection and at 95% tissue preservation. One could also presumably hasten the drowning process by first narcotizing as stated, then increase ethanol to 20% for another hour. See this article https://avmajournals.avma.org/view/journals/ajvr/86/12/ajvr.25.06.0218.xml
Personal observation: Removing the snail from the shell truly requires gradual heating. Placing directly into water already at 56C carries a high risk of viscera to separate leaving tissues within the shell unless you have done this numerous times and know just how long to maintain the snail in the water based upon its size. The rate at which to perform the heating process efficiently is an open issue. Starting to ending temperature heating time?
Detail from Miller's step 1 answers my question regarding the nature of the tissue flap from Naranjo-Garcia's step 1.

A Technique for Preparing in toto Mounts of Molluscan Anatomical Dissections by Dr. Wendell O. Gregg. Annual Reports of the American Malacological Union. 1958, pg. 39.

https://archive.org/details/annualreports19541961amer/page/n283/mode/2up

[NDW I have numbered the steps]

Abstract

In a study of the comparative anatomy of the reproductive systems of our western helicoid snails, the need arose for a method of preparing in toto mounts of these structures which could be preserved indefinitely, since preservation of these dissections in liquid preservative has proven unsatisfactory. The in toto mount permits ready re-examination and comparison. It may be photographed at any time after the preparation is completed. The process is typically one of dehydration and clearing the specimen, and mounting in one of the common mounting media between two 50 by 75 mm. slides. Canada balsam and permount have been used as mounting media, the latter being found to be the most satisfactory. By using a stain, internal characters of the organs, such as the plicae and papillae on the inner surface of the penis tube of Oreohelix or the double-tube structure of the penis of Helminthoglypta, may be seen in the cleared specimen.

Method

The stained dissection is arranged on the lower slide.
The preparation is then covered with a second 50 by 75 mm. slide which has received a coating of vaseline on the under surface.
The preparation is gently compressed with weights placed on the upper slide and the two slides are tied together at both ends while the weights are still in place.
Dehydration and clearing is accomplished as follows: Place in 25% isopropyl alcohol for 24 hours,
... then allow to remain for three days in each of the following: 50%; 70%; 80%; 90%; 95%; and 100% isopropyl alcohol;
... toluene and isopropyl alcohol, each 50%;
... followed by four changes of toluene, three days in each.
The specimen normally adheres to the lower slide. The thin coating of vaseline on the under surface of the cover slide prevents the specimen from adhering to it and it does not interfere appreciably with dehydration.
The first change of toluene dissolves the vaseline. When removed from the fourth change of toluene, the ties are removed, the slides separated,
... the mounting medium added,
... and the upper slide replaced,
... after which the mounting medium is allowed to harden.
These mounts are then stored in a horizontal position.

Comment: Dr. Berry stated he had been familiar with Dr. Gregg’s , work and that it had been developed only after the most painstaking experiments with various techniques over a period of several years. He said he considered that Dr. Gregg had accomplished one of the most important advances in malacology that has been made in many years, and that Dr. Gregg should be accorded great credit for this contribution.

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