Molluscan Research: Techniques for collecting, handling, preparing ...

40
Carbon dioxide, CO 2 . CAS no 124-28-9. Used for
narcotising, by bubbling the gas through the water with
specimens or by placing animals in water saturated
with it; mainly for fresh-water organisms.
Chloral hydrate (= chloretone). CAS no 302-17-0. Soluble
in water and alcohol. Used for narcotising by slowly
adding a 0.1% solution to the animals. Chloral hydrate
is a scheduled prescription drug in some countries.
Schroll (1968) indicates that nicotine may be used as a
substitute.
Chloretone. See Chloralhydrate.
Chloroform. CAS no 67-66-3. Used for narcotising, by
sprinkling a small quantity on the surface of container
with animals. Repeat if necessary. Note that chloroform
is poisonous and flammable and is a restricted
substance in some countries.
Chromic acid (chromium trioxide). CAS no 1333-82-0.
Chromic acid and its salts, chromates or dichromates
are valuable fixatives, but the acid is considered
carcinogenic and the salts are allergenic.
Cocaine hydrochloride. CAS no 53-21-4. Used for
narcotising. Excellent for chitons and heterobranch
gastropods. Slowly add a few crystals to the container
with animals. The possession of the chemical is
generally illegal and it is difficult to obtain permits for
its use.
Cold. Many animals, especially tropical species, will die
when the temperature approaches freezing point.
Usually they do not retract to cooling so it can be
combined with the addition of some narcotising agent.
Do not allow the animals to freeze, which will destroy
most histology (unless quick frozen, e.g. in liquid
nitrogen). Freezing is suitable for DNA sequencing
work.
Cyanoacrylate, methyl. CAS 187-05-3, super glue. Can be
used for repairing small, broken shells, but only in
cases where two pieces need to be glued together and
you can do it without much adjustment, since you only
have a second for this. If using it on a scale larger than
milligram, consult safety information since it is highly
toxic, much more than formaldehyde, but also more
treacherous since its smell is less deterring. It glues by
polymerisation induced by moisture, and sticks to skin
and any other tissue.
Dichromates. Fixatives. See chromic acid.
Diethyl ether. CAS no 60-29-7. Used for narcotising, by
sprinkling a small quantity on the surface of container
with animals. Repeat if necessary. A good solvent for
many organic compounds but due to the tendency to
form explosive peroxides it should be stored in a dark
bottle, preferably cold. It is highly flammable.
EDTA (Ethylene diamine tetra acetic acid). CAS no 60-00-4.
A 0.1 M (MW = 292.24) solution has been used for
narcotising purposes. Also used for decalcifying when
development of carbon dioxide may rupture tissues.
The process is very slow, up to several weeks. For
molluscan shells use 1–5% hydrochloric acid in 80%
GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27
ethanol instead. It takes a few minutes to a day only and
leaves the tissues in very good condition. EDTA is a
permitted food additive in small quantities, and not very
poisonous.
Ethanol. CAS 67-17-5. Has been used for storage of
biological material for more than 300 years (Boyle
1666; Waller and Strang 1996). It is by far the best
storage medium for material for general use and the
only one for long term use (Jones and Owen 1987: 60;
Levi 1966).
Avoid denatured ethanol for museum storage; the
denaturants will accumulate as the ethanol evaporates
and the jars are refilled. Many denaturants have severe
side effects on the stored material, ketones and
aldehydes react with the tissues; finally the poor human
that has to work with the denatured ethanol is affected
as is the intention with the denaturation. Some
denaturants (methanol, glycerol, isopropanol,
aldehydes) contribute to dissolution of micro shells by
forming complex ions with calcium. Most governments
accept use of tax-free ethanol for museum purposes
although the bureaucracy may be intimidating. Ethanol
is only sold as >99.5% solution and no manufacturer
guarantees 100% concentration because ethanol is
hygroscopic. When we refer here to 100% ethanol, we
indicate the purest form available. Hygroscopic beads
may be added to ultrapure ethanol, which will bind
excess water in the ethanol.
Micro shells may be affected by storage in
ethanol. This is not caused by acidity and buffering
does not help. (Actually you cannot even properly
measure the pH in alcohol since the ion product of [H + ]
x [OH - ] is no longer 10 -14 .) The reason for the
dissolving power is formation of a complex ion. A
calcium ion surrounded by five ethanol molecules is
slightly water soluble and the calcium no longer stays
precipitated as calcium carbonate. Since the complex
ion is water soluble, this effect can be reduced by
storing in 80% ethanol instead of the usual 70%. When
stored in 95% the effect is not noticeable (an advantage
with saving specimens for DNA (Carter 2002)), but
regrettably the specimens become brittle and less useful
for anatomy. At SMNH 80% ethanol is used as standard
for this reason. Presence of other organic compounds
like fat can give the same result, which is why tubes
with micromolluscs should not be stored with large
specimens.
Ethanol may be used for narcotising animal by
slowly adding it to the animals.
Ethanol is flammable and ignites at 363°C. Its
flash point is 13°C and it forms explosive mixtures with
air. The density of ethanol vapour is 2.1 g/l, 1.6 times
that of air, which means that you can walk in explosive
concentrations without smelling it, since it is
accumulated along the floor. The upper hygienic limit is
usually given as 1000–5000 ppm (1.9–3.8 g/m 3 ). It can
be smelled already at 10 ppm (0.02 g/m 3 ). Even