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

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44 based. Magnesium sulphate (Epsom salts). CAS no 10034-99-8. (MgSO 4 x 7H 2 O, MW = 246.48). Sometimes used instead of magnesium chloride. The chloride is easier to handle since it is less hygroscopic and requires smaller amounts (131.5 g/litre of the sulphate). The physiological mechanism, blocking synapses, is the same, as is the procedure. Smaldon and Lee (1979) presented 12 variations for the use of magnesium sulphate and chloride, for use with marine invertebrates, concentration between 0.1% and 20%. Menthol. CAS no 2216-51-5. Used for narcotising mainly fresh-water molluscs, by slowly adding a solution in ethanol or a few crystals to the surface of the water. May be irritating for skin, eyes, or respiratory organs, but is not considered very dangerous. Smells of peppermint. Mercury chloride. CAS no 7487-94-7 (sublimate). A component in many good fixatives, e.g., sublimate alcohol (Romeis 1989). Due to mercury being a severe pollutant and its toxicity, even at skin contact, these fixatives are less in use nowadays. Residues must not be discarded, but saved and labelled for special treatment according to local laws. Methanol. CAS no 67-56-1. Used for denaturing (5–20%) ethanol, stabilizing formalin (1–20%, by the reaction 2 CH 2 O + H 2 O ⇔ CHOOH + CH 3 OH) and storage of biological specimens (Horie 1989: 21). Its other drawbacks are that it is poisonous (threshold limit for allowable air concentration 200 ppm) and speeds up dissolution of calcareous material. MS 222 TM (Tricaine methanesulphonate). CAS no 896-6-2. Used for narcotising, usually fish, but also invertebrates. http://www.argent-labs.com/ argentwebsite/ms-222.htm. Has the advantage of giving reversible narcotisation and may be used on fish intended for later consumption (FDA approved). For invertebrates, slowly add some crystals to a few ml of water. Nembutal (= sodium pentobarbitone). CAS no 57-33-0. Used for narcotising by slowly and repeatedly adding a 5% solution. Test 1 ml solution per 100 ml sea water. Osmium tetroxide. CAS no 20816-12-0. Used for stabilising and contrasting tissues to be used for TEM and SEM of critical point dried specimens, when very high resolution is needed. It has not been found necessary for SEM of critical-point dried specimens, even at 10.000 times magnification. Osmium tetroxide should always be handled with utmost care, used in a well-ventilated area (under a fume hood) and special care should be taken to avoid eye and nasal contact. Osmium tetroxide vapours will react with any proteins, including the cornea of the human eye, where black deposits may be formed. The solution penetrates poorly (maximum 1 mm) and leaves the tissue soft and difficult to use for wax sectioning. When fixation is complete, excess osmium tetroxide GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27 must be washed out of the tissue or it will reduce to an insoluble precipitation of metallic osmium during treatment in ethanol. Paraformaldehyde. CAS no 30525-89-4. See Formaldehyde. Phenoxetol® (propylene phenoxetol = phenoxy isopropanol = ‘nipa ester’ = B-phenoxyethylalcohol = propylene glycol monophenyl ether). CAS no 122-99-6. Used as 1–2% solution, often with 2–5% propylene glycol to dissolve it more easily, for storage of zoological specimens (Lincoln and Sheals 1979: 136; McKay and Hartzband 1970; Mahoney 1973; Steinmann et al. 1975). Phenoxetol requires heavy fixation a priori. Phenoxetol is commercially used in disinfectants, preservatives and cosmetics. It is not considered harmful. More information at www.clariant.com. After having poured out several squids so stored 30 years ago (after proper fixation), I am not fond of the method. Propylene phenoxetol seems more promising as a tranquilliser and has been used by slowly adding a 0.5–2% solution to sea-water. Picric acid. CAS no 88-89-1. Picric acid is an excellent protein coagulant, forming protein picrates that have strong affinity for acid dyes. However, it penetrates slowly, causes extreme shrinkage and offers no protection against subsequent shrinkage. It is used in Bouin’s fluid (see above). Picric acid crystals are explosive, but need a heavy shock to explode. However, salts with heavy metals, e.g., iron, are shock sensitive and heavy metals must not come in contact with picric acid. To store it more safely, it is commercially handled in water. Be sure your supply has not dried and top it up with water if necessary. Polyvinyl acetate. CAS no 9003-20-7. Together with polyvinyl alcohol CAS no 9002-89-5 this forms the basis for many brands of glues used for wood and paper. They are usually white and may be thinned with water. A dry surface of such glue is good for mounting wet radulae and other small wet objects, where the moisture will soak the surface enough to make it sticky. Do not try to mount small objects in an excess of glue; the glue will use every possible crack to soak your specimen by capillary forces. Small dots of glue are good for opercula on a SEM stub, but will corrode small shells. For the same reason, an organic based glue should be used for repairing shells, not PVA glues which are too acidic and will cause damage. Use glues based on nitrocellulose or other polymer dissolved in acetone, ethyl acetate, butyl acetate or similar solvents. Potassium hydroxide. CAS no 1310-58-3. Used for radular preparation and maceration of tissues. Be sure to use analytical grade to avoid unwanted precipitations of impurities. Potassium hydroxide is said to be less hygroscopic than sodium hydroxide, which is an advantage, as only small quantities are needed and a jar lasts for a long time. The quality with pellets is preferable since it has a smaller surface and therefore
TECHNIQUES FOR STUDYING SMALL MOLLUSCAN SPECIMENS 45 reacts more slowly with carbon dioxide from the air. Prepare the solution directly in the vessel to be used by adding a few pellets or a quarter of a pellet when small quantities of water, e.g. 2–6 droplets, are used. Potassium- and sodium hydroxide are highly corrosive and all tools that have been in contact with them should be cleaned as soon as possible. Even in small quantities it is harmful on the skin, starting with redness and a burning sensation. It destroys clothing. Rinse with lots of luke-warm water, even with 1–2% acetic acid or vinegar, then further washing. A splash in the eye should be rinsed immediately with lukewarm water for several minutes; then obtain medical attention. Strong hydroxides quickly dissolve fat, which is the main reason why strong hydroxides are at least as dangerous as strong acids. It is a good preventive measure to apply hand lotion before preparing radulae, to reduce the effect of small splashes. Dissolution of potassium and sodium hydroxide in water develops enough heat to cause explosive boiling if large quantities are dissolved in a narrow vessel. Never dissolve it in warm water! Propylene phenoxyethanol. See phenoxetol®. Propylene glycol. CAS no 107-21-1. Can generally be used instead of ethylene glycol (Presnell and Schreibman 1997: 9), but is much more expensive. Propylene glycol is sometimes added (2–5%) to ethanol preserved specimens because it is assumed to reduce brittleness (Boase and Waller 1994). It contributes to rapid dissolution of micromolluscs. Sodium hydroxide. CAS no 1310-73-2. See potassium hydroxide, which is used for similar purposes and presents the same hazards. Sodium hypochlorite. CAS no 7681-52-9, commercial bleach. Commercial bleach is a solution of sodium hypochlorite in water, usually with some silicates added for preventing suspended particles sinking to the bottom. Often perfume is added to conceal the smell of chlorine. Due to the presence of the silicates, all equipment, shells and radulae must be thoroughly rinsed; otherwise the silicates will form insoluble precipitations. Commercial bleach, diluted with 1–5 times as much water, is a good oxidant for destroying organic dirt. Weak heating (30–50°C) speeds up the process. At the same time the high pH facilitates removal of the dirt. During the process chlorine (gas) is produced, causing a distinctive, unpleasant smell. Working with millilitres of the solution this does not present a danger. Be careful not to get splashes in your eyes; if this happens thoroughly rinse with lukewarm water; get medical attention if problems remain. Read instructions on bottle. Sodium lauryl sulphate. CAS no 151-21-3 (=sodium dodecyl sulphate). Used as a 5–50% solution for dissolving tissues and cleaning inorganic material. Can be used for radular preparation when potassium hydroxide disintegrates the radula, e.g., patellogastropods and chitons. This is a longer procedure and takes up to a week at 30–50°C. Much tissue remains afterwards and cleaning with a fine paintbrush in warm water and a short (a few seconds) rinse with bleach before the final rinsing is recommended. Due to the dissolution of fat and cell walls it should be handled with care. Sodium salts. Sodium phosphates are not good for buffer use in combination with sea water or calcareous material since the calcium in sea water will precipitate as phosphate, or recrystallisation of calcareous tissues may take place. Sodium salts of organic acids seem poor as buffers for protection of calcareous elements since the organic ions often form complex ions (even chelates) with calcium ions. The carbonates seem acceptable (see below), but the resulting pH of sodium bicarbonate alone is high enough to precipitate paraldehyde in formalin. Sodium carbonate. CAS no 497-19-8. MW 105.989 (also deca- and monohydrates exist) and sodium hydrogen carbonate, CAS no 144-55-8, sodium bicarbonate, (MW 84.007) have been used extensively for buffering formaldehyde and give a pH of 8 and 11 respectively in a 0.25M solution over a rather wide range of concentrations. In an aqueous solution 21 g/l (= 0.25 M) NaHCO 3 has a pH of 8.0; 26 g/l (= 0.25 M) Na 2 CO 3 gives a pH of 11.4. In formalin, however, a range of mixtures carbonate:bicarbonate 1:10–1:1 produce a pH of 9–10, which soon starts precipitation of paraformldehyde and means that sodium carbonate – hydrogen carbonate buffers should be avoided for buffering formalin. Sublimate. See mercury chloride. Superglue. See cyanoacrylate. Urethane (ethyl urethane, ethyl carbamate). CAS no 51-79- 6. Used for narcotisation by adding 1% solution of urethane in sea water. (Urethane is actually the name of the whole group of carbamates. For use as narcotising agent, the ethyl ester is usually employed: Dudich and Kesselyák n.d.). Not recommended since it is classified as a carcinogen and easily replaced by less dangerous compounds. Water. Freshwater is often recommended for narcotising marine animals, especially for echinoderms. Slowly (1/10 per minute) add it to animals in sea water until they stop reacting. Leave them for 5–10 minutes and fix. This method destroys all epithelia and is suitable for animals saved for identification only. Water used for washing material for use with the SEM should preferably be distilled.
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