The otoliths must be removed as soon as the fish dies, barring which the fish must be frozen or duly fixed to avoid the loss of the growth structures present in the otoliths. Larvae and juveniles must be handled with great care, because the area to otolith volume ratio makes them much more susceptible to deterioration.
There is a certain amount of body loss in fish storage, which is of particular importance for larvae and juveniles (Radtke, 1989; Kruse and Dalley, 1990). The corrective factors should be calculated by measuring and weighing a series of specimens, before and after preservation, covering the size range to be studied. Shrinkage is proportionate to fish size, and depends on storage time and preservatives (Kruse and Dalley, 1990). The period of time between death and fixing must also be reckoned for juveniles and larvae (Theilacker, 1980).
Being calcified, otoliths are broken down by acid fixatives such as formalin. The pH of buffered formalin can vary over time and it should therefore be used only for short periods. The fixative which produces the best results is ethanol, which should be used in a concentrated, 85 percent solution to compensate for dilution by the passage of bodily fluids into the storage medium. The alcohol should be changed from time to time for better storage. The addition of marble chips to the fixative will stabilize pH and improve otolith storage (Brothers, 1987).
Freezing is a good storage method where there is no risk of partial thawing due to temperature changes, which would degrade otolith quality.
In beginning the study of a species, the in situ position and orientation of the otoliths should be noted (asteriscus, lapillus, sagitta), so as to determine their relative morphology and orientation by their position in the saccule (Fig. 1). The size of the otolith may change throughout the fish's life, and so despite the fact that the sagittal otolith is the largest in many species, size should not be the sole means of differentiating the otolith.
The sagittal otolith is the one most commonly used, although some studies have employed the lapilli, which is smaller and requires less preparation (Brothers, 1987). In ageing, the same type of otolith must always be used as increment growth is not simultaneous in the three pairs of otoliths.
Both left and right otoliths of each fish should be collected and kept separate until their morphology can be differentiated (Hecht, 1978). The growth rings are usually identical in both otoliths: if one has been damaged during handling, or is crystalline, the other can be used.
To remove otoliths the cranium must be sectioned to reach the chambers of the inner ear. Cranial shape is filogenetic and therefore the removal technique must be modified to suit the species studied (Holden and Raitt, 1975). In round fish a transversal cut is usually made in the head a little behind the eyes. The cut must be deep enough to open the skull without damaging the otoliths. When the saccule is exposed the otoliths are withdrawn carefully with forceps so as not to break them. With small otoliths, it is better to remove the semicircular canals and separate the otoliths under a binocular microscope. Any adhering tissue can be removed from the otoliths by rubbing them gently between the fingers or with tweezers under a magnifying glass. Immersion in a five percent sodium hypochlorite solution facilitates the cleaning operation.
Fig. 1. Transverse section of right inner ear. am: ampulla, as: asterion, cs: semicircular canals, l: lagena, lp: lapillus, ml: macula lagenae, ms: macula sacculi, mu: macula utriculi, no: auditory nerve, pi: pars inferior, ps: pars superior, s: saccula, sg: sagitta, u: utricle.
The otoliths must be stored in such a way as to ensure they occupy the minimum space, save money, and are well-preserved and easy to identify. As otoliths are acellular bodies with a small proportion of organic matter, the risk of decomposition is minimal. Growth structures are nonetheless more easily visible in newly collected otoliths. They should therefore be read as quickly as possible.
Otoliths can be stored in test-tubes or vials, dry or with a clarifying liquid when they are to be read immediately. Another method in use is to stick them with transparent nail varnish to a slide or with two-sided sticky tape to a sheet of acetate. The more sturdy otoliths are usually stored dry in properly labelled envelopes.
The small and transparent larvae can be mounted in a clear medium for direct viewing of the otolith interior. The slide cover is pressed gently to split the cranium and expose the otoliths, freeing them from the surrounding tissue.
It is relatively easy to remove larval otoliths of larger size when the larvae are transparent. Read under a lens with polarized light, the otoliths appear as points of light against a dark background. The larvae are fixed on a slide with a drop of water and the otoliths removed with a fine needle.
Leave the otoliths to dry for a few moments after removal and place them distal side down on the slide, covered with some neutral mounting medium like Euparal, Flo-texx, Depex, Permounta1 to brighten the structure, and cover with a slide cover. The mounting medium shrinks as it dries and slide cover pressure can break the otoliths. To avoid this, place a piece of fishing line or other neutral material on both sides of the otolith in order to maintain a constant distance between the slide and the cover. The result should not be too thick as the microscope's working distance is small at the blow-up needed to read daily increments. When otoliths are mounted in Flo-texx-type media, which stiffen as they dry, the slide cover can be eliminated.
Mineral oil and immersion oil are mounting mediums which do not solidify and which allow later handling of the otoliths. The otoliths may, however, become unreadable after some time in such media (Brothers, 1987). The edge of the slide cover should be sealed with nail varnish or glue and the slides stored horizontally in a dust cover.
Canadabalsam is acidic, or can become so with time, and should therefore not be used to mount the delicate larval otoliths.
