Optimising gonad preservation whilst retaining otolith microstructure
I am collecting populations of very small fish from remote locations, and
need to extract two important pieces of information from them for my study.
First I need to access the cellular morphology of the gonad for sex and
maturity estimates, and second I need to access the age and growth rate
information held in the calcified structures called otoliths (fish ear
bones). These are Calcium carbonate concretions deposited in concentric
layers daily or annually.
My problem is that ideally i need a fixation method that I can leave my
field samples in indefinitely, but that will not react with or degrade the
otoliths. I used Bouins last time, but its acidic and degraded the otoliths.
Normally otoliths are stored in 95% ethanol when still embedded in tissue,
but the ethanol sometimes needs to be changed as it becomes acidic on
reaction with the tissues. Also this concentration of ethanol would
dehydrate the gonads and make them hard and not very good for histology.
The best thing I can think of is NBF, but I am not sure if this will react
with the otoliths, or how long I can leave the specimens in it. If I simply
fix and then transfer to 50 or 70 % ethanol (as would be normal), then I
stand to lose the otoliths (50 and 70 % ethanol is too acidic). What can I
buffer the Formalin with that is NOT a calcium chelator? And can I leave the
samples indefinitely in this until I get home and process them? All other
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