Osmoregulation as a Homeostatic Mechanism using the Comparitive Melting-Point Method for two Crab Species.

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Osmoregulation as a Homeostatic Mechanism using the Comparitive Melting-Point Method for two Crab Species.

Two crab species, Plagusia and Cyclograpsus, were collected from a local estuary in the littoral and deep water zone for osmoregulation studies. To examine differences in osmoregulatory mechanisms among the species, haemolymph of the specimens was extracted once they were acclimated to varying concentrations of seawater. Using the comparative melting-point, capillary tubes were filled with small samples of seawater and blood then frozen and melted in a -15˚C ethanol bath. The melting time of each was observed thereafter. Subject’s time range fell over 17 minutes of which the majority of the most salinated samples melted
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b) Experimental Design (Laboratory Manual, 2013)
The fourteen collected samples of fluid included distilled water (0% seawater/ionic concentration), 25%, 50%, 75% and 100% seawater; 20%, 40%, 60%, 80% and 100% seawater in Cyclograpsus blood; and 60%, 70%, 80% and 100% seawater in Plagusia blood. 1.0 mm bore capillary tubes were cleaned and dried. Approximately 3.0 mm of each fluid was drawn into 14 different tubes, and gently tapped to ensure the fluid was moved to the middle of the tubes. The tubes were marked and sealed using Vaseline, then placed strictly in an adjacent parallel order, using Vaseline as an anchor, across a capillary level holder. Dry ice was carefully placed on the capillary tubes to freeze the fluid in the capillary tubes.
An alcohol bath was prepared with an ethanol solution and dry ice, the solution was measured using a thermometer, at approximately -15˚ the bath was placed under a Polaroid filter and above a stable jar upon a white piece of paper. The capillary level holder with the frozen capillary tubes was placed carefully into the bath (at an angle to reduce effervescence). A lamp was placed 20 cm away from the setup and shone on the Polaroid filter. This setup ensured the ethanol solution didn’t heat up too rapidly.
The ethanol solution was stirred at regular intervals using an unreactive rod, whilst a stopwatch was used to accurately determine the melting-point of each capillary tube, by clear visualisation of the meniscus
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