METHODS
The Weeverfish chosen for the study was the Lesser weever, Trachinus vipera.
All the specimens used were caught in the Irish sea and surrounding waters around Anglesey by trawl and were preserved by one of two methods.
The venom glands were then dissected out when required and put through a number of processes. These processes varied according to the method of study.
A number techniques were used, such as Light microscopy and electron microscopy to study the nature and structure of the venomous spines along with the associated venom glands. In this study all of the observations were carried out on the opercular spine.
Preservation Techniques.
Two preservation solutions were used during the study;
10% Formalin/Sea water Solution (Light Microscope)
Glycerine Alcohol Solution (All other Studies)
The Weever fish specimen was placed in a screw top jar containing one of the above solutions and then left for a period of at least 24 hours before any dissection took place. The weever fish specimens used for these process were dead when they were captured using a trawl net, But any fish that had been in untreated sea water for longer than two hours was not used, due to the tissue structure would have been affected by natural breakdown processes and of no use for microscopy study.
Fixation of tissue.
The relevant areas of the Weeverfish were dissected out and placed in a fixative. The tissue samples were then left for a period of time (time depended on the fixative used ).
FOR THE RECIPES OF FIXATIVES USED SEE FIXATIVES PAGE.
Decalcification.
Decalcification was carried out using a commercially available solution (CALEX). The instructions supplied with the solution were carried out to the letter although the fixatives and preservatives were the ones described above and not those recommended in the text.
Light Microscopy Technique (Outline)
All the slides used in this paper were prepared in a similar way, (general outline described below). Only the staining methods used deferred, all other stages were the same in every case.
Stage 1: Wax embedment.
1) After fixation and decalcification the tissue samples were dehydrated using various strength alcohol solutions until the tissue samples were in 100% alcohol.
2) 100% alcohol-Toluene (1 hour)
3) Toluene/wax solution in a 1:1 ratio (1.5 hours)
4) Wax 1 (100% wax (1.5 hours))
5) Wax 2 (100& wax (1.5 hours))
6) Embed in wax and allow to set.
Stage 2: Sectioning
The tissue samples were cut into slices (5 microns) using a Microtone and placed onto glass slides. The sections were allowed to spread using an Albumen/Glycerol solution. The slides were then left to dry out on a hot plate.
Stage 3: Removal of wax from sections.
Before staining all wax was removed from the sections. The glass slides were inspected before they were allowed to enter the next stage to insure all the wax was removed from the slide.
1) Immerse slide in Histoclear (5 mins)
2) Transfer slide to absolute alcohol (5 mins)
3) Transfer to 90% alcohol (5 mins)
Stage 4: Removal of mercury from slides.
This stage was only carried out if the fixative used was SUSA (see FIXATIVES)
Stage 5: Staining
The tissue samples were dehydrated to the same alcohol concentration of the first step of a particular stain chosen.
Stage 6: Treatment of slides after staining.
The slides were dehydrated using various alcohol solutions until the slide was placed in 90% alcohol and then the following steps were carried out.
1) Transfer slide into 100% alcohol (2 mins). This step was carried out at least twice for each slide.
2) Transfer to Histoclear. At this stage the Histoclear was checked for any signs of clouding (presence of water in the slide) if this was seen the slide was transferred back into 100% alcohol.
Stage 7: Mounting.
The slides were left in the Histoclear and only removed one at a time for mounting. DPX was placed onto the slide (covering the stained section) onto which a cover slide was then placed. The slide was then allowed to dry (at least overnight).
Stage 8: Examination
The slides were then placed under a light microscope and examined in the usual way.
Frozen Sectioning.
One of the slides was prepared in a slightly different manner. Instead of embedding the material in wax as before, the material was frozen using liquid nitrogen. The chosen material was placed onto a drop of embedding fluid on top of a hollow metal specimen holder. This was then placed into a flask of liquid nitrogen and allowed to cool down (slowly) until the material become frozen solid. The holder and specimen was then placed in a cryostat and sectioned. The sections were then stained.
Electron Microscopy: Technique
The electron microscope used was a Scanning electron microscope. The microscope was used to assess the form and structure of the opercular spine in order to relate this to the method of venom delivery. The steps below detail the spine preparation.
Step 1: Flesh removal
The opercular spine was dissected free from the surrounding tissue taking care not to damage the spine structure in anyway. The spine along with the surrounding tissue was placed in a glass beaker along with 50 mls of distilled water which had approximately 2 grams of Potassium Hydroxide dissolved in. The tissue in the solution was heated gently to start the process of flesh removal, taking care not to allow the solution to boil. Once most of the flesh was removed from the spine. The spine was placed in a fresh solution and allowed to stand overnight to insure that all the flesh was removed from the spine.
Step 2: Mounting and coating in gold.
The spine was placed onto the mount using double-sided cellotape and coated in gold. The machine used for this process was a Sputter-coater.
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