The worms will often still move a lot when it is in a squeeze preparation (see Observation), and this can make it hard to make detailed observations. Worms can be anesthetised with a solution of MgCl2 in distilled water (tap or mineral water will often also do). For marine samples I usually us a 7.14% solution (i.e. 7.14g per 100ml), and I place the worm into a 50:50 (or even 60:40) mixture of MgCl2-solution and sea water. In contrast, fresh-water species are much more sensitive to magnesium chloride, and so in these I will often use only a few percent of solution (too high concentration will stop the beating of the cilia, and make the epidermis crumply and less transparent, which is not nice for observation).
Once you have the worm in a nice squeeze preparation go through a series of magnification steps to document more and more detailed structures of the worm. I usually start taking pictures with the 4x objective, even if the worm is very small. This makes it easier later to reconstruct the magnification at which the different pictures were taken. I try to take pictures of the following structures: habitus, eyes, brain, pharynx, pharynx glands, testis, sperm in testis, ovary, forming eggs, eggs and/or sperm in female antrum, eggs, vaginal cilia, shell glands, false seminal vesicle, seminal vesicle, copulatory stylet, prostate glands, vesicula granulorum, male pore, male pore cilia, sperm, epidermis, epidermal cilia, sensory cilia, rhabdites, rhammites, adhesive glands, and food items.
For taxonomy the most important structure is of course the copulatory stylet, but also the structure of the female antrum and the sperm morphology is important. However, the sperm morphology is a little tricky to document, because you would like to document the ready sperm from the seminal vesicles, rather than the forming sperm form the testis. This means that you have to somehow make sure what you are looking at. With a bit of practice you will be able to recover worms from the squeeze preparations if you want to do some other things with it. To do this place a drop of water (usually the stuff the worm was held in before you looked at it) next to the cover slip. The capillary force will draw in the water and the strength of the squeezing will be reduced. Generally the worm will start moving around again below the cover slip and you can then lift the cover slip with pair of fine watchmakers forceps.
What I then usually try to achieve is to amputate the tail plate, and to make another squeeze preparation with this, while I'll place the frontal part of the worm into 100% Ethanol for a DNA sample. (Details on this to follow)
- Clean slide and bino plate
- Take new yellow 40µl pipette tip
- Place the worm in the centre of slide while observing it
Anesthetize the worm
- In freshwater use Phenoxypropanol and water. 0.015% is a rough guide but concentrations vary with species. Use less and add more if needed.
- In Saltwater add 7 % MgCl2 in 1:1 or lower concentration
- Clean cover slip and put plastilin feet on it.
- Place coverslip with tweezers on slide. Move the slide to orient the worm with the ventral side towards the observer
Take pictures of the worm under the Microscope. Use all Magnifications (4x,10x,20x,40x,100x). Use DIC only for 20x,40x and 100x)
- 10x orient the camera, pic with all layers and maybe movie, rhabdites
- 20x DIC, movie, sensory hairs
- 40x special focus on testis, ovary, stylet, seminal vesicle and antrum
- 100x same plus focus throught the area around the eyes
- Cut off the tail plate with one continuous rolling of the scalpel
- With small pipette suck up anterior part with 1µl and transfer to PCR tube with EtOH (for DNA samples) or RNAlater (for RNA and DNA samples). Don’t touch EtOH with tip or worm will get stuck!
- Check if worm is in PCR tube
- Change tip and clean new slide
- Transfer tail with 1µl onto slide
- If freshwater add small quantity of 10‰ ASW. Otherwise osmotic shock will change the shape of the sperm.
- Place coverslip without feet to smash tail. This releases the sperm.
- At 40x take picture of stylet, locate sperm
- At 100x take pictures of sperm. If you have phase contrast use it.
- Add some drops of Lactophenol on the side of the slide (Don’t get it on fingers!)
- Label slide
- Label PCR tube on 3 places and tape the sides with scotch tape
- Enter information into datasheet, archive the images
Tips and Tricks
- Use enough fluid to place the worm on the slide to avoid smashing it
- Clean everything nicely to avoid something cutting the worm or obstructing the view
- Lift the coverslip with tweezers to turn the worm
- Orienting the worm with the ventral side towards the observer facilitates imaging the antrum
- Have as many informative structures on a picture as possible
- Think about orienting the worm in a way that pictures could be stitched together
- After cutting the worm with the scalpel make sure you have both parts