Slide Prep Techniques for Nematodes

[bsat6815]

Hi all,

I would be grateful for guidance as to technique for fixing, staining and mounting nematodes such as Ascaris (larvae and ova) and filarials such as Dilofilaria (heart worm) and Strongyloides (threadworm?). These structures are on the order of 100-800 microns long.

Without being fixed, the specimens dry out and crack open, leaving nothing but rice crispy-like pieces of the exoskeleton cuticle.

Is there such a thing as a "general purpose" polychrome stain for amateur biological microscopy? I realize that such stain would likely be a compromise in many cases, as a specially-purposed stain would likely exist that would better serve specific types of specimens. But a single general-purpose staining technique would be nice for this amateur. How about Diff-Quik? Is there a polychrome stain out there that would be better for what I describe than Diff-Quik?

I am a bit confused about how to temporarily stick a specimen to a slide in order to dip the slide with specimen in fixant and in one or more stain baths. Only after fixing and staining could I use fingernail polish or other permanent mounting substance that would block fixant and stain if applied before the fixant/staining steps. I suppose that I could space a slip cover over the specimen laying on the slide using spacers at the four corners to hold the slip cover in place (using superglue at the four corners?) and then dip the assembly in fixant and stain solutions. I would need to work quickly before the critters become dessicated. Please tell me if there is a better way.

Then I could drip a permanent mounting medium in under the slip cover from each of the four sides. Would clear nail polish be best for the organisms mentioned above, or some other mounting medium better?

Many thanks in advance for thoughts on this.

[MicroBob]

Hi Bruce,
you might have a look whether Hoyers mix would be applicable here. Otherwise you probably have to bring your neamtodes through an alcohol row into xylene and from there into canadabalsam or Malinol. Classic nail polish contains a lot of solvent and shinks too much for thicker objects.
Staining is always object specific and varies a lot. O.K., Bolognese sauce stains everything....

Bob

[bsat6815]

Thanks for the pointers, Bob. Bolognese sauce it is then, for the "universal stain."

[BramHuntingNematodes]

I use formalin but hear that glutaraldehyde on its own or as an adjunct works better(for nematodes). I clear with histoclear as it smells better than xylene, and omnimount pairs perfectly with it. The H-E stain would be about the closest thing to a general anatomical stain-- it's all some microtomists ever use. Even so, there's much art to the dyers trade and other stains have uses.

Sticking the specimen to the slide can be performed with a mixture of egg white sometimes. Consult Gray on this. More frequently I have seen the specimen just placed on the slide, sometimes from a water bath, and let dry either at room temperature or on a heater.

[BramHuntingNematodes]

Oh also you introduce the specimen to the fixative immediately, like as in the creatures are still alive and in the root knot