Aiptasia with Mallory Trichrome

Images made through a microscope. All subject types.

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discomorphella
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Location: NW USA

Aiptasia with Mallory Trichrome

Post by discomorphella »

Aiptasia are an invasive pest of many reef aquaria, mine included.
Typically they intercalate themselves into the crevices of rocks and I can't pry them out without cutting them up into pieces that will regenerate into more Aiptasia. But this one wasn't so clever and adhered to an old shell. I was able to let it expand in a small petri dish of seawater and then rapidly pipet nasty Bouin's fixative over it, fixing (well, that's a nicer term for it...biology is the art of killing with finesse...) it before it could contract completely. It was then washed to remove the Bouin's, dehydrated in ethyl and isopropyl alcohols, and embedded in polyester wax. I sectioned it at 5 microns on my 1935 Spencer microtome. The sections were stained with the simplest of the Mallory trichrome protocols; I am going to try the more elaborate and supposedly better AZAN technique next. Photos all taken on the BX-60 using either 20X/0.7 UplanApo, 40/1.3 UPlanFl or 60/1.2 UplanApo objectives and standard thickness (U-DICT upper prism) Nomarski optics. The image was relayed through a Leitz Variozoom (5 to 12.5X) eyepiece, Leitz 0.32X converging lens to a D300s. I've shown some cross and longitudinal sections of a tentacle and some of the main body.
Results are interesting. You can see unexploded nematocysts as red-stained coils in the outer layer of the tentacles.
The first image is a low mag shot of a cross-section, you can see the outer layer of nematocysts, and the extended tubes of some of the exploded ones sticking out. The next 3 images show longitudinal sections of a tentacle. You can see the red nematocysts with their coiled thread tubes, muscle and other fibrils stained blue, and zooanthellae (commensual algae) and other structures and cells stained shades of purple, orange or blue. Nuclei are red to purple depending on the cell type. Following images are from the main body, which show internal muscular and elastic structures in blue along with ciliated cells on the inside of the body cavity and internal partitions of the animal's body.
I am puzzled by the fact that what I am interpreting as muscle is staining blue; for all mammalian and fish tissues I've stained using Mallory and other trichrome techniques muscle cytoplasm is red and only collagen is blue. Maybe cnidarian tissues are different. I am going to try a more sophisticated trichrome technique (AZAN) on more slides and see if
this holds. For now, there's at least one Aiptasia that I am actually glad to see.

David

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Charles Krebs
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Post by Charles Krebs »

A lot of work!... but you're showing us tremendous detail here.

(I find the detail even more obvious if I "open up" the images just a bit with a basic highllight "levels" adjustment)

discomorphella
Posts: 607
Joined: Sun Oct 01, 2006 7:26 pm
Location: NW USA

Post by discomorphella »

Thanks Charlie,

Yes, I really didn't pay enough attention to the post processing of these images. Which I really should have considering that the whole point of a trichrome stain technique is to get many different tissues stained with many different colors. After some discussions with a friend who is a real biologist, I realize that the blue fibrils are most likely collagen, and that cnidarians may not have the usual looking myocytes like vertebrates. There could be myofibrils that stain red but don't look like the muscle cells I am used to, and the blue-stained fibers are in fact collagen. That anemone does need something to hold itself together after all. If anyone out there is familiar with lower invertebrate histology please correct me. I am going to try what is supposed to be a much more vibrant trichrome technique next, just need to get the time to run a batch of slides (its 6 hours instead of just 30 minutes). I'll post that if it works (with better post processing.... :) ).
David

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