Duck Intestine

[discomorphella]

Hello Amateur Histology Fans Everywhere,

In continuing with my "hunter's histology" samples, today's menu
features some thinly sliced duck intestine. A mallard (Anas platyrhynchos) to be specific, from Oregon. The tissue was collected when the bird was cleaned, fixed in the field in phosphate-buffered formalin (AFIP formula), and then dehydrated using ethyl cellosolve and ethanol, embedded in polyester wax and sectioned at 5 microns on an old (very old, 1935) AO model 820 microtome (with a disposable blade holder being a concession to modernity, sharpening knives is a pain...).
The sections were stained with either hematoxylin and a mixture of eosin Y + Phloxine B or Toluidine Blue O and Phloxine B. The TBO sections were treated with ammonium molybdate after TBO staining. The first three images show cross sections of some villi, with muscle fibers and lamina propria. Note the empty-appearing "goblet" cells in the villi in the first image. They are far from empty, in fact their mucus content shows up as a green fluorescence in the second image of the same section under epi-fluorescent violet illumination (~400-455 nm excitation, > 460 nm imaging) where the phloxine and eosin fluoresce in different colors depending on the dye's chemical environment; by contrast note how the muscle and other structures fluoresce reddish-orange.
The third image shows a similar region stained with TBO / ammonium molybdate. Here a chemical reaction with the contents of the goblet cells gives them an entirely different color (an example of metachromatic staining) in regular brightfield. The fourth image shows many mitotic figures; the gut tissue is rapidly dividing. See how many you can find in the 4th and 5th images. The fifth image shows the mucosal, submucosal and muscular layers of the intestine. Note what I believe is a bit of nerve tissue (Meissner's plexus) in the submucosa (white arrow).
The final image is a compilation of 3 higher mag shots of dividing cells (mitotic figures) to show how varied they can appear depending on what stage of mitosis the cell(s) is at.
Optics: Orthoplan with NPL Fluotar Phaco 3 40/1.3 and Pl Apo Phaco 4 60/1.4 objs., variozoom eyepiece, 0.32X relay and D300. D cube used for the epifluor shot.
Yes, it would be better to use non-phase objectives for brightfield and fluorescence, but not having to move nosepieces filled with expensive objectives every time I want to use phase makes up for in convenience and lower stress what it degrades in contrast...
Enjoy, I am slowly processing my way through this duck's viscera. Of course, given my slow pace of specimen preparation, it will be duck season again (and again) before I finish.

David

[Cactusdave]

Excellent histology sections of a professional standard, well stained and photographed. It's unusual to come across an amateur interested in this area these days. Access to some of the (often toxic) chemicals needed in staining and processing would be very difficult in the UK without suitable 'contacts' in professional histology.

[discomorphella]

Thanks Dave,

I've tried to establish an embedding / staining process flow that minimizes use of toxics. In the US you can buy the solvents you need for paraffin or polyester wax embedding at standard home improvement stores (they sell acetone, various alcohols and xylene) and obtain dyes through ebay. The PEW is especially good for this, you can process the tissue with 95% ethanol or isopropanol, no aromatic hydrocarbons required at all for embedding. I do dehydrate tissue samples using cellosolve (toxic) for convenience, but I have proper gloves and a small hood to protect me. You can get away with just ethanol or isopropanol easily. You can also use nontoxic solvents like Histoclear and Histoclear II for paraffin embedding instead of the usual xylene. The formalin is the only really unavoidable toxic compound. My embedding "oven" is an old slow cooker controlled by a proportional controller using a thermistor probe. Hematoxylin (the blue nuclear stain) is a natural product and is pretty safe despite its ominous sounding name; Eosin Y, Phloxine B and Toluidine Blue O are also safe (relatively nontoxic). So you can process histological samples without undue risk or really serious expense. Mostly its the time required that limits my slide making. Modern labs have automated processing stations that transfer samples from one bath to another in the work flow (e.g, 50% alcohol to 70%, 90%, 95%, 100%....) automatically every few hours. I have tissue holders made from old coffee strainer mesh, and must have the free time to do things manually. But its worth it to be able to see what's really inside of animal, plant, fungal and even some material samples.
David