Special Stains: Giemsa Stain, Silver Stain, Reticulin Stain, Van Gieson Stain and more

by Geoffrey Meyer, PhD

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    00:02 I think we should be happy with H&E staining.

    00:05 That's going to be the bulk of your sections as they'll say it a number of times.

    00:09 But it does have limitations.

    00:12 Have a look at this slide.

    00:14 On the left-hand side is a partial section or a partial transverse section through a very small artery.

    00:25 In the middle, you see a whole heap of little, tiny dark bright pink, bright red in fact, stained cells.

    00:32 They're red blood cells passing through the lumen of this artery.

    00:38 The pink-stained area forming the wall of the artery actually contains a number of different tissue components.

    00:48 Going into higher magnification as you see on the smaller right-hand image doesn't give you detail of what these components are.

    00:57 In fact, in the wall of that artery there, there is smooth muscle, there is collagen, there is connective tissues, there is even elastic tissue.

    01:12 And you can't differentiate those tissues in a normal H&E staining section, and that's why I say at the top it has its limitations.

    01:22 But have a look at this slide.

    01:25 Using a different stain, you can get differential staining.

    01:30 Here, a trichrome stain is used, 3 stains are used together.

    01:36 And now look at the blood vessel.

    01:38 It's a similar section of the blood vessel and again, the red blood cells are a browny red in the middle, but you start to see bluish and reddish components in the wall of the artery and even on the outside of the artery, you see this rather flimsy bluish tissue.

    02:00 That trichrome stain is distinguishing between collagen which is blue and smooth muscle which is stained that browny red color.

    02:15 The smooth muscle in the artery wall is going to contract or relax and alter the dimensions of the artery and therefore, the amount of blood flow through it.

    02:24 The collagen is supporting the artery, making it strong so it doesn't burst under excessive blood pressure hopefully and normally.

    02:34 If you look clear into that, you'll see a wiggly little line towards the center or at least towards the lumen of the artery.

    02:42 That's the elastic lamina.

    02:44 So this differential staining introduces me to tell you that certain other stains that I'll briefly describe show you various components of tissues that enable you to describe in more detail what tissues are composed of rather than just relying on an H&E description.

    03:10 Masson's trichrome is a common stain that we use to distinguishing some components, particularly to distinguish muscle from collagen and you can read through that quietly on your own if you like.

    03:26 It just gives you the basis for what a Masson's trichrome stain does show you.

    03:33 Here on the left-hand side is a high magnification of what I described previously.

    03:40 Collagen or connective tissue is blue and the smooth muscle is the pinky reddish color.

    03:49 On the other side, on the right-hand side, a different trichrome stain is used, a different combination of stains.

    03:58 It doesn't matter what they are, but again, they show you collagen as blue, smooth muscle is a greenish yellow color, but it does distinguish that the artery wall is made up mainly of those 2 components.

    04:17 Here is a similar trichrome stain.

    04:20 This happens to be a very high magnification picture or image of the wall of the gut.

    04:29 The bottom part shows you 2 layers of smooth muscle, staining a light browny pink color.

    04:36 The connective tissue that supports the epithelium which is above the image and out of view is stained a greeny color, and the red blood cells can be seen in a blood vessel.

    04:49 They're staining an orangey reddish color.

    04:53 Another example of a trichrome stain and a different combination is used, again, the details I don't want to go into to show you this differential staining.

    05:07 Here is another example of a different stain used.

    05:11 In this case, a Silver stain is used and it's commonly used in looking at nerve tissue.

    05:20 It demonstrates processes of neurons and it also shows you the neuron cell bodies.

    05:29 This is a very dark stain section, but all those black thready-like structures you see scattered and stained a dark black color are nerve cell processes, axons and dendrites.

    05:44 You'll learn about that when you look at the Nervous System.

    05:47 Towards the bottom, you see a row forming an arc of very large stained Purkinje cells, and they're obvious because of the silver stain that shows off this dark black type appearance of their cytoplasm.

    06:06 And the browny stain shows you the nuclei of the neurons and other supporting cells in the background.

    06:16 Another Silver stain is used, it's not so common now, but it's used to visualize nerve fibers, particularly nerve fibers that are in the cerebellum, interneurons in the Nervous System, interneurons of other bulk of cells in the Nervous System that connect different neuron-transmitting networks together, and in this slide, you can see the neuron cell bodies and supporting cells, or glial cells, are stained a dark color, but you can make out very fine threads of the nerve fibers using this particular Silver stain.

    06:58 On the left-hand side is a section through the spinal cord.

    07:02 It's stained to show up myelin, and myelin is insulating, fatty material that goes around neurons—not blood vessels, neurons.

    07:18 It's just similar to the very fine piece of plastic you have wrapped around your copper wires.

    07:26 The copper wire transmits the electrical impulse and the plastic sheath around it is similar to the myelin sheath you see here.

    07:37 It insulates the nerve fiber just as the plastic lining insulates the copper wire thread.

    07:45 That myelin is stained in this section and on the left-hand side, you can see a central butterfly-shaped structure and an outer part of the spinal cord.

    07:57 It shows you, it distinguishes you 2 parts of the spinal cord, the inner gray matter and the outer white matter.

    08:08 And on the right-hand side, you can see all that dark stain material represents the myelin wrapping around cell processes.

    08:19 You can see a large neuron in the center staining a pale color and all that dark staining is the myelin stain, again, around all the neuron cell processes.

    08:33 Osmium is sometimes used to stain the lipid layers, particularly around myelin.

    08:42 If you look at all those little round circles in this image, they're in fact axons or cell processes.

    08:49 This is a section through a peripheral nerve, and know cell processes or axons could be sensory or motor nerve fibers travel long distances.

    09:00 I've already explained in a previous lecture.

    09:02 All this surrounded by this myelin which is secreted by a supporting cell called a Schwann cell, and as I said earlier, this myelin helps to insulate the cell process, the axon.

    09:17 It's retained, preserved by osmium whereas normally, it would be flushed out during processing if you didn't use osmium as first of all a fixative and later on, you can use it also as a stain.

    09:37 On the right-hand side is a large nucleus belonging to a Schwann cell and it's wrapping layers of myelin around that very clear structure, the nerve axon.

    09:54 And a particular nerve axon passing all the way down a peripheral nerve is myelinated by many, many Schwann cells.

    10:04 On the left-hand side, you can see a whole host of axons through a peripheral nerve.

    10:11 The axons appear as a tiny, little black dot and they have this white halo around them.

    10:19 That white halo represents where the myelin would be if it was fixed with a fixative such as osmium that retains that myelin.

    10:30 If it did retain that myelin, then you'd see an image similar to what you see on the right here.

    10:37 Inside, you'd see a black layer of myelin wrapping around those individual axons.

    10:45 Another stain commonly used in neural tissue is a Luxol Fast Blue or a Kluver stain, and it's also commonly counterstained with H&E.

    10:56 So sometimes, you can use a number of stains together or in combination to show up various components.

    11:05 And here basically, all the blue stained material you see belongs to the fibers, the myelin around nerve processes or it could be red blood cells and the violet or pinky, reddy, purply stained components represent the neuron cell bodies.

    11:29 Again, that's been counterstained.

    11:32 Here is a marvelous slide.

    11:36 On the left-hand side is a section through the prostate gland.

    11:42 It's a gland in the male reproductive system and prior to ejaculation, the prostate gland is going to contract and offer its contents, prostatic secretions, into the seminal fluid.

    11:58 Well, to contract, it needs muscle contracting within the gland itself and if you look carefully into that section on the left-hand side, you can see some epithelial cells that are making the secretory product, but you'll also see yellow-stained smooth muscle cells supported by collagen or connective tissue which stains pinky red, and that stain is brought about using the Van Gieson staining technique.

    12:29 Probably better shown on the right-hand image, this is a section through perhaps part of a limb.

    12:37 What you see on the bulk of this section on the right-hand side is yellow-stained skeletal muscle enclosed by little fine strips or coverings of red stained collagen or connective tissue, particularly on the right-hand side, and those thin pink stained connective tissue elements divide these skeletal muscle into muscle bundles or fascicles.

    13:10 You'll learn about this when you do muscle in more detain.

    13:14 But in this central area, you can see 4 or 5 transverse sections through blood vessels, arteries, and the wall of the artery again is stained yellow because the wall of these arteries, these are muscular arteries.

    13:33 The wall is predominominantly smooth muscle, so muscle, whether it's smooth or skeletal muscles, stain similarly in this particular section.

    13:43 But what's important is that you can tell the difference between the muscle around the wall of these blood vessels and collagen which supports the blood vessel, and that collagen is showing up in that red-stained component you see in this particular slide.

    14:00 So it's another example like we saw earlier with the trichrome, distinguishing muscle from collagen, and they are 2 tissues that are often very hard to distinguish in normal H&E sections.

    14:14 And that makes histology sometimes difficult to learn if you don't have the use of sometimes using these differential stains.

    14:27 Lymphoid organs such as the spleen, lymph nodes, consist of a very delicate network of fibers that are houses or platforms for a lot of immune cells to sit or attach to and do their job.

    14:45 You can see those fibers running through this tissue using what we call a Reticulin stain.

    14:52 It stains the reticulin fibers running throughout this particular organ, you know, the aorta shown on the left-hand side, the round circular structure using H&E.

    15:09 The aorta is a very important part of the Cardiovascular System.

    15:16 It's the blood vessel that accepts blood from the left ventricle and then it carries that blood to the rest of the body, the systemic circulation.

    15:29 When the heart pumps blood into the aorta, the aorta expands under the pressure of that contraction of the heart.

    15:38 And then when the heart is going through a resting phase and not exerting a high pressure, then the aorta recoils like an elastic band, like a stretched elastic band.

    15:52 It recoils and maintains pressure inside the aorta and therefore maintains flow of blood through the aorta when the heart is going through a resting or filling phase.

    16:06 You don't see that.

    16:08 The histological basis for that when you see the H&E section of the aorta, you see in the middle of this image.

    16:15 But you do if you stain it on the right-hand side with an Elastic stain.

    16:21 It shows you that the bulk of the middle layer of the aorta is stained with elastic tissue which takes up that black stain you can see.

    16:31 We earlier saw that the muscular arteries were mostly smooth muscle to change the dimensions of the lumen and therefore, the flow.

    16:41 Here, a lot of that smooth muscle is replaced by elastic tissue to do that recoil function I just mentioned, and the red staining material you see there is going to be collagen as well as some smooth muscle.

    16:58 The Elastic stains are very important stain in some tissues to expose all the elastic tissue within that particular tissue.

    17:07 PAS Reaction staining, as you can see on the left-hand side, is used in a number of situations.

    17:15 We saw it in an earlier slide to show you, in another lecture, to show you glycogen accumulating in cells in the liver.

    17:23 Here, the PAS staining reaction shows you the very fine basement membranes running and supporting these epithelial cells.

    17:36 Not so much that red structure in the middle.

    17:38 That's the glomerulus in a kidney and all the red staining and pink, there is basically basement membrane, but in the more peripheral part of this image, the light brown colored areas is the cytoplasm of the tubules within the kidney and these tubules made up of epithelial cell, are supported by that pink stained basement membrane you see.

    18:04 And sometimes if you look closely at the luminal side of these tubules, you'll also see the pink staining because the PAS reaction also stains parts of the glycocalyx that lines these epithelial cells.

    18:21 Finally, the last couple of stains I just want to show you is a blood stain.

    18:28 The Giemsa stain is used for staining blood cells and here, you can see red blood cells are stained and you know they don't have a nucleus, but the other 2 cells you see, you can see various stains show up various components.

    18:47 So nucleus is that bright blue, dark bluey color to violet color, whereas the cytoplasm is really a paler color and the erythrocytes stain is pink color, but the Giemsa stain is used to show up really the nuclei because you can see that the nuclei are quite different in some blood cells and that enables them to be distinguished, the nuclei.

    19:14 The Giemsa stain aloso shows often the different granules within blood cells and that again is used to differentiate some of the blood cells.

    19:25 It's a common stain for blood cells.

    About the Lecture

    The lecture Special Stains: Giemsa Stain, Silver Stain, Reticulin Stain, Van Gieson Stain and more by Geoffrey Meyer, PhD is from the course Histological Techniques and Staining. It contains the following chapters:

    • Special Stains
    • Masson’s Trichrome
    • Silver Stain
    • Myelin and Osmium Stain
    • Kluver Stain
    • Van Gieson Stain
    • Reticulin Stain
    • Elastic Stain
    • PAS Reaction Stain
    • Giemsa Stain

    Author of lecture Special Stains: Giemsa Stain, Silver Stain, Reticulin Stain, Van Gieson Stain and more

     Geoffrey Meyer, PhD

    Geoffrey Meyer, PhD

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