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Hot Topic: Identification of Hyaline Filamentous Fungi - Part 1 (Common Zygomycetes)



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Summary

Published: March 2014

Dimorphic fungi cause several common diseases including histoplasmosis, blastomycosis, and coccidioidomycosis. Dr. Roberts discusses the distribution and ecology of these fungi, as well as their pathogenesis and cultural characteristics. This is part 1 of a 6-part series.

Presenter: Glenn D. Roberts, PhD

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Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Our speaker for this program is Dr. Glenn Roberts, a professor of laboratory medicine and pathology and microbiology at Mayo Clinic as well as a consultant in the division of Clinical Microbiology. In this series, focusing on hyaline fungi, Dr. Roberts discusses perhaps the most common fungi that you will see in the clinical laboratory and a significant cause of disease in immunocompromised patients including transplant patients. This module examines common Zygomycetes. Thank you Dr. Roberts.

Thank you, Sarah for that introduction. I have nothing to disclose.

First, we’re going to discuss how to make mounts for a fungal culture so that we can make an accurate identification. This image you see here actually is representative of what happens in the laboratory. Many times where a plate is contaminated with many organisms and you need to figure out how to identify those that are present.

The next image shows you a schematic of what you might see. This is a drawing with fungi exhibiting all the different kinds of spores that might be produced, maybe not all, but a lot of them. It gives you an idea of what you might expect to find with certain other cultures. And it’s got, I kind of call it the universal fungus because it has everything there. You can notice in the center there is a tall stalk with a round sac at the top. This is a sporangium of a Zygomycetes. Then we go from there down to Penicillium, which is like about 2 o’clock, which has different types of sporulation and you can just look around in there and see that many of these fungi that sporulate differently and so we will begin to look at some of these as we go along.

Scotch Tape Preparation

The first preparation that can be used in the clinical laboratory and probably the most widely used is the scotch tape preparation. Basically, what you do is to take a piece of scotch tape and tear off a piece and fold it up so that the adhesive side is facing downward and what you do is you touch the colony with that piece of scotch tape, you stretch it out and place it on a slide that has a drop of lactophenol aniline blue on it. This scotch tape will then stick to the slide and it will allow the fungus to be stained with the dye that you see in the center there.

This is an example of where you might end up taking a scotch tape prep from the wrong place. Many times if you take it from the very center of the colony, that’s the oldest part of the culture and that’s where it sporulates the most heavily. In this case, that is what happened. You see all of these spores in here and it’s difficult to see because there are so many of them. The bottom line is what you need to do to make a good mount, is to make the mount from an area that is in between the outside, the advancing edge of the culture and the center of the culture growing up so it is kind of in the middle.

This is an example of what you would like to be able to see. This is an organism that has all of the spores attached to the conidiophore the way they were grown up. This is what the scotch tape prep allows you to be able to do. If you get it from the right place in the culture, you will see the spores that are attached just as they were growing in the culture. They are attached to this scotch tape, but then you can see exactly how they are produced and exactly how they look. In this way, you can get an idea of what it is that you are dealing with.

Wet Mount

Another kind of time-honored preparation is the wet mount. This is where you take a little bit of the colony, you cut it out of the agar with that wire that’s been bent at a right angle and you take a little bit of the colony along with some of the supporting agar and you place that on a slide with some lactophenol aniline blue. This is an example here of where you see the piece of agar that has been taken up along with the culture. One of the things you have to remember is that it’s easy to get too much of the supporting agar on the slide. If you do that, when you put the coverslip on, if it’s too large, what it will do is it will fly way out from underneath the culture slip onto the top of the bench where you are working and that’s not what you want it to do. So you have to take a smaller piece.

Here you can see the coverslip is going on there and it will be flattened out, unless it’s too large, and you’ll be able to see the culture, kind of as it’s been growing. The problem is, with a mount like this, is the spores don’t stay connected to where they were attached. The pressure that you put down where the pencil eraser or some other object on there to flatten that out, causes them to disassociate from the hyphae or the conidiophore that they’re produced on.

Slide Culture

Probably the scotch tape prep is the most universal one right now and the wet mount may be second. And then as a last result, we have in the past used what’s called a slide culture or micro slide culture. This is an example of what that is. Basically when you have a problem with a culture and you need to see how it produces the spores in detail, what you do is you take a plate of 2% agar, just water agar, and you place a glass rod that is sterile in there or you can just lay a slide on top of the agar like you see here. The slide is sterile and what you do is take a little bit of the culture and you cut out like a circle or like a square with a wire or with a sterile test tube, place the agar plug on the slide in 2 places, at either end. And then you inoculate the 4 quadrants of the plug with the culture. Then you put a coverslip on top of it and as it grows and produces spores just the way it does in a culture but they will be underneath that coverslip. And then what you can do when you think the culture is mature, is you can remove the coverslip, take it off, put it on a slide with some lactophenol aniline blue and look at it underneath a microscope and you probably will see the spores just as they have been produced underneath that coverslip. Sometimes you happen to look at it too early so that you don’t see things that you need to see and that’s why we have a second plug on there. You can go back and put a coverslip on top of that first plug and let it grow longer if you like too.

Here’s where you take the coverslip off and put it on a slide with a drop of lactophenol aniline blue and then take a look at it underneath a microscope. This is the cheap way to do it. It works well. You take a piece of filter paper, put it in a sterile petri dish, break an applicator stick that’s sterile in 2, and then put the slide on there with a couple of agar plugs and inoculate it. Put a coverslip on it and let it grow. And you put some water in the bottom so that there is enough humidity in there and that filter paper will absorb the water.

Common Zygomycetes

This presentation will focus on how to identify the common Zygomycetes. There is a movement afoot that has changed the name of the Zygomycetes back to what we used to call them. And they were called Mucorales. You will see in the literature and textbooks, some of the textbooks the term Mucorales and they are really referring to the Zygomycetes. We continue to use the term Zygomycetes because it has been used for so many years and it’s going to be difficult for everyone to change that, but regardless of that, at least you know that it’s going to be changed to Mucorales.

Pauciseptate Hyphae

One of the things that is the hallmark of these Zygomycetes is the fact they produce these rather large pauciseptate hyphae. And pauciseptate means that they have few septations, not very many, but occasional septation and they are usually described as being nonspeptate but they are really pauciseptate because we can with most Zygomycetes find a few of the septations and so that should not confuse you. This is an example of a wet mount that shows you these rather large hyphae that have no septate anywhere in them. If you just look at that 1 field, you would say, well, they are nonseptate. If you moved around in there you probably would find a few septations and we would describe it as pauciseptate. But that’s the point of looking at this is that you make sure that when you look at examining a slide that you need to look at many fields before you can get a consensus idea of what’s there. Don’t just look at 1.

Septate Hyaline Hyphae

There are some of these fungi that will produce septations regularly and we will talk about those in a subsequent presentation. This is an example of what the hyphae look like. They are smaller than the hyphae of Zygomycetes and there are a few septations in there. They’re a little difficult to see. There is a line dividing up the hyphal strands into compartments. You can see them in there about 6 o’clock up toward the middle, it’s curved a little bit toward the right and above that in the same hyphal strand and up at about 3 o’clock there is another septation. So, septations are easy to spot once you start looking for them.

This is an example of the small hyphae of the dimorphic fungi that are very difficult to interpret sometimes because they are so small. It’s hard to tell if there are septations there, but there are septations within this hyphae. We have hyphae that range anywhere in size, and the large ones are the Zygomycetes, down to the small ones that are the dimorphic fungi.

Mucorales (Zygomycetes)

Let’s talk about the Mucorales or the Zygomycetes. These have been defined in past years as the bread molds. They don’t grow on bread as often as they used to because we have too many preservatives in the bread, but they’ll grow on just about anything, on fruit, on vegetables, on lots of things that sit around for a while, even things that sit in your refrigerator.

Zygomycete: Macroscopic View

This is an example here of the fruiting head and the supporting structure of a Zygomycetes. The big round black balls that you see at the top are called the sporangia. Down below is the stalk that supports those and it’s called the sporangiophore. If you were to look at this underneath the microscope, you would be able to see this. You might not be able to see where it’s attached to the agar. It probably would not be present. This is a culture of a Zygomycetes. And it’s kind of defined as being wooly. It’s kind of a course looking appearance and has those black dots in there and the black dots that you see are these sporangia of these Zygomycetes. And here again you see the hyphae. They are large. This is to remind you that this is what you are going to see, large pauciseptate hyphae.

Now, we in the past have called these organisms Zygomycetes because they produce these spores that you see here as a result of sexual reproduction, that produce these spores called zygospores. Consequently, people named them the Zygomycetes. And I think the term Mucorales preceded the term Zygomycetes and so they have gone back to the older term and that’s kind of the rule of thumb in naming things in mycology. This is a drawing showing you what you might see with any of the Zygomycetes. It shows all of the structures that could be possible. The big sac-like structures that you see are the sporangia. Each one is a sporangium. The stalk that supports that is called the sporangiophore and then these organisms are attached to the agar surface or any substrate by, some of them by structures that look like roots and they are called rhizoids. If you find some that have rhizoids, it’s the way they are produced and it’s where they’re produced that helps you identify them. And so we will go into some detail about that as we go along.

Basic Structures of a Zygomycete

This is a schematic showing you the basic structures of a Zygomycetes. This is a real photomicrograph of an organism that we recovered from the back of some wall covering when some remodeling was being done near an oncology ward. This is something that you don’t want to see in that kind of a setting but, luckily, we were able to pick up on it early and contained that before too much organism spread around the environment. Down at the bottom are these rhizoids. Those are the rootlike structures that you might see with certain of these. The big black structure is the sporangium, the big sac that produces the spores and the stalk that supports those is called the sporangiophore. The term phore means supporting.

Zygomycete Sporangium

This is a scan of an electron micrograph that Dr. Dennis Kunkel allowed me to use for teaching. And this shows you what the sporangium looks like. It’s rough on the outside. The interior of that is filled with spores and you can see the stalk there, that is the sporangiophore. And here is a better photo micrograph scan, we have a scanned electro micrograph for this and basically what you are seeing here is the sporangiophore coming up and at the top of that sporangiophore is a structure that we call the columella that’s found in certain Zygomycetes. It looks like here that the sporangium is ready to pop and what you are seeing there are all of the spores that are inside that sporangium. It’s almost as if the wall of that sporangium has been removed and I don’t know quite how he took this photograph but it is very good because it shows you that what’s inside of that sporangium are billions of these spores that pop out into the environment.

This is another photograph that shows you the hyphae and notice that, you notice that most of them are very large. Every once in a while you will see a smaller hyphal strand as you see on the left hand side that does happen. What you are looking at it here, the kind of golden color thing is a sporangium that’s been popped open. The spores are sitting around on the outside and that thing sticking up inside of that big sporangium sac, that golden thing is called the columella. And you see those, when you make a mount sometimes of the organisms. This came from Dr. Kunkel as well. This is another example of a culture of a Zygomycetes. They don’t always produce these dark black spores as time goes along, some of them don’t do that. One of the hallmarks of these cultures of these Zygomycetes or Mucorales is that they grow very quickly. If you inoculate a culture at 4 p.m. in the afternoon and you come in the next morning, you will notice that the culture dish is probably already filled with the organism. And sometimes it will actually push the lid up and so the colloquial term we use for these things, they are called the lid lifters, because they push the lid right up, they grow so quickly. And I think the thing to remember about this is that they grow in this culture dish and also in the patient at the same rate, so it’s important to make a diagnosis as quickly as you can and to be accurate about it because they invade host tissue just like they grow in the culture plate, as quickly as that.

This is an example of a rhizoid. It’s just to show you in great detail. They are like root-like structures that actually adhere the culture to the agar surface.

Rhizopus

So the first one we are going to talk about is one called Rhizopus. Rhizopus has these sporangiophores, the long stalks that are produced singly or in groups. And the rhizoids are produced at the base of a sporangiophore. The long stalk at the bottom of the, you see the sporangia at the top of the big sac-like structure, you follow the stalk down at the bottom of the stalk will be these rhizoids. And when you see that you know you are dealing with Rhizopus. And the sporangia produce these columella that are kind almost like umbrella looking things inside of the big sac that you see at the top, the sporangium. This is an example here of Rhizopus. We’ll start at the top where you will see the big black sac, that’s the sporangium. Follow the stalk down, that’s the sporangiophore to the bottom and you will see the rhizoids or the root-like structures coming up right at the base of where the sporangiophores are. You see that, you know you’re dealing with Rhizopus. But the problem is, there are a lot of species of Rhizopus that are out there, and to try and put a species name on them is out of the realm of our discussion.

This is another Zygomycetes. This happens to be another Rhizopus and you can see that the rhizoids come off right at the base of where the sporangiophores come and is produced. This is Rhizopus. Another one of Rhizopus where the sporangium is actually broken open and you don’t see the sporangia wall any more. What you see are all of these spores that are popped out of that sac at about 10 o’clock. If you look closely, you will see a darkened area there, it looks almost like an umbrella, it’s shaped, that is the columella. And then going all the way down to the bottom, it’s a long stalk, that’s the sporangiophore and there the rhizoids are being produced right there at the base. So, we know that is Rhizopus because of those features.

This is the world’s largest rhizoid and I’m not quite sure why I decided to put it in here but it gives you an idea of what a rhizoid looks like. There are times when it’s difficult to try to determine what the organism is and these Zygomycetes. It’s difficult to make a scotch tape prep to identify them. So, what you end up doing is making a wet mount and try to take a small portion out. But when you do that, you still end up with a lot of organism, it’s all, they are all intertwined among each other and so you have to find the sporangium and the sporangiophore of one and follow it all the way to the end to see if there is a rhizoid produced at the base. In this case, they were. And this is another example of Rhizopus, but it’s kind of hard to tell that.

This is one where you just see the collapsed sporangium at about 3 o’clock, follow it but down there the rhizoids, and so the rhizoids are at the bottom of the sporangiophore and that’s an example of Rhizopus, again. And if you see something like this, you know you are dealing with that particular organism. Another example of a culture that is growing rather rapidly and you can see the black spots in there and those are the sporangia. This is a Zygomycetes and you can notice that this culture is getting filled already. This is a fairly young culture. This is another one that doesn’t show you those black dots, the sporangium.

Lichthemia (Absidia)

This is a culture we are going to talk about which is called Lictheimia and the old name for it is Absidia. And so the taxonomy of some of these Zygomycetes or Mucorales has changed and this is one example. This organism has branching sporangiophores, in other words the stalks that are produced that give rise to the big sporangium are branched. There are many sporangiophores and these are the stalks that produce these that have a funnel-like shaped base at the tip. And I will show you what that looks like. And it has a conical columella, with many of them, and particularly 1 organism, that this one, this Lictheimia, there is a septations that is produced down below the columella on that sporangiophore. And more importantly, this organism produces the rhizoids but it doesn’t produce a rhizoids right at the base of the sporangiophore. And when this organism is attached to the agar surface it produces a cluster of rhizoids and then there is a big branch that goes over and it forms another cluster of rhizoids. The rhizoids are actually produced in between the 2 big sporangiophores. And so you will see them not at the base of the sporangiophore, but you will see them in between 2 clusters of sporangiophores.

This is not a very good example, but you can see that there are sporangiophores and sporangias sitting on the right side and on the left side and in the center you have this cluster of rhizoids. So they’re not produced right below the stalk that gives rise to the sporangium, but they are produced off to the side and this is Lictheimia. This one here, you have to follow it down a little bit to be able to tell what it is. But if you start in the kind of off center where you see the rhizoids, you follow that long stalk all the way, you can see that there is a sporangium coming off in the middle there that’s just a very short one and there’s another one coming out pointing downward, coming at about 5:30. The rhizoids are produced way away from there. They are not produced down below. And that’s what you call, it’s called internodal production of these rhizoids. And this is Lictheimia or Absidia.

This is another example here, a little bit harder to follow, but if you see this sporangium in the center and if you follow it down to just a little ways, you will see there is a septations across there and then you follow it on further down you will see the stalk that goes into a hyphal strand and it goes off down to the right, somehow or other, it curls around and there are the rhizoids off to about 5:30 over there and there is still a part of that long structure and so the rhizoids are not produced right at the base, they are produced down way off to the side. Hard to see this organism, sometimes, and know what it is. This one shows you another example here. There are 2 sporangiophores, the sporangia at the top and you follow it to the left-hand side out near the rhizoids coming off, not at the base of those, but off to the side.

Mucor

And then we have another one which does not produce any rhizoids at all and this is the one that we see fairly frequently, it’s called Mucor. And Mucor has sporangiophores that are unbranched to branched and when they’re branched, the branches are produced on one side and then the other side and it’s called sympodial arrangement of the branches. There are no rhizoids with Mucor and so that helps you a little bit to be able to tell what it is. The name of the infection now that’s caused by these Mucorales is mucormycosis. It used to be called zygomycosis and again they have gone back to the old terminology and so it’s called mucormycosis and so we’ll call it zygomycosis just for traditions sake but know that it would be called mucormycosis in today’s terminology from some people. This is the culture plate of another Zygomycetes or Mucor. Now they don’t all look alike, some are gray, some are brown, some are black, so on. And this is an example of the 2 sporangia that are there and if you look around that’s all you are going to find are sporangia and sporangiophores. You’re not going to find rhizoids at all. And this is one where you see that big sac-like structure full of spores that’s the sporangium. The columella is that structure, dark structure in the center and then, at the base of it is the sporangiophore that produces it.

Sometimes you see these spores that look almost like chlamydoconidia in there and they’re just part of the Mucorales or Zygomycetes. They’re not diagnostic for them. Notice the spores in the background. Those are the spores that come out of those sporangia. There are numerous of those present and when one of those sporangia gets popped open, millions or sometimes billions of spores are released into the environment and this happens during the fall of the year, for example, when you are out raking leaves. These things are found in soil and on top of the grass and so on. As things begin to dry in the fall of the year and you are out raking leaves and everything, these things pop open and these clouds of these spores are just everywhere. In a normal patient, it doesn’t make any difference if you inhale those. If you are an immunocompromised patient, it makes a huge difference because this is a very important pathogen of immunocompromised hosts.


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