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Identification of Hyaline Filamentous Fungi - Part 3 (Septate Hyaline Hyphae)

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Published: May 2014

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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 3 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 septate hyaline hyphae. Thank you, Dr. Roberts.

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

First, we are 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 of 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 of the 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 and 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 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 this 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 it a good mount is to make the mount from an area that is in between there, the outside advancing edge of the culture and the center of the culture growing up so it’s 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 the scotch tape and then you can see exactly how they are produced and exactly how they look and in this way you can be able to 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 and you cut it out of the agar with a wire that’s 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 can 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 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 will be able to see the culture kind of as it’s been growing, but 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 with a pencil eraser or some other object on there to flatten that out causes them to disassociate from the hyphae or the conidiophore that they are produced on. Probably the scotch tape prep is the most universal one right now, and that wet mount may be second, and then as a last result we have in the past used what’s called a slide culture, a microslide culture.

Slide Culture

This is an example of what that is. Basically, when you have a problem with a culture you need to see how it produces the spores in detail. What you do is to take a plate of 2% agar, it’s just water agar, and you place a glass rod that’s sterile in there or you can just lay a slide on top of the agar like you see here. The slide’s sterile and what you do is you 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, 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, it produces spores just the way it does in the culture but they’ll 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 the 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 to.

Here’s where you take the coverslip off and put it on the 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 top and let it grow. And you put some water in the bottom so that there’s enough humidity in there and that filter paper will absorb the water.

One approach to identifying the hyaline septate molds is to look at the way they sporulate and how, see how the conidia are produced and how they are arranged. And we’re going to start off with the simplest mode of reproduction of the fungi and that is with the production of arthroconidia. We will look at some of the fungi that will produce arthroconidia and see how they are produced and see how they are grouped together. So we will start off with septate hyaline hyphae and if you see conidia or arthroconidia produced in clusters, we will go that direction.

Septate Hyaline Hyphae

The first one we will talk about is Geotrichum. Some people pronounce it Geo-trick-um. In this case you see hyaline septate hyphae and you find arthroconidia that are produced within those hyphae and there are no budding cells produced at all and just all you are going to see are rectangular arthroconidia lined up in a row.

Geotrichum

This is an example of just that where you see the rectangular arthroconidia lined up within the hyphae and this is the simplest mode of reproduction of the fungi is to produce arthroconidia. And it’s a nonspecific trait for most of the fungi, but you find consistently large numbers of these like we’re seeing here then you know you are dealing with Geotrichum. Here you see arthroconidia that are more rounded rather than rectangular and that does happen with some of the cultures, however, you can see about 6 o’clock you can see one that is rectangular, some are round, some are oval, some are rectangular but nevertheless they’re in chains, they’re at least in a long linear fashion within a hyphal strand.

Here you see an example of rectangular arthroconidia. They’re very long actually and they are just lined up. They’re lined up one after another. You’ll see in the dimorphic fungi that there are alternate arthroconidia where there’s a space between each arthroconidia and in this case with Geotrichum, you do see a little space between them but it’s not what we call alternate arthroconidia, they don’t produce a dead cell in between them, these are just arranged in a linear fashion. Here you see some more arthroconidia and they just kind of disassociated from each other but this is an example of Geotrichum.

This is a cornmeal agar plate. If you have looked through a microscope, look at a cornmeal agar plate. Some places still look at yeast as they are grown on cornmeal agar and you have to be very careful when you’re doing this because you never know what it is you are working with and it’s kind of risky to look at a culture without working on them under a hood. However, the cornmeal agar you have to just look at it through the microscope or look at the morphology through an objective without a coverslip and sometimes you put a coverslip over an area that’s been streaked. But what you see here are arthroconidia in chains and that would be an example of Geotrichum. One of the difficulties is that we still see an organism called Trichosporon and Trichosporon is a fairly common cause of disease and it produces not only arthroconidia but it produces blastoconidia, and blastoconidia are just budding cells so you would look around and see if you see any budding cells. And this would make a difference in what you would call the organism.

This is an example here where you see the arthroconidia that have been totally disassociated and some of them have germinated. And they almost look like a hockey stick and this is a nonspecific trait but it does help to put you in a ballpark when you’re looking at one of these organisms and it’s kind of characteristic of Geotrichum. This is another example of the arthroconidia of Geotrichum and you can see here that these here are more rounded, yet they are still arranged in a linear fashion within the hyphal strand. So don’t be surprised if you happen to see some that are not rectangular.

This is a culture of Geotrichum and many times it is mistaken for being a yeast because it has this glabrous smooth appearance to it, a yeast-like appearance to it, and people get confused and think that it is a yeast. And it’s a yeast-like organism. And here you see what happens if the culture grows a little longer. It becomes very powdery. And so it is truly is a mold. It’s not a yeast. But initially, it does look like a yeast.

This is another plate. You can see this plate is a totally confluent growth in the whole plate and it looks a little feathery around the edge. I don’t think it’s necessarily characteristic of Geotrichum, but at least you can see that they don’t all look alike. And here’s another one where it’s kind of powdery. And when you see cultures that are a bit powdery like this one, it usually means that they are producing large numbers of spores.

Let’s talk about the fungi that produce conidia in different ways than what we just talked about other than arthroconidia. Some are going to be producing clusters, some that are produced along the sides of the hyphae and this diagram you see shows you a long stalk of a conidiophore with a cluster of conidia at the tip and there are a number of organisms that produce conidia in clusters like you see here. And there are ways to differentiate them from the hyaline molds. There are times you see nothing more than just conidia being produced right off the sides of a hyphal strand, sometimes in the same culture you will see some of the spores are large and some are small, hence, you call them macro and micro conidia so you have to look at everything when you try to identify these organisms.

So we’ll look and see what we can come up with. This is a culture plate, here, of an organism. And what’s happened is, you can notice that it looks kind of yeast-like. But in the center, you see some things that are kind of look like micro-colonies in there. Those are probably colonies of bacteria that are sitting on top there. And here you see clusters of conidia, just like you saw in the diagram a minute ago, that are produced at the tip of a long conidiophore if you will. Conidiophore is a general term. When we see an organism like this, the long stalk more than likely is going to be a specialized structure called a phialide and the phialides gives rise to conidia and they are produced in clusters like you see here.

Acremonium

What we are going to talk about is Acremonium. Acremonium has these long tapering phialides that sometimes arise just from a hyphal strand or from bundles of hyphae. Collection of hyphae that you see, the hyphae that you see are very small, very delicate, sometimes they are all grouped together in bundles. You find conidia that are spores that are produced in balls at the tip of these phialides and if you look closely, you will see that there at the tip of the phialides. They are produced in slimy balls. There is some gelatinous type of material that holds those spores together in that cluster and they are easily disassociated when you make a mount trying to look at them underneath a microscope.

So this is an example of Acremonium and sometimes what you see is, what you see here is, you are looking down on top of these phialides with the clusters of conidia at the top. They look like balls sitting there. And if you follow it down, there is a long phialide going all the way down to a hyphal strand and they don’t always look so good.

This is a better example of what it would like typically. These are long phialides and at the tip there are clusters of these elongated conidia that are in a gelatinous mass that holds those spores together. If you look at the hyphal strands that go across from about maybe 7 o’clock to maybe towards 3 o’clock or 3:30, you’ll notice that some of those there are grouped in bundles right there, if you look closely you will see that there are several hyphae that are just kind of laying on top of each other. And that’s what we talk about with them bundling and these are small hyphae and they look like the hyphae of a dimorphic organism and there is some association between these organisms molecularly and in terms of genetics of these organisms. This organism, Acremonium is a known pathogen and we have recovered it actually from blood cultures and so it’s caused some systemic disease.

This is a larger view showing you the bundling when we were talking about going all the way across and left to right and right to left and then you see the phialides that give rise to the clusters of conidia at the tip. And when you make a mount like a scotch tape preparation, those conidia generally stay together in that cluster, but if you make a wet mount and try to put a coverslip on top of a piece of the agar and so on and push the coverslip down, those conidia actually spread apart and it’s hard to tell that they ever were in a cluster at the tip.

This is another one showing you a long stalk, a long phialide, and then you see the conidia sitting there in a cluster and they’re elongated. Now we have to worry about another organism; one called Fusarium that will produce similar type arrangement of spores or conidia in there. And so we’ll have to deal with that one. If you notice this culture has a bit of a lavender tint to it, kind of depending on the angle that you look at it.

Fusarium

This is a culture of an organism called Fusarium, which also produces these conidia in clusters at the tip of a long stalk or a phialide, and Fusarium produces 2 types of conidia. One group would be larger and the other is smaller. Macroconidia are the large ones and they are kind of canoe-shaped and the microconidia are small and oval, a bit like you see with Acremonium. The conidiophore is kind of short and has a phialide. It has a taper at the tip of it. Sometimes you look at the end of it you will see that there is a little bit of a collar or a ring around it but that’s not very obvious and they are called collarettes. The macroconidia, the large ones have 1 to 5 septations in it so they are divided up into compartments, and they have a foot cell that attaches it to the conidiophore. And microconidia are kind of kidney-shaped and they’re much smaller and they have 1 or 2 septations, so, they’re different then Acremonium but sometimes they are confused.

Not here’s a better one. You can see the lavender color of this. Fusarium produces kind of a reddish to purple color and sometimes it just produces a colony that’s yellow. Another one of those examples where looking at the colonial morphology doesn’t always tell you the story, it doesn’t always help you all that much because they vary so much. This is Fusarium. And at first glance, it would look like Acremonium. These conidia that you see at the cluster, in a cluster at the tip of this long phialides, are more elongated in shape. And they are like macroconidia of Fusarium.

This is a long phialide and if you look at it you will see that it’s pointed at the tip and then at the tip of that phialide you’ll see this cluster of conidia, macroconidia that are kind of canoe-shaped, they’re not so much so in this particular view but they are in real life. But you see they’re in a cluster and so at first glance somebody would say that looks like Acremonium, well, it’s not, it Fusarium. And this is Fusarium and there is a good example of the bundling of those hyphae. You see they’re small and they’re as small as a hyphae of a dimorphic organism and you can see the phialides in there some of them do not have anything at the tip of them at all, the spores have been knocked off. At about 8 o’clock, you can see that there are some tapered phialides and the conidia are in a cluster at the tip of those.

And here is Fusarium that shows you a very good example of these canoe-shaped macroconidia. These are very long as a matter of fact. Notice the hyphae are septate, but when you start looking at the conidia, the spores that are in there, you will see 2 things. One is you see those chlamydoconidia, those ones at about 8 o’clock getting toward 9 o’clock, the 2 rounds cells and they hyphal strand, those are nonspecific spores for the most part. Although, there is one Fusarium that is characteristically produces a lot of these chlamydoconidia. But for the most part what we are going to see are macroconidia like you see here and these are very elongated and canoe-shaped as we mentioned before. And so this is what you would see with Fusarium. Fusarium is a very important pathogen and we’re seeing it more and more often in immunocompromised patients, particularly those who have had organ transplants, and cause some very serious disease and they have a lot of tissue invasion and then a lot of necrosis and dying of cells occurs as a result of this organism being present in the tissue. So this is one you need to really know about and if you find it from a normally sterile site, you need to make a phone call real quick.

This is a cluster of those macroconidia at the tip of the phialide. It’s kind of hard to see what the phialide, the top of the phialide looks like, but they are the macroconidia and they are not as obviously canoe-like as the ones you just saw. But here you see some that are. These are just macroconidia of a Fusarium.

There are many species of Fusarium and if you’re asked to put a species name on these, I would probably try to avoid doing that at all cost as it’s too much trouble and it takes a lot of time and effort and if you don’t have the expertise, you probably won’t get it right anyway. So, there’s no need to probably do that clinically unless someone wants to write a case up. What you see here are just examples of these macroconidia that are actually multiseptate and they are canoe-shaped and they vary in length. And you can see this one is very detailed macroconidia. They are kind of pointed on the ends. You can see the number of septations there.

And this is a slide that’s been stained with another dye that’s not lactophenol aniline blue and you can still see the same structures in this one. You can see that those macroconidia are extremely long, but they’re curved on the ends and sometimes they’re pointed on the ends too. Fusarium is one that you will see in everyday life. It’s a common organism to find in the respiratory tract specimens and so you will see it. It’s not always the cause of disease but I think from a normally sterile site you need to make sure that the clinician knows about it so that they can determine if it is significant or not. This is a culture where it looks like one that is dark and almost lavender in appearance. And here you see one better with kind of the lavender appearance to it too.

And here’s one that has some yellow present and also some red. The reverse side of these plates, sometimes, it is important to look at those, at the back of the culture, and with one of these Fusarium cultures that produces that kind of red colonies, you can see their backside is red. And more times than not, Fusariums some of these Fusariums will be red.

Trichoderma

That’s what Fusarium looks like. And now we’re going to look at another one that produces conidia in clusters, however, it is not one that we see all that often. It’s one called Trichoderma and Trichoderma produces green colonies, but it produces conidia that are in slimy masses at the tip of a phialide kind of like what we talked about with Acremonium and Fusarium. Conidiophores are hyaline and they’re branched. The hyaline just means they are nonpigmented. The phialides are inflated at the bottom and the conidia are produced in clusters attached at right angles to the conidiophore and then the conidia are produced in slimy masses at the tips of these phialides. So we’re going to look at it. This is slightly out of focus but what happens is, these phialides are produced on the long stalk, come off of a long stalk, and they’re pointed on the end and then they give rise to a cluster of these conidia like you see here. They’re more rounded than anything else, not elongated like Fusarium.

And here you can see Trichoderma. It almost looks like Trichoderma produces these phialides in a whirl. They’re coming off kind of all the way around that whole thing almost like a tree like you see. The one on the right-hand looks almost like a tree, if you have a vivid imagination. This is a culture of Trichoderma and it is lawn green. And that’s characteristic of that organism, so if you see a morphologic feature like you just saw and you look at the culture and it is lawn green, you’re going to know that it’s Trichoderma.

This is another one showing you just another colony variation. And now were going to look at another organism. This is one that produces conidia in clusters in a gelatinous mass at the tip of some structures and it’s not a single phialide, it’s a cluster that look like phialides that are produced that didn’t give rise to this cluster of conidia. And this is one called Gliocladium. And it’s one that’s not very often seen, but it is kind of one that I think if you see it once, you won’t forget what it looks like.

Gliocladium

It has conidiophores that are kind of branched in the upper part and so it gives rise to a cluster of these phialides rather than single ones. And the conidia that it produces at the tip are one-celled, they’re produced in slimy masses just like the other 2 things that we talked about at the tip of these phialides. So we’re still in that group. And here you can see at the top, there are a cluster of these phialides and the phialides are not long tube-like structures at all, they are more vase-shaped. They are rounded and inflated at the bottom and they’re kind of tapered at the tip and they come off all the way around. There is a cluster of those, and then what’s happened is a cluster of conidia is produced at the tip of all of those phialides. And I think once you see Gliocladium you won’t forget what it looks like.

And here’s more of the cultures produced a huge number of conidia. Down below, the mass of conidia would be those phialides that are arranged kind of in a cluster. And you can see here, this is another one that is kind of lawn green.


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