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Identification of Hyaline Filamentous Fungi - Part 5 (Aspergillus species)

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Published: July 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 4 of a 6-part series.

Presenter: Glenn D. Roberts, PhD

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Welcome to Mayo Medical Laboratories Hot Topics.  These presentations provide a 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 Aspergillus species.  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 of the different kinds of spores that might be produced, may be 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 what I call 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 zygomycete.  Then we go from there down to Penicillium, which is seen at about two o’clock that has a different type of sporulation and you can just look around in there and see that many of these fungi sporulate differently 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 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 making a scotch tape prep from the wrong place.  Many times if you make it from the very center of the colony, the oldest part of the culture and that’s where it sporulates the most heavily.  In this case what happened, you see all of these spores in there 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; 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 make 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’re 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 onto 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 cover 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 is too large and you’ll 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 the wet mount may be second and then as a last resort we have in the past used what’s called a slide culture, a micro slide 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 two percent 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 take a little bit of the culture and you cut out a circle or like a square with a wire or with a sterile test tube, place the agar plug on the slide in two places, at either end.  And then you inoculate the four 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 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 two and then put the slide on there with a couple of agar plugs and inoculate it, put a coverslip on top of it and let it grow.  And you put some water in the bottom so there enough humidity in there and that filter paper will absorb the water. 

Aspergillus species

This presentation will address the issues of identifying Aspergillus and some other organisms.  Aspergillus species - the number of species of aspergilli are common and you will encounter those in the clinical laboratory, so we will present the ones that are the most commonly encountered and the others probably will not be covered.  So Aspergillus species, just a general description of the organism, the conidia the spores are produced in chains at the tip of a phialide and a phialide is a structure that actually produces the spores within it and then pushes them out and they remain in chains.  Sometimes the phialides are on top of a structure called a metula and so there will be some species that will produce these metulae, these branches or these extra pieces before the phialides and some of them will not be produced and that is helpful in the identification.

Aspergillus fumigatus

So we will start off with Aspergillus fumigatus and that is the most common species of Aspergillus that is recovered in the clinical laboratory.  It is a known pathogen but it’s also an organism that colonizes our respiratory tract and often times, doesn’t cause any problem at all unless you happen to be immuno-suppressed.  So with Aspergillus fumigatus and some people pronounce it as Aspergillus fum-i-got-us, I think it’s entirely up to you as to how you decide to pronounce this organism’s name..  This organism, Aspergillus fumigatus, has a vesicle that is hemispherical.  The aspergilli produce a vesicle at the tip of the long conidiophore and some of them are round, some of them are hemispherical, some of them are other shapes.  This organism is uniserate , which means that it produces phialides on top of the vesicle without anything underneath them, any branches, any metulae at all underneath them.  The phialides cover the upper one half to two thirds of the vesicle surface for this organism and if you would like to determine if it is thermotolerant or not, you can place it at forty five degrees centigrade and it will still grow.  This is an organism that is known to grow in moldy hay and moldy grain and produce problems for people who encounter the organism and inhale it. This is a slide of Aspergillus fumigatus and it turns out that most of the time it’s kind of blue-green but there are times when you see colonies that are a little different in color and some of them are actually white with just a tinge of green.

Another one here you can see is kind of gray-green.  Depending upon the medium, you’ll see different colors for these colonies. This is an example here of Aspergillus fumigatus on three different media containing blood and you can see that the colonies look almost glabrous or smooth and they are not so fuzzy like you see on other media and this organism of all of the aspergilli is susceptible to a compound called cycloheximide.  And cycloheximide inhibits the rapidly growing molds so that one can recover the slower growing molds like Histoplasma and some of the other dimorphic fungi.  The problem is that cycloheximide actually inhibits the aspergilli and that may be the very thing we are trying to look for so you have to use a medium with it and without it so that you ensure that will be able to detect this pathogen. This is another one here, that’s almost turning a little bit white.

Typical fruiting head of Aspergillus

And here are structures of the Aspergilli, just in general.  And if you look on the right hand side you will see the structure is labeled conidiophore.  That is the long stalk that supports the fruiting structures for this organism.  At the tip of that structure, the fruiting head is that vesicle and you can see that vesicle where you see fruiting head, if you look right down and you’ll see vesicle is at the tip of that conidiophore is hemispherical and then there are these phialides that may be produced right on the top of that vesicle that produce the conidia, the spores. This is a slide showing you the structures of Aspergillus fumigatus as it begins to grow and it starts off with the really tiny structures but you still can see the vesicle there in the darkened area there in the center of those two heads. And as time goes along, they begin to form these phialides and the phialides are bottle like structures and then the spores are produced within those and then are produced in chains.  But you can see here  on the right hand side of the vesicle is, it is hemispherical and you can see the whole half or two thirds of that vesicle is covered with those phialides, those are the structures that go around it there.

And you see that one that is growing a little bit more, it is a little bit more mature in the center and then you see the large vesicle there with the phialides on top. The upper one half to two thirds of that vesicle, is covered with phialides. And here you see the mature fruiting head of Aspergillus fumigatus and if you follow the conidiophore, you can’t really see it well, but you can see the enlarged area at the tip there, that’s the vesicle and then the phialides are on top of that and then the spores are produced in chains.  And notice in the background, that there are many conidia sitting out loose out there floating around in this mount.  If you use the scotch tape preparation, you probably will be able to see some of the conidia that are produced in chains. Otherwise, if you use a wet mount,  you are going to destroy the integrity of the structures as they are produced.  So this is a good example of the scotch tape prep.

Aspergillus flavus

We’re going to talk about another Aspergillus and this one happens to be an organism called Aspergillus flavus, an organism that has a couple of different things about it.  One is that it seems to infect people with leukemia and we see it fairly frequently.  The other thing is that it resembles a Zygomycetes in some ways and that is that the hyphae are generally very large and sometimes they don’t produce a lot of septations and so when you look at a direct microscopic examination of a specimen that has Aspergillus flavus in it, you have about a fifty percent chance of getting it right or wrong when you try to identify it.  So, Aspergillus flavus also has a characteristic in common with the zygomycetes and that is, that it grows at the same rate, which is rapid.  So Aspergillus flavus has these vesicles that are large and they’re spherical so they are nice and round.  They produce biserate structures.  There are  metulae underneath that phialide that’s produced on top of the vesicle so you have a vesicle surface, you have this branch and a friend of mine one time described Aspergillus as this, he looked at the structure, and he said, “It looks like you have a basketball that would be consistent with the vesicle and then on top of that,” if it was biserate”, he mentioned, “ you would have a brick or a two by four and then on top of that you would have a pop bottle and on top of that you would have tennis balls coming out.”  And that’s a good way to remember what Aspergillus flavus looks like.  So this is biserate, which means it has like a brick or a branch before the phialides are produced and the conidia of Aspergillus flavus are often times rough and they’re often times pigmented too, a yellow-green kind of a pigment.  This organism is one that you will see in the clinical laboratory.

This is a culture of Aspergillus flavus and typically it’s described as kind of a yellow-green culture or khaki color.  It grows very quickly just as I mentioned before, like a Zygomycetes.  It will grow very fast and in the next day or two you will see the whole plate get filled up with the culture itself. This is a good example of Aspergillus flavus and you notice how rough it is.  The reason it’s rough is, if you look closely you will notice there are little dots all over that whole culture plate that are the color of the organism.  Those are the heads, the fruiting bodies of Aspergillus flavus.  They are that big. Aspergillus flavus also causes what is called external otomycosis and it will involve the external auditory canal of the ear.  And what you see, really, is those heads,  the fruiting bodies, protruding from the side of the ear canal and if you look with a stereo microscope, you can see them sticking out and it’s growing just like it grows on this culture.  And as a clinician breathes, you can see those things move, so they are there just like they are in a culture.  Now this culture is a typical, pretty typical for Aspergillus flavus and what you need to do is to make a mount to determine exactly what the morphology is and you would make that mount from somewhere between the center and the outer border of the culture because if you look at this particular plate, you’ll see it is very kind of granular, that means it has lots and lots of spores. 

This is another culture of Aspergillus flavus and it looks about the same.  A little bit of white on there and sometimes that white is described as being pleomorphic and if you subculture it from that area it will remain and grow as a white mold and not as the mold as you see it here. Here is just another one.  This actually came from a respiratory tract.  If you look in the background you will see there are many colonies of other things in there and some of them are probably are Aspergillus flavus, but maybe not.

This is what you would see if you looked underneath a microscope.  Sometimes it is very difficult to see the vesicle on these because there are so many phialides surrounding the vesicle that give rise to chains of conidia that it obscures the view of the vesicle.  So any way, you can look at it and if you look closely you will notice that it’s round, darkened area in the center there is round and all those things you see in the background there are the conidia or the spores of Aspergillus flavus. And they are usually rough-walled.  And they are usually in chains.  But when you make a mount, a wet mount, then it destroys the integrity of the way the culture is growing and the spores are just sitting out in the background and you can’t see exactly how they’re produced.  Look on the left hand side, lower left hand side.  You notice the size of that hyphal strand there.  That’s the same size as a Zygomycete. But here you can see in the center you can actually see the round vesicle and it’s difficult to see if there are metulae or not but you can certainly see the phialides.  If you look closely, actually the metulae are there.

This is another slide showing you kind of the color of the culture when you look at it underneath the microscope and you can see that it’s kind of a golden color or maybe reddish brown. And there is the head of that Aspergillus flavus.  The vesicle is in the center and then you can see it’s obscured.  It’s really difficult to see the morphology of this organism because it’s a big round ball and when you make a mount of it you are looking at it and trying to look at it in another dimension.  And it’s hard to get everything in focus at the same time.

Aspergillus flavus

This is a slide that I have put together that shows the biserate nature of Aspergillus flavus.  You can see the round structure of the vesicle.  If you look at the left hand side you see the round vesicle, there is a structure that is kind of like a brick called a metula and at the tip of that if you look at it you’ll notice there is a phialide which is a bottle like structure and that gives rise to spores and there is a single conidium or spore at the tip of that phialide.  So this is what we mean by biserate.  You look at all the keys for Aspergillus and if someone asks you to key one of these organisms out, it’s difficult to do.  But the key usually starts off to ask you if it’s uniserate or biserate.  This means you have to know what these structures look like and how they’re produced.

Aspergillus niger

Another one, Aspergillus niger.  This is one that you will see in the clinical laboratory very often.  It has conidia that are roughened and they’re black.  They’re jet black.  The vesicles are round, they’re spherical.  The conidiophores that produce this and the vesicles at the tip of that are colorless.  And this organism is biserate.  This organism is kind of unique too in that it may cause disease in humans.  It generally colonizes an old pulmonary cavity.  That’s about all that it does, it’s not something we see every day.  Another place you can see Aspergillus niger is if you were to culture the black pepper that you have on your kitchen table, you would find Aspergillus niger is in that very commonly but that’s not what makes it black. This is a lower power view of Aspergillus niger and you can see that it has those big round vesicles again and when you see those, it’s really difficult to see the morphology because you are obscured by so many spores and phialides on there you can’t tell anything about the morphology on here.  However, if you look at the big round structure, the fruiting body on the left, you can see a small chain of conidia there to show you they’re produced in chains and then on the right hand side, they’re also produced in chains.

This is a plate of Aspergillus niger and when it starts off growing, it starts off as a yellow colony and as time goes along then it begins to turn dark and you can see it looks granular and the reason for that is that’s where you see the other fruiting bodies with all the spores being produced. Another Aspergillus niger, and the border looks white which means it’s young, the youngest part of the culture and in time it will turn black as well. This is one growing in a blood containing medium and you can see the colony looks the same.  It has the same outer border and it has the dark center.  And then here’s another showing you the black nature of Aspergillus niger.  There are several species of Aspergilli that will turn black.  This is the most common one.  And then this again shows you the big vesicle up at the top of this thing.  It’s round, it’s covered with the phialides and metulae but you can’t tell on this slide.  My point is it’s hard to see. And here you can see it better.  If you look closely you can see the vesicle is round in there.  It’s very difficult though to see beyond that.  You may be able to see the phialides there a little bit but it is also very dark to see, and all the conidia are in the background.

And this one is likely what you would see.  It’s an obscured view of what we’re looking for. And here’s another, you can see the round vesicle and if you look closely at the peripheryy of that round vesicle you can start to see some structures.  And if you have the ability, which we don’t here, to focus up and down, you would see the brick like structure and then you would see the phialides being produced. I took this photograph to show you what you would see.  I knocked these phialides and metulae off of a fruiting body.  I guess if you’re bored, this is something that you could do.  So anyway, I took a wet mount and I knocked the structures off of the round vesicle, and you see them here in the center.  And what you are seeing there, start at the outer edge, there is a phialide that looks like a soft drink bottle and then down below it, you would see on the right hand side there is some other structures there that don’t look like that bottle like structure, those are the metulae.  And they were attached to the round vesicle and then on top of that would be the phialides, and you can notice that  there are two of them, you can see that one of them has a spore, on the right hand side, that is coming out of it and the other one has a spore probably sitting at the tip of it.  This is what we are talking about are these kind of structures that you would see with Aspergillus niger and the other aspergilli.

Aspergillus terreus

This is Aspergillus terreus;  we are going to talk about and it has vesicles that are fan-shaped, kind of like of the old paper fans some used to use.   It has a biserate head.  The upper two thirds of this vesicle is going to be covered with spores and they are generally in chains like you see with all of the Aspergilli except this one has something different.  It produces these oval to spherical aleurioconidia or aleuriospores that are found down on the hyphae that are submerged in the medium.  It’s difficult sometimes to be able to see these and when, it‘s also difficult to know when to go look for them because you don’t look down in the agar for the spores of many of these.  But with Aspergillus terreus you probably would and it happens to be one that is, looks like powered cinnamon. And you can see this culture here has a cinnamon like appearance to it. And here you see the fan shaped vesicles on the left hand side; you see two of them.  One facing about maybe ten o’clock and the other one is down below it.  You can see that the vesicle is fan-shaped and it has phialides and metulae around it.  You’d have to focus up and down to be able to see that.

And here you see them better.  You can see the fan-shaped vesicle on there and if you focused up and down you would be able to see what kind of conidia there are that are present. Now, this next slide shows you essentially the same thing but I believe you can see the vesicles a little bit better.  What we are going to be looking for with this organism is if we see its cinnamon color, we are going to be looking for the aleurioconidia. So you make a mount down in the agar and there is what you’re going to find.  You’re going to find hyphae that are submerged into the medium.  You’re going to find spores that are produced on the sides of these hyphae that are bigger than the conidia that are produced on the fruiting head.  So, they’re larger and they’re found only submerged down in this agar and they are called aleuriospores or aleurioconidia.  And they are produced on the sides or the ends of the hyphae that are there and whenever they are produced and when they break off, they take part of the back wall of what they are attached to along with them.  That’s why they call them aleurioconidia. And here you can see one in the center, then you can see the base it is attached to and when it breaks away from that, it will break part of that supporting structure off with it.  So you can see these are aleurioconidia.  They’re found down in the agar and they’re not anywhere on the fruiting head and you can see here these are still aleurioconidia.

And this is a culture of Aspergillus terreus. And another one showing the powderered cinnamon appearance of it. And this shows you something that you see with some of the aspergilli.  This happens to be one of the fruiting heads of Aspergillus terreus but if you look at it from the top to the bottom, notice that the bottom, it has a structure that looks like a foot, and that’s called a foot cell.  And Aspergillus is known to produce foot cells.  Aspergillus terreus is one of those ones where you see it well and Aspergillus nidulans is another where you see it very well. And here again is a structure that you can see the fruiting bodies at the top and if you follow all the way down to the bottom, there is another foot cell.  This is what we see with Aspergillus terreus.

Aspergillus nidulans

We’re going to talk about Aspergillus nidulans, which is one that is a bit different from the others that we’ve talked about.  It has conidiophores that are small, kind of brown pigmented and the foot cells are really pretty obvious.  It is biserate.  It produces the metulae and phialides on the upper one half of the vesicle surface.  It also produces ascospores as a result of sexual reproduction so you will see cleistothecia, big round balls full of ascospores in this culture and there are some other structures in there that we don’t know quite what they are, what they mean but they are present with Aspergillus nidulans and they are called Hülle cells. So this is an example of a foot cell again, a very good one.  And then if you look in the background of that foot, so you see there are some round structures and we are going to look at those with some more detail.  Those are the Hülle cells but notice the hyphae are small and the conidiophores pigmented and has a foot cell.

And this is an example of the foot cell. These are round structures are called Hülle cells.  I don’t know what their purpose is but they’re found with certain of these with Aspergillus nidulans and Aspergillus versicolor and so they’re much, much, larger than the conidia that are produced.  And they sometimes are in different shapes instead of just round. Here you see a whole collection of these Hülle cells and the conidia are in the background to give you a size comparison. Here are some Hülle cells that are a bit distorted.  They’re not round.  This is what you might see. So that’s what Hülle cells look like and Aspergillus nidulans looks like and if you see that organism, I suspect you would not have much trouble trying to figure out what it is.

Aspergillus clavatus

The next one is one that is not seen all that often but I put it in because it’s a totally different looking organism and I think you might want to see it.  It’s called Aspergillus clavatus or cla-vah-tus.  It produces a club-shaped vesicle different from anything that we’ve seen. And there they are.  They are club-shaped and they contain, produce a lot of spores around the perimeter of that club-shaped vesicle. And you can see it here.  You can see the pigment on there but if you look at the vesicle, it’s not round, it’s not fan-shaped or anything, it’s long like a club and then around the perimeter of it are all the spores that are being produced.  This is what Aspergillus clavatus looks like, or cla-vae-tus looks like.  It’s green and you can see it’s very dark green here.

Aspergillus glaucus

The next organism is Aspergillus glaucus.  This is an organism that will not be very common in the clinical laboratory but I included it because it produces some different structures other than what we’ve seen.  The vesicles produced by Aspergillus glaucus range anywhere from being club-shaped to being round and spherical.  The phialides cover the whole surface of the vesicle no matter what the shape.  And some isolates have these fruiting bodies or fruiting heads that produce secondary conidiophores directly from the vesicle.  And that’s a unique thing to see and sometimes you will see cleistothecia.  These are the big sac-like structures, big round balls that produce ascospores inside of them because this is a product of the sexual reproduction. Here you see the cleistothecia that we are talking about and if you were to take a pencil eraser and push down on the coverslip that would pop them open and you would see the ascospores come out.  And so this is Aspergillus glaucus; what it looks like. And here’s another one.  It’s hard to see what the interior of this cleistothecium looks like.  The outside of it looks like it is almost like organized tissue but’s really not.

Aspergillus versicolor

The next organism is one that is included because of its uniqueness.  And this is Aspergillus versicolor.  It has vesicles that are round to oval.  They are biserate which means they have the metulae and the phialides.  If you look closely at this culture and move around the slide around you probably will end up seeing something that looks like Penicillium and your first thought is, this culture is contaminated with Penicillium.  Ahh, It’s not.  Aspergillus versicolor also produces Penicillum-like heads in addition to the heads that look like Aspergillus and even to complicate the thing, sometimes they even produce Hülle cells.  So this thing does it all.

Here is the head of Aspergillus versicolor.  And it’s kind of green looking.  You can notice that the phialides are there and at the tip of the metulae are there.  You see the vesicle, you actually can see the metulae and then if you look at the top of those you will see the phialides and then there are the chains of conidia.  So you look around in there and there is what looks like a little phialide coming off the side. But you look around more and you see here about maybe six or about seven o’clock ,a Penicillium head is present and then right next to it is one that you might think is Penicillium but it does have a vesicle in it.  So, you see the Penicillum-like head and the typical head of Aspergillus it belong to the Aspergillus versicolor group.  There are a lot of structures in there.