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Identification of Hyaline Filamentous Fungi - Part 4 (Hyaline Monomorphic Fungi)



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Published: June 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 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 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 miscellaneous hyaline fungi with conidia produced in chains by phialides or phialides with metulae and no vesicle present. 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 Zygomycetes. 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’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.

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 resort we have in the past used what’s called a slide culture, a micro 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.

Hyaline Monomorphic Fungi

The next group of organisms that we are to talk about are the hyaline monomorphic molds that are just like the others that we have talked aboutwith the other hyaline molds; however, the way the conidia are produced are different in this group than with the others that we have discussed.  In this group we are going to speak about today is has conidia that are produced in chains and generally thay have phialides that produce these conidia that are produced in these phialides.  There may or may not be metulae present and metulae are nothing more that branches that occur right down below the phialide.  You will see a branch sometimes and a phialide on top of that and that phialide gives rise to a long chain of conidia.  And sometimes you do not see metulae at all.  Now at the tip of the conidiophore in this particular group we are are going to talk about, there is no vesicle present. Sometimes with some of the fungi, they have a swollen area at the tip before the metulae are produced or before the phialides are produced but we are going to talk about organisms that don’t have a vesicle right there.

Pencillium

So we’ll talk about Penicillium first.  Penicillium for the most part, everyone thinks about the fact that it looks like the fingers on a hand. Produces a conidiophore that is simple or unbranched and the phialides are grouped in a brush like cluster and the whole fruiting head is called a penicillus.  The whole structure there with all the branches and all the conidia that are produced, in chains and if you look at them, some of them are smooth-walled and some of those actually are rough-walled. So this is an example of a compact head or a penicillus of Penicillium.  It’s difficult to see on here and be able to tell what this organism is because it is so darkly stained and so tightly compact.  So what you would do, would be to look around and look at all the different fields to see if you could find some that are at earlier stages of development or maybe you would find one where you could see the structures better than on here.  But this is commonly what you would see.  They just don’t magically appear to have all the features when you start looking at them. If you try to take a picture of one that is textbook perfect, it is very difficult- it is very difficult to find those.

This is a culture of Penicillium and it is the organism that produces penicillin.  And if you look at some of the colonies of Penicillium you will see some drops of exudate on top of the colonies that are kind of amber colored.  And probably what’s in that exudate, is penicillin, but only a certain species produces penicillin. This is the an example now of where we looked at an early culture and what we’re looking at here on the left hand side about eight o’clock, is a long stalk and off to the left and to the right are other structures that are being produced and there you see there are some phialides and you see a chain of conidia coming out of those phialides. Here is a better example.  If you look at the tip of those, you will see some round conidia in chains, follow them back down to where they are attached and you will see they are attached to a bottle-like structure and that’s the phialide.  They are produced inside the phialide actually and pushed out and they remain in chains and there is no vesicle anywhere in there like Aspergillus, you just see this head of Penicillium with all these phialides sitting around, they look like the fingers on a hand and the phialides produce the chains of conidia.

Here you see some that are more mature and you can see what I am getting at with the fingers on the hand with this type of arrangement here.  And notice that the conidia are totally in chains and so when you make a wet mount, one of the things that happens when these chains of conidia get disturbed then you don’t see them in chains very often.  But if you make a scotch tape prep and look at it you will be able to see the chains of conidia just like you see here in a culture that was growing. These structures where the arrows are show that these are mature heads of Penicillium and basically what you are looking at are these phialides that give rise to the long chains of conidia and down below the phialides are some branches that are called metulae but they are not so easy to see on here.

Here’s where you can see the chains of conidia and you follow them back down to the phialides and then down below the phialides where the arrow is, right down below that cluster of phialides you see two branches, those are called metulae. And this is Penicillium again with long chains of conidia; it is very common organism.  It’s probably the second most common organism that you see in the clinical lab. This is a culture of Penicillium and it’s dark green.  Some of the cultures have a red pigment to them and there is one that is dimorphic and this one that we know is called Penicillium marneffei and it will produce phialides and chains of conidia but it produces a colony with a red reverse.  It’s red pigment may be on the top too but not all culture that produce that a red pigment are Penicillium marneffei either.

So here’s another one, Penicillium.  You can see that it has some growth rings almost on there. And here’s one where actually you can see some exudate produced on there in the center, but for the most part, the colony is kind of green. Well, the next one here is what some people describe as looking like a small Penicillium and it’s one called Paecilomyces.  And Paecilomyces can produce phialides and chains of conidia, however, they are much smaller than you see with Penicillium.  And they’re very delicate.  The chains of conidia are extremely delicate and break apart very easily.  So if you look on the right hand side you will notice that the phialides are kind of a vase-shaped and it goes up to the top and it becomes very narrowed and it actually has a tapered tip.  And then it gives rise to these chains of conidia that are produced there and it often starts off what looks like a yeast-like colony and then it kind of becomes brown or tan suede color as time goes along.

Paecilomyces

Paecilomyces produces these Penicillium-like conidiophores but they’re much smaller.  It has long chains of elliptical conidia and they are kind of oval shaped, irregularly branched heads that terminate in a long, tapering phialide.  The phialides have a long tip on them and sometimes you will find that these phialides being produced singly and not in clusters as well.  And that’s something that we will show you. Here is an example here of the head of Paecilomyces and if you look at the arrow, you will see those ovoid conidia and then down below there you will notice that there is a dark structure and that’s the phialide and that comes off of another stalk.  And so you see these chains of these elongated phialides like you see here with the elongated tip on them.  You pretty much know you’re dealing with Paecilomyces.  It’s very tiny and very delicate.

And here is where you see the long chains, long flexuous chains of conidia with a very long and a delicate phialide producing them. And here you see one that just occurred singly without being in a cluster like a head of Penicillium or a other things.  You can find them  sometimes you have to look around a bit. This is what Paecilomyces might look like at the beginning.  It’s a young culture and you see that it has a tan appearance to it and often times it looks almost suede-like when you see it early on. And then you see one when it gets older and it begins to get more dry.  It kind of still has a bit of a tan color to it.

The next one shows you something else that produces a something like a penicillus but it’s much larger than Penicillium and much larger than Paecilomyces.  It’s one called Scopulariopsis and Scopulariopsis sometimes is referred to as being a large Penicillium.  It’s not but it kind of reminds you of that and it produces phialides that have a specialized name for them and they are called annellides.  If you look at about maybe five thirty, you will see phialides where the conidia that are produced downward and if you look at the conidia you will see that the one on the left hand side has a flat base on it and it’s rough-walled.

Scopulariopsis

This slide shows you some of the features of Scopulariopsis and what it produces are these Penicillum-like conidiophores or thes heads of Penicillium- like a penicillus but they’re much larger.  The conidia are very large, they’re round, they are rough-walled and they’re produced in chains.  They almost look kind of lemon shaped.  If you notice at the base of those conidia, there is a flattened area because they are produced in chains and every one of them is connected.  There is a flat base so they have these short, branched heads that terminate in these phialide-like cells that we call annellides.  These long conidiophores give rise to the spores called annellids because every time a spore is produced, a little bit of material is left behind and it starts to form rings that look those on an earthworm, if you get right down to it.  Earthworms have these rings and they are called annellids because the do.  When a spore is produced it lays down some material behind it and it aggregates it together and leaves what looks like a ring.  And every time a spore is produced there is another ring and another ring and those are called annellids and so we call the structures that produce these spores, annellophores or just call them conidiophores, that’s just fine.  It gets complicated when you are trying to use all the terms trying to distinguish between one organism and another.

This is an example of what Scopulariopsis looks like.  It looks like powdered cinnamon for the most part.  It’s supposed to be about that color and it usually has the consistency of the powdered cinnamon as well.  This doesn’t exactly look like the powdered part of cinnamon you see here. Here is a smaller view of one of the heads of Scopulariopsis.  What you are seeing here are some conidia that are floating free and then you see some that are attached to the structure below it and there’s a flat base on those spores.  And the structure that produces those conidia are called conidiophores, if you want to call them that or, if you want to call them annellophores, you can certainly do that to be more specific.  Sometimes people describe those conidia as looking like light bulbs because they will have a  base on them, a little bit of an extension and then a flattened area on there and that’s a way to remember what they look like.

This is an example here of Scopulariopsis where you see chains of conidia that have been broken off and on the left hand side about nine o’clock, you see a whole bunch of them in chains and look at the bottom of each one of them, see it’s flat and you can see the rough-wall in nature of these spores of Scopulariopsis so they’re very rough-walled.  And there is not much else that looks like this.  There is, however, a dematiaceous or pigmented Scopulariopsis that you might see in the laboratory and it’s called Scopulariopsis brumptii.  It’s a dematiaceous organism and it has morphology very similar to this except it is pigmented. Here you can see another one, where you can see all the chains of conidia and just a flat base on the bottom of those things and the rough-wall and you can see they are produced on a what looks like a phialide or if you want to call it a conidiophore or if you want to call it annellophore, whatever you want to choose to use to remember.

Here you see some examples of the conidia.  There are some of them are free, some of them are still in chains.  And when you make a wet mount, you knock those apart; you can pretty much tell what it is by looking at the morphology of the spores microscopically, they don’t really have to be in long chains for you to be able to tell that it is Scopulariopsis. Another one showing the extreme rough-walled nature of these cells, Scopulariopsis is an organism that is found in the environment but it is certainly becoming pretty much a pathogen.   It has been associated with sinusitis in transplant patients.  A very serious disease, in fact it is reported in literature that a patient has actually died as a result of being infected by this organism and it started on the sinuses.

And you can see here there are a couple of these annellophores or conidiophores giving rise to the chains of conidia and they have that flat base like we talked about. And then you can see this one here where these annellophores or these conidiophores come off in almost a whorl but if you look at the conidia you can see that  they are rough-walled on this one but they are usually very rough-walled and they have that flattened base on them. This is a colony of Scopulariopsis and it doesn’t look really all that much like powdered cinnamon but sometimes they don’t. And you can see this one.  It has a suede like appearance to it. You see better on this one. And this is still Scopulariopsis.

And then this came from one case of sinusitis that we saw.  The plate on the lower left hand side is one that contains cycloheximide that actually inhibited Scopulariopsis, so you notice that the other plates allowed it to grow and so one of the problems with the cycloheximide is that if you’re going to use it to try to inhibit some of the rapidly growing molds, you have to use a plate that doesn’t contain it to recover the very thing that you are looking for because sometimes cycloheximide will actually inhibit the very same thing that you are trying to recover.  So these are colonies of Scopulariopsis, you can see how suede -looking they look.


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