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Identification of Hyaline Filamentous Fungi - Part 6 (Miscellaneous Septate Hyaline Fungi)



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Published: August 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 6 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 miscellaneous septate hyaline fungi.  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.

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. 

Miscellaneous Septate Hyaline Fungi

In the next presentation, we will deal with miscellaneous septate hyaline fungi that you don’t see in the clinical laboratory all that often but it’s kind of nice to recognize them if they happen to be present.

Trichothecium

This next one is called Trichothecium.  Generally, the species name Trichothecium roseum because it produces a rose colored colony.  It produces conidiophores that are single.  It produces conidia that have two cells in them and that’s all.  And they’re kind of club-shaped.  The conidia when they’re produced are produced in what we call a zigzag column at the tip of the conidiophore.

Then next slide shows you the culture kind of a rose colored culture. 

These are the spores of Trichothecium.  They’re a little bit dark and just not quite crisp in terms of being able to see them.  But the spores are kind of elongated and they are divided in two and they come off one side then the other one side, then the other.   It’s one that’s easy to recognize.

And there you see the spores much better.  They are two- celled and they come off in a zigzag fashion.  The funny thing about mycology is that the rule is if it’s something that is really easy to recognize you don’t ever see it.  That’s one of the problems that we have.  But you can see this, and I think if you saw it you would know what it because characteristically, it has the two cells.

And here you see two cells again.  And you can see the one on the left hand side at about nine o’clock, the point of attachment is kind of a flat area. 

Beauvaria

The next one is an organism that is very tiny and very delicate.  And it is called Beauvaria.  The cells that produce the spores are inflated at the bottom or the base.  These cells terminate in what we call thin, zigzag apices.  So at the tip, you will see the spores come off of one side then the other side, one side, and then the other side at the tip of a little point.  We call this sympodial production of conidia where they come off one side and then the other.  And the conidia are round to oval and they are tiny and delicate.  This happens to be an insect pathogen.  Sometimes you will see this, a dead wasp or a bee and you may see there is mold growing out of it but this is what killed the wasp.

This is a culture of Beauvaria and it’s kind of unique in itself.  The colonies are heaped up and they’re like a round ball.  And they are not flat on the top; they are very heaped up.

And then the next one you can see where there is a lot of organism present, it fills up the culture plate but the colonies are still domed on the top.

Here you see a microscopically how it produces the spores or the conidia.  The conidiophore is a very short, kind of curved, a very delicate structure and at the tip there is a little denticle that protrudes out from the apices where the spores are produced. It is like a little protrusion connected to the top of the conidiophore and is very delicate.  They are twisted back and forth in terms of producing conidia.  It is hard to see unless you oil immersion because the spores are so small and they are produced in that zigzag fashion and that’s what Beauvaria looks like.

And here you can see it again.  Now this one has many more spores.  If you look at about maybe four thirty, you can see that the spores are produced kind of a one side and then the other and if you look closely you’ll see they are attached by a little tiny structure.  It is not the easiest organism to recognize because it is so small and you do have to use oil immersion to be able to recognize the structures.

Scedosporium prolificans

This is another one that is seen in the clinical laboratory and it’s one that is an important organism because it actually is a totally drug resistant organism for the most part and when it infects someone, it is very difficult to treat.  This is one called Scedosporium prolificans.  This organism had another name before it was called Scedosporium prolificans.  It was called Scedosporium inflatum because the base of the conidiophore was inflated or swollen and it’s very obvious and you’ll see it here as we go along.  It produces numerous dark brown conidia formed from short annellophores.  The annellophores are the structures that have the little growth rings around them and if you look down farther, farther down at the bottom towards the base, you’ll see that the base of the structure is inflated.  And the conidiophores are arranged singly or in branched clusters.  This organism produces two kinds of spores, some that are brown and some that are not.

This happens to be Scedosporium prolificans.  And if you look at the spore and then see what almost looks like a phialide and  you follow it down, look at the bottom, you see that it’s swollen, a very tiny delicate organism.  And if it’s swollen at the base like this it is Scedosporium prolificans.  There is actually one at the top at twelve o’clock with a swollen base.

This is one where you see it very well.  You notice at the top there are a cluster of conidia you can see the brown pigment in those, follow it down and look at the conidiophore.  It is swollen before it is attached to the hyphal strand.  So some of these spores in here, they happen to be brown in this particular mount, in others you will see that some of the conidia are not pigmented at all.  They’re hyaline.  So this is Scedosporium prolificans.  This is a very important one to let the clinician know about because it causes sinusitis, it causes disseminated disease and it requires a lot of attention to detail to treat these patients with this organism.


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