Hyaline Fungi Part2
4/1/2014
This is the written version of our Hot Topic video presentation available at:
MayoMedicalLaboratories.com/hot-topics
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 unusual Zygomycetes. Thank you Dr. Roberts.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
1
Hyaline Fungi Part2
4/1/2014
Thank you, Sarah for that introduction. I have nothing to disclose.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
2
Hyaline Fungi Part2
4/1/2014
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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
3
Hyaline Fungi Part2
4/1/2014
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 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 so we will begin to look at some of these as we go along.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
4
Hyaline Fungi Part2
4/1/2014
The first preparation that can be used in the clinical laboratory and probably the most
widely used is the scotch tape preparation.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
5
Hyaline Fungi Part2
4/1/2014
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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
6
Hyaline Fungi Part2
4/1/2014
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, 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 growing up so it’s kind of
in the middle.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
7
Hyaline Fungi Part2
4/1/2014
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’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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
8
Hyaline Fungi Part2
4/1/2014
Another kind of time honored preparation is the wet mount.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
9
Hyaline Fungi Part2
4/1/2014
This is where you take a little bit of the colony and you cut it out of the agar with a
wire that’s been bent at a right angle and you take a little bit of the colony along with
some of the supporting agar and you place that on a slide with some lactophenol
aniline blue.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
10
Hyaline Fungi Part2
4/1/2014
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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
11
Hyaline Fungi Part2
4/1/2014
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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
12
Hyaline Fungi Part2
4/1/2014
Probably the scotch tape prep is the most universal one right now and the wet mount
may be second, and then as a last result, we have in the past used what’s called a slide
culture, a micro slide culture.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
13
Hyaline Fungi Part2
4/1/2014
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 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 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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
14
Hyaline Fungi Part2
4/1/2014
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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
15
Hyaline Fungi Part2
4/1/2014
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 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.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
16
Hyaline Fungi Part2
4/1/2014
This presentation will focus on some of the uncommon or the unusual Zygomycetes
that we see in the clinical laboratory.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
17
Hyaline Fungi Part2
4/1/2014
One of those that we see is called Apophysomyces elegans. An Apophysomyces is, we
are starting to see it more often as a cause of infection and it’s an organism that has 1
very distinct structure. It produces these straight to slightly curved sporangiophores
that are produced as singly. And it produces sporangia at the tip. And these
sporangias are kind of pear-shaped and they’re grey looking. When you look at that
sporangium, you will find that the base of the sporangium is a funnel-like structure
called an apophysis. That’s very obvious when you look at it with Lictheimia, you also
find an apophysis is present. It’s not quite as obvious as it is with Apophysomyces,
that’s where the name of Apophysomyces comes from.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
18
Hyaline Fungi Part2
4/1/2014
And here is an example of an organism, and if you look at the dark structure there that
looks like a funnel, that is called the apophysis. That whole thing would have been a
sporangia, it would have been a sac full of spores but the top part of it is broken off
and you can see the spores that are still in there so there’s part of the sporangium and
then at the base of it is that apophysis. And Apophysomyces is one you wouldn’t have
to work at identifying.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
19
Hyaline Fungi Part2
4/1/2014
This is a culture of a Zygomycetes, not necessarily Apophysomyces, but it looks wooly,
very rough in texture.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
20
Hyaline Fungi Part2
4/1/2014
And here’s another one that we see infrequently and it’s called Circinella. And it
produces curved sporangiophores. These sporangiophores are always kind of curved
and toward the inside part of the hyphae. I have never seen, I don’t think, this
organism be associated with disease. But it’s an easy one to recognize if you ever see
it. The problem with identifying these Zygomycetes is that if they are easier to identify,
you don’t see them very often.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
21
Hyaline Fungi Part2
4/1/2014
So with Circinella, you have these sporangiophores that are distinctly curved
downward and you have this sporangiophores produced within these sporangia.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
22
Hyaline Fungi Part2
4/1/2014
And this is the example here. A larger view of Circinella and you can see the sac-like
structure and notice that the sporangium or the supporting structure is not straight,
it’s curved. And you might see some septations in there as well.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
23
Hyaline Fungi Part2
4/1/2014
And here you see a better one of Circinella.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
24
Hyaline Fungi Part2
4/1/2014
And very large and full of spores.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
25
Hyaline Fungi Part2
4/1/2014
It’s another Zygomycetes or Mucorales showing just a culture itself.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
26
Hyaline Fungi Part2
4/1/2014
This one produces spores in a whole different way. This one produces a kind of a
vesicle at the tip of the sporangiophore and there may be some secondary ones being
produced. And they have this structure that is dark. You see there that is kind of
swollen and it’s called a vesicle. And then if you notice the spores are produced here
under kind of a thread-like attachment to that vesicle, and the whole head of those
things is covered with those, and they’re called sporangiola.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
27
Hyaline Fungi Part2
4/1/2014
If you notice that this organism is different from the ones that you’ve seen and it does
produce those sporangiola. This is one that you might see as a cause of disease
particularly in a patient who has been burned. And the organisms name is
Cunninghamella. The sporangiophore ends in kind of an oval terminal vesicle, which
you saw a minute ago, and in some of those vesicles may produce some secondary
ones in there and it kind of produce in a whirl. The sporangiophores are smooth,
sometimes they are kind of rough, and you can see that on these mounts. The
sporangiophores are produced on these short denticles over the surface of these
vesicle and we call those sporangiola.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
28
Hyaline Fungi Part2
4/1/2014
This is an example of a very high-powered view of these sporangia spores and if you
look at the one almost to the left of center and notice that it is light in color but if you
look closely you will see little tiny spikes coming out of that thing and it’s roughwalled. You can see it if you focus up and down. If you look at one at about eight
o’clock down at the bottom, you’ll see that it’s darkly stained but it has those spikes
coming off of it, very finely roughened spores.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
29
Hyaline Fungi Part2
4/1/2014
And this is an example of how those spores are produced and sometimes they just fall
apart. So what you end up having to do is look around and try and find all of the
structures that are there to get an idea of what it is. In the center, you can see that
there is a long sporangiophore but it’s difficult to tell any more than that so it would be
hard to know if this is what this is or not.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
30
Hyaline Fungi Part2
4/1/2014
And on here, the same thing holds true. These are more like chlamydoconidia.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
31
Hyaline Fungi Part2
4/1/2014
And this is an example of one where you actually can see the 2 on the right-hand side.
Those are the kind of vesicles that are produced. And on the left-hand side, those
sporangiola are attached to the vesicle that is in the middle of that. It’s hard to see
because it is so dark and then the spores are attached to the tip of that structure that
adheres them to the vesicle.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
32
Hyaline Fungi Part2
4/1/2014
Here’s where you can see it well. The left-hand side there is a vesicle and on the righthand side, if you look down deep you can see the vesicle but you can see the little tiny
pegs attached to it and then there is the spore at the tip of that. Those are called
sporangiola and the spores are what you see at the tip of that. And that’s a good
example of Cunninghamella.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
33
Hyaline Fungi Part2
4/1/2014
Now this organism here is just another Zygomycetes that is wooly in texture and you
end up having to make a mount of this to get an idea of what it is, but you certainly
can’t tell from looking at the culture.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
34
Hyaline Fungi Part2
4/1/2014
Here is another one that shows you the kind of a brown pigmentation.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
35
Hyaline Fungi Part2
4/1/2014
This one is a different looking organism. This is one called Syncephalastrum. Some
people say that when they look at it under low power, it reminds them of Aspergillus.
Well, that may be the case, but then what you do is you look under a higher power. So
we’ll look at some of those and see if we can figure out what the organism is.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
36
Hyaline Fungi Part2
4/1/2014
Syncephalastrum, there is a terminal vesicle that’s produced just like you saw the
Cunninghamella but around that vesicle there are some cylindrical structures called
merosporangia that contain the spores. So the merosporangium is the sac-like
structure that holds the spores but it’s not round, it is like a tube-like structure and so
the sporangiospores are formed in its linear fashion within this merosporangia, which
looks like a tube that surrounds this kind of vesicle. And you may find a rhizoid with
this too.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
37
Hyaline Fungi Part2
4/1/2014
This is an example here of the tube-like structure of this sporangia that you see called
merosporangia and you can see the spores inside of there and they are arranged in a
linear fashion.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
38
Hyaline Fungi Part2
4/1/2014
This is Syncephalastrum and you can see the central vesicle there and surrounding it
would be all of these merosporangia that are kind of tubular in fashion inside of those
are the sporangiospores.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
39
Hyaline Fungi Part2
4/1/2014
And here’s one where you see a very large hyphal strand and if you look above at the
dark structure you’ll see that there’s a round vesicle and then up around that vesicle
are the cylindrical merosporangia containing all of the spores in that’s
Syncephalastrum and that’s what it would look like.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
40
Hyaline Fungi Part2
4/1/2014
This is just another one showing you a better view of what it looks like. Sporangia
actually are those cylindrical structures.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
41
Hyaline Fungi Part2
4/1/2014
This is another Zygomycetes or Mucorales. It’s kind of brown.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
42
Hyaline Fungi Part2
4/1/2014
And this is an unusual organism to see except that we are starting to see it more often
than we have in the past. And it’s one that produces a different looking sporangium.
This organism is called Saksenaea vasiformis. And it produces a sporangium that is
kind of flask shaped or vase shaped and has a long beak on it. Notice in the center we
just start off about 4 o’clock, there are some rhizoids on this thing. There are rhizoids,
there’s a sporangiophore at the base and all of a sudden it swells up and becomes kind
of vase shaped and it has a long beak on it. And you notice at the tip of the beak,
there are spores that are coming out of that thing.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
43
Hyaline Fungi Part2
4/1/2014
And here is just a culture.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
44
Hyaline Fungi Part2
4/1/2014
To look at Saksenaea vasiformis in some detail is what you see is the distinctive long,
flask-shaped sporangium with a long beak present and you find rhizoids that may be
brown.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
45
Hyaline Fungi Part2
4/1/2014
Here is that sporangium. You can see that it is kind of vase shaped at the bottom on
the right-hand side facing 4 o’clock and look at the left-hand side going up at about 11
o’clock. That is where the beak was that has broken apart and all of these spores are
coming out of it and they are rhizoids that you see at the base there. So this is
Saksenaea vasiformis.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
46
Hyaline Fungi Part2
4/1/2014
And here’s one that’s intact. You see the vase-shaped part of the sporangium and you
see the long beak on this and this is an unusual one to see, but it’s been associated
with brain infection and a number of other things and we had a case of osteomyelitis
associated with this organism. It’s found in the environment. It’s found in the soil.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
47
Hyaline Fungi Part2
4/1/2014
Another one of these Mucorales or Zygomcetes. And they pretty much all look alike in
culture. You have to rely on a microscopic identification to be able to identify these
organisms.
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
48
Hyaline Fungi Part2
©2014 Mayo Foundation for Medical
Education and Research. All rights reserved.
4/1/2014
49