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Clinical Mycology: Direct Examination Series


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Published: October 2012

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Direct microscopic examination of fungi in clinical specimens relies on both bright-field and phase-contrast microscopy, as well as multiple stains to optimize visualization of the organism. This presentation includes an extensive collection of specimen photographs to assist you in identifying these organisms. Each presentation in this 11-part series addresses 1 or more genus or group.

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. Dr. Roberts discusses the features of specific organisms under direct microscopic examination using multiple preparations. This module examines Aspergillus. Thank you, Dr. Roberts.

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

This is an ongoing presentation that focuses on the individual or groups of organisms as seen using direct microscopic examination of clinical specimens. It allows us to see the fungi before a culture can actually be done and often times we can make the diagnosis early.

The next slide shows you some of the methods that are used for detecting the fungi even though some of them are not designed specifically for that purpose, they still can be used if you think about the fact that fungi can be seen in these stains and you look for them. You often times will find for example a Gram stain you can see fungi well with and with the Wright stain used in hematology you can see fungi particularly in Cryptococcus and cell counts (CSF). So those are some of the methods.

The next slide shows you the continuation of the methods that can be used and these are other stains, some of which are used for staining acid fast bacilli, the pap smear used for looking for malignant cells in the respiratory tract and then histopathologic sections, the methenamine silver stain, the PAS stain are designed for detecting fungi. And the H&E stain is used for detecting tissue architecture but it still stains fungi and you can see them there so the moral of the story is you have to think clinical microbiology when you are using any of these methods because you have to find fungi there.

The next slide we are going to look at some of the features of organisms as seen in a direct microscopic examination of clinical specimens. And basically, if we have a sample of hair, skin, or nails and we see hyaline septate hyphae, we are probably going to know that it represents a dermatophyte.  Not all of the time but most of the time. And so I think we just have to remember that, it simplifies things a bit. Also, in other specimens besides hair, skin, and nails you might see branching hyphae in there that are dichotomously branching at 45 degree angle. They would be septate hyphae that show some form of branching and they are organisms like Aspergillus, Fusarium, Pseudallescheria, and a whole host of other organisms can be represented there because they present with the very same features. It’s impossible to tell microscopically which organism it is when you just see dichotomously branching hyphae. But based on the odds, if you see that, it is probably going to be Aspergillus.

The next slide shows a hair, an infected hair from the scalp of someone with tinea capitis. And what you are looking for there are the hyphae that penetrated that hair, that go the length of the hair shaft and those are septate hyphae.

And then the next slide shows that there are some arthroconidia. If you look carefully, on those cells are kind of coming out from the edge of the hair at about 3:00 o’clock, you will notice that some of those cells are rounded and some of those are elongated. Those probably represent the arthroconidia along with a hyphae that you can see within the hair shaft. So this would represent a dermatophyte which represented an infected hair in a case of tinea capitis.

The next slide shows you a phase contrast photomicrograph of branching hyphae, they are not really branching hyphae, those are hyphae that are twisted amongst the squamous cells that you see, but you could see branching cells there in a skin sample. And when you see that, you pretty much are dealing with a dermatophyte. So you can use a bright field microscopy, Calcofluor white, any of the stains to show this. This just happens to be phase contrast microscopy.

The next slide shows a representation of Aspergillus. And basically, the features of Aspergillus are that the hyphae are 5-10 microns in diameter. They have septate acute angle branching, dichotomous branching and some of those Aspergilli may have hyphae that are large enough to represent a Zygomycete. And so there are times when one has very much difficulty and you may not even be able to tell the two apart. Zygomycetes, Mucorales, and Aspergillus.

The next slide shows a very low power view of the sputum. And if you notice the background in the upper portion of the slide is red, that is because there are a lot of red cells in there in this particular specimen. And down a little bit lower you see all of the hyphae and there are some of those cells those hyphae that branch dichotomously and they show what looks like a Y. And that is acute angle branching and that is what you see with Aspergillus but you can see it with Fusarium and you can see it with Pseudallescheria and other things so just looking at this does not tell you what the organism is but again, based on the odds it probably would be Aspergillus.

The next slide shows you a higher power view of some of the dichotomous branching from a previous slide. You can see at about 6:00 o’clock that that cell there that hyphal strand is showing a Y and in some specimens you will see that dichotomous branching is really prominent. This one happens not to be quite that way.

The next slide shows you basically just hyphae in a respiratory tract. But if you look carefully, at about 9:00 o’clock, you will notice that there is a Y and that tells you that it is branching acutely and if you look closely along the hyphal strand you will see there are septations there. And so this is just another example of what an Aspergillus or one of the other fungi might look like using phase contrast microscopy.

The next slide is one that shows you what real life is like. It is where the hyphae don’t show so well. They are very faintly stained and in the background and you can see a couple of areas where the acute angle branch is prominent. Otherwise you see just hyphae in there. It is hard to tell really if they even represent septate hyphae. Sometimes it is very difficult to see all of the features you would like to see so you do the best that you can. And you look at all of the fields to get a consensus of what is there.

The next slide shows you a number of dichotomously branching hyphae. If you notice the way this slide is oriented, it almost looks like there is a mass that runs from about 1:00 o’clock down to about 7:00 o’clock. Longitudely what you are looking at actually is a mucous plug from a patient who has allergic bronchopulmonary aspergillosis. This mucous plug is coughed up and then if it is caused by Aspergillus it contains all of the hyphae of Aspergillus. And what you are looking at in there are acute angle branching throughout that mucous plug. If you culture that, you would probably grow Aspergillus out of that. So we do see this in patients who have acute allergic bronchial pulmonary aspergillosis. And this is not an infection, it is actually a response to the organism and the patient produces mucin to eradicate the organism so the patient will cough it up and expectorate it and get rid of the organism but it is treated by corticosteroid therapy rather than a fungal therapy. So you do see this but it is not as common as it used to be commonly have allergic broncopulmonary aspergillosis for farmers and now they wear masks or respirators and they protect themselves and so we do not see that very often. It used to be that the farmers would become acutely ill and some of them had to actually stop farming because of the hypersensitivity to things like Aspergillus like you see right here.

Next slide shows you phase contrast photomicrograph of the hyphae of an Aspergillus. If you look in there throughout the field you will notice that there are a few places where there are some Y’s and notice there are septations in there that are prominent. And then there are swollen cells present all throughout that. This is an Aspergillus that we grew and this is what it looked like under the microscope from a clinical specimen you may see all of those features of an Aspergillus at one time or another maybe not all in the same field. But to see those swollen cells, is not an uncommon thing. So, this is basically what you are going to see and it is not anything dramatic but it is helpful to know if those are present, particularly in a sample that is sterile, normally sterile sample. In a respiratory tract it is difficult to know what this means. You could even grow it out and if you grow Aspergillus out you still would not know what it means so what has to happen is a biopsy has to be done and you have to prove that that organism is invading the tissue and causing infection otherwise, it might just be a contaminant if you will and we have seen that happen. So looking at something in a sputum for example and finding something like this doesn’t necessarily prove that the patient has Aspergillus infection.

The next slide is one that is a bit difficult to read. But if you look at it closely, you will see that there are hyphae in there of two sizes. Large and small sized hyphae, you notice there are swollen cells in there and you can see the septations in several places in there and this represents an Aspergillus as well in the same kind of a sample. Aspergillus and Fusarium and some of the other things could look like this in a clinical specimen.

The next slide shows you more of the same. It shows you actually longer filaments of hyphae in there that exhibit septations and not so much branching in there and you notice that some of the hyphae are larger than others and we do see this with Aspergillus and with some of the other molds and so looking at this would not tell us for sure what this is.

Then next slide shows you again some of the same features. It shows you that at 12:00 o’clock you see dichotomous branching and there is some acute angle branching with a Y up there. You’ll see the hyphae down below that area and going over to about 2:00 o’clock you see the Y there and you see septations, you see the hyphae of a couple of different sizes, some are small, some are large. And down about maybe 7:00 o’clock or so, you see two areas where the hyphae are very large and septation between them. So Aspergillus can present multiple presentations when you look underneath a microscope and some of the other fungi as well.

The next slide shows you a bit more of the same. It shows you the septate hyphae in there and some of them appear to be fractured on the ends and with Aspergillus, for example, particularly in a biopsy the hyphae fragment very easily belong with the Zygomycetes or Mucorales as well. So it doesn’t tell us what it is. There is some dichotomous branching even on this slide. So, I see it at about one, two, three areas where I see, at least some acute angle branching in there. You see the septations that are present and this will tell you that it belongs to one of the septate hyaline molds, which ever one it might be, I have no idea which one this represents.

The next one shows a bit of the same thing. A lot of fragmented pieces of hyphae there and it just shows septate branching hyphae there and not much branching in there, actually. So this may be all that you see. And so you are not going to see everything that’s textbook perfect.

The next slide shows what you might find inside of a mucous plug. And you can notice that the way it is arranged it’s almost in the shape of a rectangular type specimen and this is a mucous plug. What you see in there is a central hyphal strand and then coming off of there are many, many branches. And this is an Aspergillus in a case of allergic bronchopulmonary aspergillosis and we cultured it from this patient and in some of those patients who we used to see this disease in and some of them actually lost their livelihood and had to move away from the farm and lose their whole source of income because repeated exposure to this organism found in moldy grain and moldy hay resulted in an increase in symptoms each time. Each time they got exposed, the worse things got. And so this is not a benign thing and you don’t see it very often in the United States any more.

The next slide shows you more of the same from the inside of a mucous plug. And there the acute angle branching is really prominent and that is about all you can say about this particular organism or this slide.

In this next slide shows you the perimeter of one of those areas of the mucous plug and you can see the Y’s and you would have to look further to find out how many septations and so on are present but if you look down to the right hand side of one of the those two Y’s on the left there is a hyphal strand with a septum in it and the one next to it is septated as well. So, this is an Aspergillus.

The next slide shows you a Calcuflour white stained slide of just hyphae. And you can see the septations on the strand on the left hand side. They are very prominent. And the one on the right does not show septations at this point. Maybe it is a young hypha. But this will show you what a Calcofluor white will stain.

The next slide shows you calcuflour white using a different filter combination. And this is actually the filter combination most people use. It stains it, makes it a pure blue white. It is a filter that allows you to use about 440 nanometers for being able to see these organisms. And this is a case where you see nothing more than just hyphae that are septate. And that is all, that is all you are going to see. It doesn’t tell you that the patient actually has disease unless it comes from a normally sterile sight but the calcuflour white allows you to see organisms much more readily because it is bright and it is against a dark background. And I would recommend this particular method in the clinical laboratory, however, the filter that you use for these types of stain, it is really not a stain, it is a fluorescent brightener but if you use that, the filters are very costly and so you would have to weigh that benefit of that in your own laboratory.

The next slide came from a brain abscess of a patient who had Aspergillus. And you can see the hyphae are all kind of twisted around in there and there are a couple of areas where there are some rounded cells. And it doesn’t really show a dichotomous branching. It turned out this one actually grew Aspergillus from that patient and we used a different filter combination to make it look blue green because people that read these slides often read the acid fast smears and they were using fluorescent microscopy so they would see the blue green bacilla and we decided to keep the filter combination the same so that is why the green is here.

The next slide came from the same patient with a brain abscess. This patient ended up having nine brain abscesses. A young 29-year-old lady who just had a baby and she had eye pain. And they gave her in her home hospital some high dose, very potent steroids. It turned out they gave her 200 times the dose that should have been given and she ended up getting brain abscess caused by Aspergillus. And became obtunded and died very shortly thereafter. And what we saw in the brain abscesses were what you see here. You see the hyphae and smaller pieces, and we see swollen cells. This is kind of a tragic case. It is something that doesn’t happen too often but it did happen and she was a normal, healthy host until she became immunocompromised with Decadron.

Next slide is an interesting one. This is an Aspergillus. It came from a patient who was treated with an antifungal agent called Amphotericin B. In Amphotericin B, weakens the cell wall and if you look at the cell wall of these hyphae you’ll notice that it is full of holes. And that is what the drug did to the hyphal strands. It attacked the hyphae and allowed the cytoplasm to be able to leak out and the cells would die. And so these are septate hyphae that are probably nonviable because all of the cytoplasm has leaked out and you can see the holes in there. It is something you don’t see very often. It just happened they come along and I photographed it.

The next slide shows something that occasionally occurs in the laboratory. A patient will come in with a cavitary lesion and expectorate sputum and in that sputum might be the whole fruiting body of an Aspergillus. In this case you can see the top, you can see the vesicle up there that is surrounded by the filage and you follow it on down and you see where the hyphal strand is. It probably anchored it to wherever it was in the fungus ball maybe or there just in the sides of the cavity but this is the head of an Aspergillus, a fruiting body of an Aspergillus. And just on this day we were making this recording, I had an email from one of our former fellows who actually photographed something using his Iphone through the microscope and he saw this very same thing in a specimen. It is not uncommon to find.

The next one shows you a bit better view of the vesicle. It is hard to see the folates but the vesicle is kind of hemispherical. You can see it in the middle. It is kind of darkly staining more green and then the vesicle. The folates come off around that vesicle. Sometimes you can see it sporulating and sometimes you can see which species it is.

The next slide is a Gram stain. This is from an eye lesion and eye lesions, particularly cornea lesions, are caused by a number of fungi including Aspergillus. And so just because you see these branching septate hyphae in here, that stain with a Gram stain doesn’t tell you it is Aspergillus. It turns out that it was but there is something else in here that is a little bit helpful. If you look in the background you will notice there are some cells that are kind of round and a little bit rough on the outside. Those are conidia of an Aspergillus as they were being produced. And that is not a very common thing to find so you look around in there and try to figure out if that is what’s really what they are. Look at all of the fields and see if everything fits together and this is just one of those cases where you see something unusual. And why it sporulated, I don’t know. It had all of the nutrients that it needed and it had an air supply and so it grew the way it grows in a culture.

The next slide shows you a Gram stain and it just shows the dichotomous branching. And you can see the basic folic stippling all around the whole perimeter of the hyphae. And I don’t think that is all that important but it’s a dichotomous branching that you see. But this could be any organism, any mold that we deal with that produces hyphae.

The next slide shows you another Gram stain. This shows gram positive hyphae that are septate. And that is all you can say.

The next one actually came from a skin lesion. And you can see that there is a lot of stippling in there.  But, this turned out to be Geotrichum and it is not an Aspergillus. And it just simply produced hyphae that have some septations in it. So, you never can tell what you are looking at until you grow it out.

The next slide shows you a Pap smear of the respiratory tract specimen.  And there you can see septate hyaline. You can’t tell if they are hyaline, but they are not pigmented in any way, hyphae and you can see a couple of places where you see the acute angle branching. And so if you see that you think about Aspergillus. And this is the Pap smear that was read by the cytotechnologist.

And the next slide shows you another Pap smear. Showing the very same thing, you can see the septations very well. And you can see the acute angle branching on the filament on the right hand side. This is a stain that is not designed to detect fungi but it stains them well. And so, it is just a matter of being astute enough when you see something like this and being able to recognize what it is and then report it.

The next slide is something that’s a little bit different. There is a large hyphal strand in there that shows septations and then a smaller one going up at about 11:00 o’clock. But down below are these rounded cells that are a bit out of focus. Aspergillus produces these large swollen cells and I don’t know if there is any other name for them or I don’t know why they are produced either, but nevertheless, they are produced in clinical specimens and you see them just like you see here.
And so this is an example of one of the Aspergilli.

The next slide shows you what they really look like. You can see some of them are produced right inside the hyphal strand and most of them probably were produced within the hyphal strand but you can see some at the top there that are kind of sitting there by themselves. So these are swollen cells of an Aspergillus.

The next slide shows you the Pap smear showing the hyphae and very large septate hyphae. One of the problems that we have when we look at Aspergillus is that we find these large hyphae like this and we look closely and we see that there are a few septations here and there and then we started thinking about the Mucorales.  And this when you start looking around, you notice there are quite a few septations. Well, the organism that’s sometimes confused with Mucorales is Aspergillus flavus. And if you look at Aspergillus flavus like this, in a Pap smear under the microscope, it will have large hyphae like the Mucorales have but it will have more than just a few septations, although, not near as many as an ordinary Aspergillus fumigatus or one of the others that you see. So, sometimes when we see these things, we don’t know if we’re dealing with an Aspergillus or if we’re dealing with one of the Mucorales and we have to sit down and look at the relative number of septations and try to then decide are there a lot more septations there that you would probably see with a Zygomycetes and if there are then there is probably going to an Aspergillus flavus and when you get done with that then you probably going to have a 50 percent chance of being wrong.

So, the next slide shows you Pap smear and some dichotomous branching. And really that is about all that you see there.

The next slide is one of those ones where you might suspect that it might be one of the Mucorales or even an Aspergillus. And I don’t know which of the two this one turned out to be. But you can see that the hyphae are very large and they’ve kind of narrowed down in some spots. It is an unusual presentation but I think the point of fact is that they are present in a respiratory tract specimen and they really probably shouldn’t be there.

The next slide shows you a Pap smear showing what almost looked like arthroconidia produced in there. And you see some large hyphae that are septate and you see some that smaller. And this turned out to be one of the Mucorales that we saw. And it is not supposed to be there. On the other hand it is in a non sterile site. From a non sterile site so the significance of it would have to be weighed by the clinician. Then they would have to decide if they were going to do a biopsy or not to confirm that that’s the cause of the problem in that patient.

The next slide shows you just another Pap smear. And it shows a hyphal strand. I really don’t see more than, you may a spot or two where there may be a septation. It could be one of the Mucorales or it could be an Aspergillus. And that’s a situation we are dealing with all of the time, trying to sort these two out and sometimes you can’t do it. Sometimes there is just no way to tell.

The next slide came from a heart valve of a patient with endocarditis. And I did a touch prep of the lung at the autopsy and of the heart as well. And this was the vegetation that was found on the heart valve and I don’t remember which valve it was. This is what we stained with methenamine silver stain and look at all of the dichotomous branching that you see there. This is from a sterile site and something like this probably represented Aspergillus which it turned out it did. This patient also had Aspergillus meningitis.

The next slide shows you a higher power of view and look at the acute angle branching and look at the septations in there. So this is, was a major problem, a major problem in that patient that probably killed that individual as a matter of fact. So, this is a case of fungal endocarditis caused by an Aspergillus and I think the features are there. The septate hyphae and the acute angel branching is probably about as good as you are going to see.

The next slide shows you another field from that heart valve. And you can see again the acute angle branching and you see a few septations in there. Sometimes we just show you different fields just to let you get a little experience looking at some of these things because they don’t all look alike and sometimes you see one thing in one field and something else in another. And this one right here you don’t see the number of acute angle branches that you saw in the previous slide but you can see some septations in there too.

This is a methenamine silver stained slide from that heart valve. And what you see there is part of the valve and then the hyphae just all around it. And generally what happens is the heart valve will then develop a vegetation on it and often times you can end up getting a blood clot formed and it will just break off and it will go some place and cause some major damage to wherever it happens to go. It will block an artery. It will stop the blood flow to an area and everything will become necrotic from that point on so a case of endocarditis these are serious matters, particularly of fungal endocarditis.

The next slide shows you just another view of what you might see on a heart valve. There is just hyphae stained with methenamine silver stain.

The next slide came from, actually from a blood vessel. And I think I have a better slide to show you here in a minute. This shows just a few, maybe one area where you can see some dichotomous branching maybe more than that but you just see septate hyphae and that is all you can report as you see septate hyphae. And the clinician is going to have to then decide what am I going to do with that. Well, if it is from a sterile site, if it probably, if it seems to be a significant finding then they will go ahead and do an open lung biopsy. But in often times, these immunocompromised patients, they have low platelet counts and to try to biopsy one of those patients would be very difficult because if a bleed might occur in that patient, and so they have to be very careful. They have to weigh the significance with some of these things even if they are from a sterile site.

The next slide shows you, actually a cavity, a pulmonary cavity. You see in the very center there, there is a mass inside that cavity. And that’s a fungus ball. And if you look at the tube that’s going all over the top about 12:00 o’clock, that happens to be a bronchus that’s been cross-sectioned. That’s where the air gets into that cavity. And so this mass in there is a fungus ball that is made up of just nothing more than a mass of hyphae and some host material. And you’ll see some dichotomous branching on there.

This is just a, next slide, is a better view of that bronchus. And you can see where the air gets into that cavity, that fungus ball has been removed at this point.

The next slide shows you what you would see in that fungus ball. And you are going to see just a mass of hyphae with the dichotomous branching or acute angle branching present. And that occurs in an old pulmonary cavity caused by maybe old tuberculosis infections, some other infection that’s left behind the cavity. That organism grew in there and it grows unrestricted because it has a round space to grow and it will grow as a ball and it will sometimes sporulate and if it has an airway, it will sporulate maybe and what you are going to see is just a mass of hyphae with maybe a few spores on it. And that’s all it is and the treatment is to go in and remove that fungus ball but the difficulty with that is that often times there is a lot of bleeding associated with trying to get rid of a fungus ball like this.

The next photograph, the next slide shows you Charcot-Leyden crystals. When you see Charcot-Leyden crystals, that means that the eosinophils are beginning to break down. Aspergillus is one of the things that has Charcot-Leyden crystals associated with it. So this would tell you, if you saw hyphae in this, this is just showing Charcot-Leyden crystals here, but if you saw hyphae in an area right close by, pretty much know you are going to be dealing with an Aspergillus.

The next slide shows you an area where an organism has caused extensive thrombus formation. And that is nothing more than just a big clot that has formed inside of a vessel. And it has formed because the hyphae have penetrated a vessel and then the neutrophils, the all white cells and red cells have actually aggregated along with a platelets onto the hyphae and then it forms a big clot and thrombus inside, occludes the artery, and all of the blood supply to that area ceases and so the area becomes necrotic. And that is not an uncommon thing to have happen.

The next slide shows you a mucous plug from one of those cases of allergic broncopulmonary aspergillosis as stained by the methenamine silver. You still can tell the orientation, it’s a rectangular type orientation going from left to right and inside that plug will be all of these hyphae. And they don’t necessarily show dichotomous branching, they can, but you see here just hyphae. If you see a mucous plug like this, it is more than likely an Aspergillus and if the patient has symptoms of allergic bronchial pulmonary aspergillosis.

The next slide shows you what these hyphae look inside that artery. And you can see that there are septate hyphae and a hint of branching in there. And you can see the wall of the vessel around the outside of there. So this is what it really looks like on higher power, nothing more than just hyphae.

The next slide shows something that sometimes causes problems in pathology. This is a biopsy from a lung that shows a lot of round cells. It shows some hyphae in there, too. You can see the hyphae but if you look you’ll see that there are a lot of round cells in there. Basically, those round cells are nothing more than cross-sections of hyphae as you would be looking down through them. If you think of a garden hose that you’re looking at it and you slice it and you are looking down into the hose itself, it’s the same sort of scenario. And so those round cells are often referred to as yeast by some people in pathology and they’re not yeast at all. They are just nothing more than cross-sections of hyphae. And I think it’s important to know that.

The next slide shows you what you might find in a case of cavitary aspergillosis. And this, you see the hyphae in there and you see the dichotomous branching but more importantly you see that there are quite a few heads of Aspergillus, fruiting heads of Aspergillus in the cross-sections. So basically, the area in the center that’s white is the vesicle of an Aspergillus and around the perimeter is going to be those phialides. And you don’t really see the conidia on there, at least on these at this power. But you can see some of the vesicles there that look almost hemispherical that are not cross-sections through the top one about maybe 4:00 o’clock.

The next slide shows you essentially the same thing. You see septate hyphae in here and you see the vesicles that are covered with phialides, just looking at it down from the top to the bottom. And these are big round spherical structures. Some of them are club shaped. But you are looking at them from the top to the bottom except the one at about maybe 2:00 o’clock, almost in the center you can see that it has some phialides coming off the top very well and it’s lighter in the center where the vesicle is and you can see that and it looks like it only covers the upper half or those phialides only cover the upper half or two thirds of that vesicle. In that case you can probably say this is probably going to be Aspergillus fumigatus because of that feature. And if you look around in the background you will notice that there are some rounded cells sitting in there. Those can even be the conidia of Aspergillus because you are not going to see this except in a cavitary lesion with a communication with an airway and they can sporulate and those may be conidia in the background, it is hard to tell from here but I think that is probably what they are.

The next slide shows you a really pretty view of a cross-section of one of those fruiting heads of an Aspergillus in a lung biopsy. And if look all around the perimeter of that thing, you see that there are conidia. And those conidia are kind of spiny. They have spines on the outside of them. And so looking at this, you would think that, well, which Aspergillus is it going to be. Well, Aspergillus niger forms, it usually is black, conidia are dark and they are rough walled. And looking on here, some of them appear to be dark but more importantly, I think you can see that the vesicle is round in the center. And you can see that there are phialides around the outside and in some cases there is a branch down below them, the metulae. And probably this is going to be Aspergillus niger. It is hard for me to tell from here and would probably not try to make that diagnosis by looking at this but just guessing that might be what it is particularly with those rough walled conidia. So this is what you would see in a cavitary lesion that has a connection to an airway.

The next slide shows you and H&E stained piece of a portion of some respiratory tract specimen and it just shows you the dichotomous branching. This is probably inside of some sort of a mucous plug. You can see the perimeter there that there are hyphae that are showing the dichotomous branching and the other hyphae are kind of matted together. You can see some septations in there.

And the next slide just shows you more of the same. And they are concentrated, and they are almost like micro colonies growing in a sample. And that does happen sometimes where not in a case of allergic bronchial pulmonary aspergillosis but in a case where you have an organism that happens to have some space and it causes these hyphae to begin to form kind of a clump. They look like micro colonies.

The next slide shows you a biopsy with a methenamine silver stain showing mainly what we have been talking about. Dichotomously branching hyphae with septations and nothing more, that is about it. So it would be hard to say if it is an Aspergillus, it probably is but it could be anything and this could be just from a biopsy from a patient with evasive aspergillosis or a patient with a fungus ball.

And this next slide is a methenamine silver stain slide showing you a little different view of what the hyphae may look like. They are kind of twisted around. You do see a Y and acute angle branching in a few spots. And the methenamine silver stains it well and there are septations in there so we we’ll look at another one.

And you see more of the same but you see some of the swollen cells in there. In this case there is one at the terminal end of the hyphal strands.

And then the next slide shows a lot more of those swollen cells and then you start to see those cross-sections of hyphae. And that’s an important thing to bring up.

I think the next slide shows you more of the cross-sections.

And the next slide shows you a large number of them. And many times as I have mentioned before they will be reported out as yeast present along with hyphae. And those are not yeast, simply cross-sections of hyphae that are oriented in a position that they are vertical looking down on top of them and we’ve cross-sectioned them. So, this is probably going to be an Aspergillus too.

The next slide is another Aspergillus and you can see there is a lot of dichotomous branching in this and this is methenamine silver stain. And that is all you may see. It looks like you see some of those cross-sections in there and so you have to look at more than one field to get an idea of what’s out there. And this case, you know, you may not have to do that, you see about every feature you want to see.

The next slide is one that represents an unusual case. We had a liver biopsy submitted for direct microscopic examination and culture. And looked at it using calcofluor white and saw this mass of hyphae. And that represents an awful lot of organism in a sample, particularly if it is a fine needle aspirate from a liver. So we looked at it and decided we had better make a few phone calls. So we called one of the residents, and the resident said, “This patient is not particularly sick.” It may not be anything that we think is going to be significant.” And we thought, boy, there is a lot in here too but it probably doesn’t make sense. So we called the attending physician and he said, “No, the patient is pretty ill. This may represent something.” So we then decided to call pathology and asked them what they saw in the biopsy and they saw nothing. So that opened up a window there trying to figure out what is this and where did it come from. So we called the area where the liver biopsy was done and we asked them, “What did you send this sample over to microbiology in?” And they said, “We sent it over in lactated ringer solution.” So, I asked for some of that and I got it and this is what I found in the lactated ringer solution and it was not in the liver, it was in the transporting solution. So, sometimes, you have to ask questions when things don’t fit. If you saw all of those hyphae in a biopsy, it just doesn’t make sense that patient would have that many in there and that patient would still be alive. So, we did a little investigating and that’s what we found in there.

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