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Published: January 2012Print Record of Viewing
An introduction to the field of clinical mycology. Dr. Roberts reviews the basics of mycology, including culture and identification of organisms encountered in the clinical practice. This is Part 2 in the series of 4.
Presenter: Glenn D. Roberts, PhD
Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.
Our presenter for this program is Dr. Glenn Roberts, a Professor of Laboratory Medicine and Pathology, and Microbiology as well as a consultant in the Division of Clinical Microbiology at Mayo Clinic in Rochester, Minnesota. Dr. Roberts provides a 4-part introduction to clinical mycology, including culture and identification of organisms encountered in the clinical practice. This is Part 2 in the series. Thank you, Dr. Roberts.
Thank you, Sharon, for that introduction.
The introduction to Clinical Mycology, this is a second in a series of four presentations on clinical mycology. Part one was a diagnosis, classification and general features. Part two reviews the basic structures of molds and yeasts. It presents a brief introduction to the direct microscopic examination of clinical specimens and also discusses media that are useful for culture. And we will cover these today.
This is a list of general terms using clinical mycology and the thing that I think most people have trouble with is the language that we use. It is not that the discipline is so difficult to understand, it is the terminology that we use to try to define what we are looking at. Well, these terms here give you examples of things that we use in an everyday basis, and we will just go through these and you will see them enough times that I think they will become clear as we go along. Hyphae- these are tube-like structures that compose a mold colony. If you think of hyphae as being a long garden hose that transports medium and nutrients to the body of the fungus; that is what a hyphal strand is. And we call them hyphae is plural, hypha is singular and we sometimes call them hyphal strands. We know that in some cases the hyphae are divided up into compartments by cross walls which we call septae. And these allow the organism to survive if these pieces break apart. Each one of those can become a separate organism. There are hyphae that lack septations and we call these nonseptate hyphae. There are hyphae that lack pigment, that are just simply clear and stain with the dyes that we use to examine them and they are called hyaline hyphae. Some of the hyphae are pigmented by a dark pigment whether it is brown, dark gray, or black and these are called dematiaceous hyphae. There are structures within the fungal hyphae and at the end of a hyphal strand that are nothing more than an over wintering, and there are round structures in there, chlamydoconidia. And then we see commonly the asexual spores produced by the molds that have septations in their hyphae and these spores are called conidia.
This is an example of what we call the universal fungus. It shows many of the different kinds of spores that are seen as well as some other structures. If you notice, at the bottom of this illustration is called vegetative mycelium. Mycelium is a collective term for hyphae that are grouped together to make a colony. It is an old term that is not used very often. But these are hyphae that can form a number of structures. If you look at the bottom right hand side, you will see what are called arthroconidia. On the right side you will see the term. This is a hyphal strand that has broken down into compartments by the production of septations. This is the simplest mode of reproduction of the fungi. On the very bottom right hand side, the term chlamydospore can also be called chlamydoconidium. This is what we talked about a minute ago. These are over wintering type spores formed on the inside of a hyphal strand or at the terminal end of one. If you notice on that same strand by the chlamydoconidium, you will see some small spores coming off the side. Those are called microconidia. There are two kinds there and on the far left side you will see one that has a green color and that is called macroconidium. These are 2 extreme examples. One is a large spore and one is a small spore, denoting macroconidia, microconidia, respectively. And we use all sorts of terms. We use the Zygomycetes. We use the sporangium that is the structure that produces the spores. If you notice the one that looks like the fingers on a hand that is branched, that is Penicillium. You will notice the spores are produced in chains at the tip of an elaborate structure. Spores are produced in a number of different ways. The morphology of the fungus depends upon the arrangement of the spores and how they are produced and we will look at some of that as we go along here.
Well, chlamydoconidia and arthroconidia may be present in almost any of the molds. Some of the fungi that we deal with reproduce in an asexual way and also a sexual way. The results of some of the sexual reproduction among some of the classes of the fungi result in some spores that we call ascospores that we see not very frequently. Some of the fungi that we deal with are dimorphic. They have a mold form in nature or in the culture and have a yeast form in the patient or at 35-37 degrees centigrade. Most of the colonies of the molds are fluffy or fuzzy. They are filamentous organisms that are made up of hyphae and when you look at them microscopically you notice that they do look like fuzzy colonies. And for example, some of the laboratories just simply call them fuzzies because of that.
This happens to be an illustration of a chlamydoconidium. It is probably the world's largest chlamydoconidium that you have ever seen. But it is nothing more than a round spore that is sitting in inside of the hyphae or at the end of one.
This is an example of hyphae with arthroconidia. On the lower left hand side about 7 o'clock, you will notice that the hyphae are beginning to break down into spores that are kind of rectangular almost like a rounded ends on them. Those are arthroconidia. And that is the simplest mode again of the reproduction for the fungi. Each one of those will break apart and germinate and form an entirely different organism. So we see arthroconidia forming with almost any of the fungi. They are not specific for any particular organism at least in this format.
Here is what it would look like underneath the microscope if you had the stained preparation. You will see that these are breaking apart. They are rectangular in shape and each one of those again is a separate unit.
With the Zygomycetes, we define them as having pauciseptate hyphae. Pauciseptate means there are a few septations or can be a few septations but for the most part the hyphae do not have septations within them. Zygomycetes produce sporangia, the spherical structures that are produced at the end of a long stalk, the sporangia produces spores. The hallmark of Zygomycetes is the growth rate, very rapid growth. They may have root-like structures called rhizoids that anchor them to the substrate surface. They may not and if they do have them, then the arrangement of those rhizoids helps with the identification.
This is the example of nonseptate hyphae. These hyphae are large. They transport nutrients to the body of the fungus but there are no septations and so if one of these hyphae happens to break apart, all of the nutrients will leak out. The cytoplasm leaks out. The whole entire hyphal strand will become nonviable. And this brings up a point about culturing which is not discussed at this point but I will just tell you anyway and that is that, if a patient happens to have an infection that you suspect is caused by Zygomycete, you need to let the laboratory know about that because in some laboratories the tissue is ground up and processed in that manner before it is cultured. If it happens to contain a Zygomycete, all of those hyphae will be destroyed, the cytoplasm will leak out and everything will be rendered nonviable and you will never recover the organism. So then if you suspect Zygomycetes there, the clinician tells you that , then you can cut up some small pieces of tissue or whatever it happens to be and culture those and hopefully, you will get a viable segment of an organism in there that will grow so you can identify the organism.
This is what the hyphae looked like underneath the microscope that are nonseptate. You can see no septations in those at all.
This is an example of pauciseptate hyphae. There are a few septations in there. Not more than about 3 or so, and with hyphae that produce regular septations you will see it much more frequently than you see are produced here. So this is an example of what we call pauciseptate hyphae.
This is a sporangium of a Zygomycete that shows you a big saclike structure. On the outside you see what looks like the wall has been removed and you see the spores that are there. At the very base of that, you see where the sporangiophore has then attached to that sporangium. In that whole big structure there will rupture and all of those spores will pour out into the environment and each one of those is capable of starting a new colony. So that is a sporangium of a zygomycete.
The ascomycetes are a group of fungi that have septate hyphae. As a result of sexual reproduction they produce ascospores that are unique to that particular group. Most of the fungi within that group produce asexual spores called conidia that we use to identify the organisms.
This is the example here of what sexual reproduction would result in a production of ascospores. The ascospores are produced inside of a sac called an ascus and usually there eight ascospores inside of an ascus. Another name for ascus is ascocarp. It just means a structure that produces ascospores. So this is what they would look like. Sometimes though those are produced in long tubular type arrangements, sometimes they are in a cyclic arrangement like you see here.
This is a good example of what one of the ascomycetes might look like. There are lots of them. Have you ever had an orange or a lemon in your refrigerator that has been there too long and you noticed it has started to mold? Generally, that is caused by something like Penicillium. That is one of the organisms that belong to the Ascomycetes. And as you know, penicillin is produced by Penicillium, and by a particular species of Penicillium. Mold on this orange, you can see that it is a green kind of mold and it is Penicillium. If you look at it underneath a microscope, on the right-hand side you will see it has some pretty elaborate looking structures and at the very tip of what looks like a little bottle- like structure are the conidia. Those are the asexual spores that are produced by Penicillium, and those are the result of asexual reproduction.
Well, another group of fungi that we deal with is called Basidiomycetes. They produce septate hyphae but they include mushrooms, the smuts like corn smut, wheat rust, and some others. They produce spores called basidiospores. When the organism reproduces sexually, and this happens with the mushrooms they produce conidia in the asexual form.
This is an illustration showing you a mushroom on the right hand side and if you look on the underside of the cap of this mushroom, you will notice this is made up of gills. On either side of a gill are these structures called basidia and they produce the basidiospores. The basidiospores are produced on little denticles off of the basidia and they hang down and then those are spread around by the wind or whatever happens as the mushroom cap begins to dry. The basidiospores are not something that you would see in the laboratory at all. Something that is interesting, though, is that we have one pathogen that we deal with that belongs to the group of Basidiomycetes; that is Cryptococcus neoformans. And the rule in mycology is that if you find this sexual form of an organism you give it one name and you give the asexual form another name. Well, the asexual form of this organism is Cryptococcus neoformans. The sexual form is called Filobasidiella neoformans. And this is a very common human pathogen. The problem with this nomenclature is that when you try to change the name of an organism whose name has been around for 25 or 50 years or longer, it is not accepted very well to change that name by people who do the work. And so nine times out of ten, you rarely hear the term Filobasidiella neoformans even though it is botanically correct. So this is the example of something that belongs to the Basidiomycetes.
We have another group of fungi that we deal with in the clinical laboratory and these are the yeasts. The yeasts primarily are made up of budding cells called blastoconidia. Blasto means bud and conidia means spore. Some of these yeasts produce pseudohyphae and true hyphae. We know true hyphae are these filaments that look like garden hoses. Pseudohyphae are nothing more than yeast cells that have elongated and remained attached to each other and they look almost like a hyphal strand except that where the yeast have elongated the ends are rounded. And you will notice that it is not one big long filament, it is made up of a number of the elongated cells that have rounded ends that are still attached to each other. They look like links of sausages. Ascospores are produced by some of the yeasts and you could use special stains like the acid pas stains to be able to show those if you need to do that. Most of the yeasts produce creamy bacterial-like colonies. If you grow yeast in the laboratory, you know there it will smell almost like a bakery. The yeasts are very important to agriculture. They are very important in the baking and brewing industry but they are also very important in the clinical setting because they do cause human disease and lots of problems from immunocompromised patients. Some of the fungi are dimorphic in that they produce yeast in the tissue or at 35 to 37 degrees centigrade in the laboratory under the right conditions that you would have to supply.
This is an example here of a yeast underneath a scanning electron microscope showing you some of the structures that are labeled. The cell is the YMC is a yeast mother cell. That is the primary cell. On the left of that, you will see what looks like is called a pseudohyphae. It is starting to form one. It is elongated and it is narrowed down where it attaches to the yeast mother cell. On the cell above it, you will see a round cell. On the right hand side it says GT. That is a germ tube and that differs from a pseudohyphal strand in that it is not narrowed down it just comes straight out of the yeast cell. And then we use a germ tube as a means of trying to identify Candida albicans. If you look on the left hand side you will see there is an elongated cell, a hyphal strand, it is a hypha. So you see these structures with yeasts and as we go along maybe you will see some of these.
This is an example of how the yeasts reproduce. They reproduce by buds, by blastoconidia. And the small cell on the right coming off of the mother cell is the blastoconidia.
Just another example of budding yeast cells, you can see the small bud coming off a number of those cells. So yeasts reproduce by budding.
These are yeast colonies. This happens to be Candida albicans. It is bright white but you cannot identify yeast based on their colony morphology or the color. You still have to do it other ways to which is morphologically and biochemically to put a name on these specifically. But these are small colonies. They are bacterial like. They are not mold like colonies at all.
An example of what we talked about a minute ago. The PH shows you pseudohyphae. You see those elongated yeast cells that have the narrowed ends. They look like links of sausages attached together and on the right hand side you see a blastoconidia or a yeast cell, big cell at the bottom with a bud at the top right at the tip of the arrow, that is a budding yeast cell, the blastoconidium. So this is what you might see a combination of those yeast cells and pseudohyphae. Any time you see a combination of a budding yeast cells, hyphae, or pseudohyphae, you know you are dealing with something that belongs to genus Candida because that is the characteristic of that particular genus.
There are times when you see yeast cells that produce by buds and also may produce arthroconidia. Now in a case like this, you would have to discern which is the predominate form that you see. This is an example of an image that shows arthroconidia and yeast cells. And there are some yeasts that produce both of these, predominately you will see budding yeast cells or you may see some that predominate with arthroconidia and a very few number of blastoconidia, or a budding yeast cell.
Terms of being able to detect fungi in clinical specimens, we do have methods for detecting these organisms in clinical samples. The KOH or potassium hydroxide preparation is probably the time honored procedure that has been used for so many years; it still works; it is old but it still works. We add a compound called calcofluor white which is a fluorescent brightener to that and it allows us to be able to use a fluorescent microscope and increase the sensitivity and be able to see morphology better of some of the organisms that are found in clinical specimens. The PAS stain, the Periodic acid-Schiff stain has been used for many years. It is not used so much now in clinical laboratories. It is a tissue stain but it still works. The Gram stain is used for scanning bacteria as you probably know but it also stains fungi and so when you are looking at some of these stains that are not specific for fungi, you need to think Microbiology a little bit because you are apt to find some of these things that don't normally stain the things you are looking for. For example, the Gram stain will stain bacteria well but it also stains fungi. And you need to be conscious of the fact that you can see other things with some of these stains other than what you are just looking for. The Wright's stain is used in Hematology. You can see some of the fungi stain in blood films or on bone marrow cells. You can also see a slide that is in preparation of a spinal fluid to look for cell count.
Calcofluor white is used as I mentioned as a fluorescent brightener. It is a nonspecific stain that binds with a chitin in the cell wall of the fungi. You have to use certain filter combinations, 440 nanometer filters. They are very costly but they work well. And the calcofluor white needs to be made up ahead of time and kept in a bottle that is dark and is covered with foil because it is light labile. But it is a very useful tool in the clinical laboratory to be able to stain to look at the fungi.
This is an example here of calcofluor white. You can see that this organism with a filter combination appears to be blue white. And there are septations where the tip of the arrow is. So this would be an example of septate hyphae in a clinical specimen. Now in a non-sterile site, it may or may not mean anything but if it is from a sterile site, then you certainly would be able to report that all you would be able to say is that you see septate hyphae in the specimen because many organisms can look like this.
In terms in the topic of microscopy that can be used to examine the clinical specimens, bright-field microscopy can be used for things like Gram-stain and so on. It can be used also if you turn the light down or you close the iris diaphragm down you can get some contrast. You can see organisms in clinical specimens. Phase-contrast microscopy is not used so often any more but it does a nice job of being able to allow you to see fungal structures. The Auramine-Rhodamine stain is used in a mycobacteriology laboratory. It can stain organisms like Blastomycetes. The Ziehl-Neelsen or the Kinyoun stain is an acid--fast stain that uses methylene blue as a counter stain. And the organisms may not necessarily stain acid- fast, but you can see them with a counter stain. So you have to look beyond the concentrated acid- fast part of the stain. The Papanicolaou's stain is used in cytology for looking for cancer cells. You can see fungi in the Pap smears, the sputum, and other clinical specimens. And then in the clinical laboratory where the pathology is involved, you notice the Gomori methenamine silver, the H&E, the PAS stain, all those stains there. Some of them are used to detect tissue morphology; others are used to detect fungi. But when you are looking at this section, you have to be thinking about anything, and the fungi are one of the things a pathologist has to be looking for. The microbiology laboratory can be of some help recognizing some of those fungal elements.
In terms of trying to make a definitive diagnosis of an organism, first you have to be able to culture and grow it up before you can make an identification. Then you are faced with which media do you use for trying to grow these organisms. There are a number of media available that have been around for many years, a few new ones. Brain heart infusion agar has been around for a long time, works fine for recovery of fungi. Sabouraud's dextrose agar is one that has been written about in text books for many, many years and is probably the worst medium for growing fungi that we have. It is a good subculture medium but is a poor primary recovery medium. Inhibitory mold agar is an agar that is very useful for the recovery and it does not inhibit molds like it sounds like it might. It has chloramphenicol in it that inhibits bacteria and allows the molds to grow. Sabhi agar is a combination of Sabouraud's and Brain heart infusion agar works well. Mycosel is Sabouraud's agar that contains chloramphenicol and a compound called cycloheximide. Works fine for recovering dermatophytes primarily, but not much of anything else. Potato flakes agar is developed in recent years at the University of Texas in San Antonio works fine for primary recovery. Yeast extract phosphate agar will be shown as an example a little later on how to recover some of the organisms from contaminated clinical specimens.
This is an example of something that is important for you to remember in the clinical laboratory. Not only are you faced with which media do I use for recovery of things but once you decide which ones you are going to use, you must become familiar with the morphology of the organisms on those particular media because they can look different depending on the medium. This is the same specimen; same amount inoculated onto three different plates of media all containing different ingredients. The upper left is Sabouraud’s dextrose agar. This happens to be Cryptococcus neoformans on Sabouraud’s agar where it is white. The upper right hand side is Inhibitory mold agar; the colonies are kind of golden. And the bottom single plate is Brain heart infusion agar that contains blood enrichment and notice the colonies are more compact and a little bit off white. So that is pretty consistent for those medium. You need to not only know which media to use but know what they look like on those media to help you with the identification.
This completes the Introduction to Clinical Mycology Part 2. The Introduction to Clinical Mycology in the future presentations for Part 3 presents specific information on the culturing and incubation of cultures for the optimal recovery of fungi and Part 4 presents methods for identification of fungi, primarily the molds, and some helpful hints for working in the laboratory.