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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 nocardia. 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 that are seen in the direct examination of clinical specimens.

This next slide shows you a number of stains and methods that can be used to detect fungi even though they are not designed to do so and an astute eye can pick up organisms using things like the Gram stain, the Wright stain and so on.

And the next slide shows you a continuation of those methods.  And there are a number of things here that you might not expect to find fungi in but if you look closely there are times that you certainly can find them.

The next slide we are going to talk about Nocardia even though Nocardia is not a fungus.  It is a partially acid-fast bacterium and is often times seen in the mycrobacteriology part of the laboratory or in the fungus lab.  And it is an organism that is a gram positive branching bacterium that actually shows coccobacillary forms and forms of beading.  So, you will see the branching of bacteria.  They are branching bacteria they are aerobic and they show beaded forms as you see on this slide.

The next slide is not very good but it does show in the background there that there are bacteria that exhibit branching.  If you look this happens to be a phase-contrast
photomicrograph which most people probably would not use but it does show, nevertheless, an organism is there and you have to look very closely in the background to see those branching bacteria.  It illustrates that same point when you look at a Gram stain.  You look at some other stains where you can see the same thing.

The next slide shows you a phase-contrast photomicrograph of a specimen, a sputum and there are numerous branching bacteria.  And it is important to remember that once you see a situation like this, it is not possible to distinguish Nocardia from Actinomyces because they are both Gram positive branching bacteria.  And this is not a Gram stain, by the way, this is phase-contrast microscopy but you would have to do a modified acid-fast stain to be able to distinguish between the two. 

So, the next slide shows you a Gram stain of Nocardia and it just illustrates the branching bacteria and they branch at all sorts of angles.

The next one, next slide shows you some material from an abscess.  And if you look closely you can see the Gram-positive branching bacteria in there.  And this is a Gram stain and it shows you very well.  And again, you cannot distinguish Nocardia from Actinomyces.  

This next slide shows you a Gram stain exhibiting numerous beading filaments of Nocardia that also exhibit branching.

The next slide shows you an acid-fast stain and there are branching acid-fast bacteria in here.  It is important, I think to remember, that when you look at these acid-fast smears that you look in an area where it is not so concentrated because in an area where the stain is concentrated, almost everything looks acid-fast and if you get away from that area out where the specimen is thin, you can recognize that some of the filaments are stained with the acid-fast stain and some of them are not.  And in this one it just exhibits good branching and it is difficult to tell if it is partially acid-fast or not.

The next slide came from a psoas abscess.  And this is showing you an elongated bacteria.  They are very filamentous bacteria in here.  You can see some beaded forms in there.  And this turned out to be a case of Nocardia

The next slide shows another acid-fast stain and the background is very faint.  But you can see the branching in there and that is what we are looking for.

The next one shows you some material from the psoas abscess again.  And there you can see the branching bacteria that are red and they are acid-fast.  But what we are looking for are partially acid-fast bacteria.  Some of the filaments will stain with the Kinyoun stain or whatever stain you chose to use -a modified acid-fast stain.  And others will take up the methylene blue stain or the counter stain. 

Now the next slide shows you what we were just talking about.  If you look in the background, you can see some branching bacteria but in the center there are some very faint red organisms in there and those are the acid-fast ones.  And there is a long one about maybe 6:00 o’clock, a little up from the bottom.  And you can see the elongated bacterial filaments in there that retain the acid-fast stain.  And then there are some that don’t.  And that is exactly what we are talking about, is the modified acid-fast stain will show you the partial acid-fastness of Nocardia.

The next one shows you a better illustration of that and you can see the acid-fast filaments and there in the background there are some that are not.  But you can see the beaded forms in this one just a bit.

The next slide shows you an even better example of Nocardia.  What is apparent here are the beaded acid-fast bacteria and then around near those would be the ones that are not stained with the acid-fast stain.  So you would see partially acid-fast bacteria some stained and some do not.  But the beading is very prominent. 

The next slide shows you an area that you would probably not want to look in.  This is an area where it is highly concentrated and almost everything is stained in the acid-fast stain.  And so I would move around to another part of the field, another part of the slide and see if I can find something that looks better than this.  This kind of re-emphasizes the point that we have made before in other discussions that you need to look at the entire slide before you make a decision as to what you are going to call something. 

The next slide shows you partially acid-fast bacteria up around 10:00 o’clock and in there are some in there that are standing kind of blue and those are the ones that take up the background stain.  So there are partially acid-fast bacteria in here.

And this is a silver stain that we use for detecting fungi.  And it deposits silver around the cell wall of organisms that it stains.  And what happens is it makes the cells look larger and they are actually easier to see.  And bacteria stained with a silver stain and this happens to be an example of Nocardia as stained with a methetamine silver stain.  And so you can’t tell if this is Actinomyces so you would have to order a modified acid-fast stain to be able to distinguish it from Actinomyces even though Actinomyces is not as common as it used to be.