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Published: February 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 3 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 3 in the series. Thank you, Dr. Roberts.
Thank you, Sharon, for that introduction.
This is another series in an introduction to clinical mycology, the third in a series of 4 presentations. Part 1 was diagnosis of classification and general features of the fungi and fungal infections. Part 2 was the basic structures of molds and yeasts and a brief introduction to culture. Part 3 presents specific information on the culturing and the incubation of cultures for the optimal recovery of fungi, and we will cover this today.
This slide shows a culture variation of Cryptococcus neoformans on different media showing that the colonial morphology is medium dependent. And that is something that we emphasized in the previous presentation. Emphasizing that you must not only select the right media, but you also must know what the organisms look like as they grow on those particular media to help with the identification.
We have shown in the past that we need certain compounds added to the culture media to allow us to recover the organism that we want to find. And the reason for that is there are bacterial overgrowths in many instances, because the specimens that we get for cultures we get are often contaminated. Gentamicin, chloramphenicol are 2 antibiotics that can be used in combination to inhibit many of the bacteria that will allow the fungi to grow through fungal media. Ciprofloxacin can be added to culture media and that is used to try to inhibit the growth in things like pseudomonas. Cycloheximide, another name is actidione, is a compound that we will see in just a moment here that is used for preventing the rapid overgrowth by some molds of slower growing molds. And then there are certain instances where we have used media that contain sheep blood enrichment because the organisms may require a bit more enrichment before they are able to be recovered.
Cycloheximide is a compound that is used primarily only in culturing. It is not an antibiotic for any treatment of any disease at all. But it allows for one to prevent the overgrowth of slowly growing molds by others that grow more rapidly. And we deal with certain things like the dimorphic fungi that grow, some for the most part, slower than things like Aspergillus or some of the other fungi. And, cycloheximide partially inhibits the growth of those rapidly growing molds so that we can pick up and see the growth of slower growing molds that are known pathogens. The dimorphic fungi are primarily the ones that we are trying to recover, and prevent the rapid overgrowth of rapidly growing molds, and cycloheximide allows us to do that. However, there is a problem using cycloheximide. That is that certain of the pathogenic fungi may be inhibited by cycloheximide, and it is necessary to use a medium that lacks that compound along with it.
This is an example of some of the fungi, and certainly the list is by no means complete, that are inhibited by cycloheximide. Aspergillus fumigatus, Cryptococcus neoformans, Pseudallescheria boydii, Candida krusei, and Trichosporon species. I will just point out a new name that is just recently been introduced, Candida krusei, has had a name change and I doubt that it will be a name that will be used by everyone, but it is called Pichia kudriavzevii and if you see this in anything you will know that this is a new name for Candida krusei. The Trichosporon species also can be inhibited by cycloheximide, so we use this compound to help us with the slower growing molds.
This is the example of Aspergillus fumigatus and on the plate on the right-hand side; the culture dish shows no growth. This is the culture plate that contains cycloheximide. If we were looking for that organism, causing aspergillosis in a patient, and we only used that particular medium, we would miss it. It would not grow and we would never see it and so we would miss the diagnosis. So the 2 plates that lack cycloheximide you can see grow Aspergillus fumigatus very well.
This is Aspergillus niger. If we had a patient who had a disease caused by Aspergillus niger, we would never recover it on the plate on the bottom because that plate contains cycloheximide. The other 2 would, since they lack cycloheximide. So cycloheximide is used for compound used in the right way.
This is Coccidioides. These are colonies of Coccidioides except for the one on the left-hand side about 9 o’clock that is bright white. All of the other colonies are Coccidioides immitis on a culture plate. They came from a contaminated specimen and cycloheximide allowed those colonies that were slower growing to be able to grow with out being inhibited by a rapidly growing mold.
Something that was developed years ago, is a medium called Smith’s medium. It simply uses a compound called ammonium hydroxide to treat contaminated specimens, and there are times when specimens submitted by mail take a long time to get to a place and to the laboratory and they contain a number of contaminants and things that will overgrow the very thing you are trying to find.
Basically, this is an example of 1 of those plates where you try to culture something and everything in the world is on that plate, probably including what you are looking for, it is just you can’t find it. You notice this has a number of notes on there. Someone saw something in the clinical specimen and said to save the plate because they were going to try and recover it but it grew everything except what they were looking for. So what Smith’s medium allows you to do is to take ammonium hydroxide, a small amount and place it on the side of the culture dish. After it has been inoculated by the clinical specimen, and as you know ammonium hydroxide is a very volatile compound, what happens is it spreads quickly across the plate and as it does it forms a concentration gradient. From the highest concentration where the drop is, as it spreads further it distally and becomes less, and it is like an antibiotic disk and it allows things to be recovered.
This happens to be Blastomyces that was recovered from a clinical specimen. And if you look at the right-hand side of the plate you can see bacterial colonies along there along with some of the fungal colonies, and as it gets further across to the left-hand side of the plate, there are just fungal colonies. This allowed for the organism to be recovered, whereas, it would have been missed probably or not grown well at all on a plate that didn’t have the ammonium hydroxide added to it.
You notice that what we have shown before have been culture dishes and there certainly are advantages and disadvantages to using these. In laboratories with little experience, this is probably not a very good idea; it is a risky situation to use culture plates. But in a laboratory that does a moderate to great volume of fungal cultures, these culture plates allow you to have a large surface area for the isolation of colonies. It provides good aeration of colonies. It is certainly easier to make a microscopic mount by just simply opening up a culture dish and making the mount. One of the problems is it is easy to get these plates contaminated. An open system is less safe to handle in the laboratory. And culture dishes are apt to dry out unless you use deep pour plates. Deep pour plates are meant to be like 40 mL of culture medium or more.
This is the example of how culture dishes can dry out, and if that happens the plates are virtually useless. So, if you happen to be in areas where the humidity is very low, you will have this problem. So deep pour culture dishes are preferred. And, culture dishes work fine as long as you work in a biological safety cabinet.
Use of tubes for cultures. The advantages and disadvantages of those, well, culture tubes certainly you have less dehydration because generally, the lids are kept tight. And, you have the lack of isolation of colonies in there because when you inoculate the tube, the substance all runs to the bottom, or the butt, of the slant and everything grows in one place. If the caps are tightened, no air is going to be able to get in, no oxygen will get in there and the cultures will become nonviable, so the lids need to be left slightly ajar. Culture tubes are certainly safe to handle, and in most instances are safer to handle because most people do tighten the caps so nothing can get out, and you can’t do that. It is very difficult to make a mount from down inside one of those tubes; it can be done, but it is not easy. And, certainly, a culture plate is easier for that to happen.
This is an example of a culture tube. You can see all of the growth is concentrated in the area down at the butt of the slant. Not a very useful situation unless you happen to have a pure culture.
The incubation of cultures – fungal cultures pretty much grow best at 30 degrees centigrade. And that is what we recommend as a temperature for growth for trying to recover these organisms. The humidity needs to be high because otherwise the media will begin to dry out. We mentioned earlier the culture plates need to be about 40 mL in volume, and once something grows, the plates need to be taped. They need to be taped at the beginning of incubation to prevent the lids from coming off, preventing a spill. And, if you use tubes, you need to use a large bore tube, it gives you much more surface area for the slant and in the butt. When you inoculate the tube with clinical specimen you need to place it on its side so that the slant is facing up, horizontally. Allow that to sit for 30 to 45 minutes so that the specimen is allowed to soak into the agar and then the organism will grow without everything running to the bottom. Most places just simply don’t take the time to do this.
Terms of incubation of cultures – you can incubate the cultures in any incubators, in any way you like. At Mayo Clinic what we use are cafeteria trays. And you notice that all of the culture dishes are placed in bags and sealed. There is a reason for this, it prevents dehydration, but it also allows us to work safely with the cultures, that we can read those plates through the bags without having to take them out.
This is the example of taking a tray out at a time. You can pick the cultures up to look at them on the front side, the reverse side. You can look at the morphology and then if you have to work with them, you work with them inside of a biological safety cabinet.
If you happen to have a culture that grows up a significant pathogen, or an organism that grows a lot of volume of an organism that is going to contaminate your incubator, you need to seal the plate with tape if you plan to keep it for any length of time. If it is something that you have already identified and it looks like it’s going to be a problem, you can simply just autoclave it. But if you are going to keep it for a significant period of time, make sure you tape it up with oxygen-permeable tape.
There is a problem that you run into sometimes, a mitey big problem. And you can see that these happen to be media mites. Media mites get into cultures and they can contaminate 1 plate from another. I often joke and say, “You know how you can tell if you have mites in your cultures? You look for their tracks.” And you can see there are tracks as they walk through a culture. It is interesting some of these mites have a preference for which organism they like to eat. In our laboratory, that happens to be Penicillium, and they will walk through a colony of Penicillium, and they will walk around the culture plate, and you can see later where they’ve walked. You will see tiny colonies appearing as they walked around. So that is another reason that they are in sealed bags.
Well, this is the finish of Part 3 Introduction to Clinical Mycology. Part 4 will include methods for identification of fungi, primarily the molds, and some helpful hints for working in the laboratory.