1. Microbiology Lab 10
Basic Mycology

2. What is special about medical mycology?
Medical mycology is a specialized branch of medical microbiology. The identification of yeasts depends both on morphologic data and biochemica tests. The identification of filamentous fungi rests primarly upon the gross morphology of the colony and the appearance of the organism under the microscope. Because of the incredible number of different species of filamentous fungi, morphologic identification can be quite difficult. Fortunately, there are only a small number of pathogenic filamentous fungi, and most obey some general rules that make identification easier.
Here, we will examine a few organisms and discuss several cases of invasive fungal disease.

3. What is the difference between molds and yeasts
What is the difference between molds and yeasts? How are yeasts identified in the laboratory?
The first division in this large group of organisms divides the molds form the yeasts. Molds from aerial mycelia (grow up into air) and form complex structures (mycelia, spores, etc.). Yeasts do not grow up into the air, and do not form specialized structures. They divide by budding and daughter cells resemble their parent. On a plate, they form smooth colonies that can easily be confused with bacterial colonies.
Yeasts are identified by a combination of morphologic and biochemical tests. There are relatively few medically important yeasts. We are primarily concerned with species of Candida, which can be pathogenic in special circumstances, and Cryptococcus neoformans, which can cause disease in both normal and immunocompromised individuals.

4. What are some features of Candida albicans?
There are a number of species of Candida;
the most pathogenic is Candida albicans.
The pathogenicity of this species is probably due to its ability to form pseudohyphae in tissue, which allows the organism to adhere to mucosa, and may be important for invasion of tissue.
In the laboratory, we identify C. albicans by its ability to form germ tubes.
If a yeast forms germ tubes, it is presumptively identified as Candida albicans. This can be confirmed by a variety of fermentation and assimilation biochemical tests.
If it doesn’t form germ tubes, then it is either another species of Candida, or a member of another genus.

5. What are some more features of Candida albicans?
Major pathogenic species of the genus
Oval yeast in culture
Tissue shows oval yeast with pseudohyphae and true hyphae
Forms germ tubes when exposed to protein solution at 37’C
This can be used to presumptively identify a yeast as c.albicans
Part of the normal flora
Opportunistic infections. Immunocompromised, and those on antibiotics are susceptible.
Normal Tcell function and normal flora are important host defenses.

6. What are some features of Cryptococcus neoformans?
Cryptococcus neoformans is the only primary pathogen among the yeasts, and the only pathogenic species of the genus cryptococcus.
It is a round yeast (Candida are usually oval) with a large capsule.
The capsule is essential for its pathogenicity.
Mutants which lack the capsule are nonpathogenic.
All species of cryptococci produce urease, and this property is used in the laboratory to separate them from almost all the yeasts.
When grown on Nigerseed agar, the production of a black pigment can be visualized.

7. What are some more features of Cryptococcus neoformans?
Typically considered one of the monomorphic fungi.
Grows as round yeast in culture.
Primary pathogen.
Capsule is major virulence factor.
Others include: phenyl oxidase production (makes it grow black on niger seed agar), urease production (like proteus species and h.pylori)
Unlike other members of the speices, C. neoformans grows at 37-39’C.
Worldwide distribution.
Associated with pigeon droppings and eucalyptus trees.
Two variants (neoformans [pigeons] and gazzii [eucalyptus tress])
Causes the following: pneumonia, fungal meningitis with gradual onset of headache with or without fever. Sometimes patients will experience symptoms associated with hydrocephalus, such as nausea, vomiting, and cranial nerve defects.
Can be diagnosed by visualization, culture or a latex agglutination test. The latex agglutination test looks for capsular antigen.

8. How do you identify filamentous fungi in the lab?
As mentioned above, most of the organisms in this very large group of fungi have a complex structure.
The basic supporting structure is a filamentous mat called a mycelium, which is made up of either septate or non-septate hyphae.
Conidiophores, specialized spore-holding branches, bear conidia (the spore or reproductive form of the organism).
In many ways, fungi resemble plants more closely than animals, and mushrooms are actually large, complex (often edible) fungi.
The morphologic identification of filamentous fungi depends largely on the specific nature of their spores (conidia) and conidiophores.

9. What are the most common pathogenic fungi?
Only a small number of fungi cause invasive disease in healthy people. In the US, the four most common are:
Histoplasma capsulatum
Coccidioides immitis
Blastomyces dermatitidis
Sporothrix Schenckii.
All of these organisms grow in the soil. They are all dimorphic, which means that they have one morphologic form on culture medium, and a different form in animals.
They grow as molds (saprophytic form) in culture and as yeast-like organisms (parasitic form) in the infected animal.
The parasitic form is more resistant to oxidative killing than the saprophytic form.

10. What are some features of Coccidioides immitis?
C. immitis is unusual in that the spores of the mold (called anthroconidia) are found within the mycelia, so there are no specialized structures (conidiophores) for holding spores.
What you see in mature mycelia are alternating barrel-shaped conidia with empty cells in between.
The parasitic (tissue) forms are called spherules, are also very distinctive. They are double-walled, round structures.
They divide internally to form endospores, which are released when the fully mature spherule ruptures.
Each endospore can grow into a mature spherule.
Spherules are not yeasts, which divide by budding.
The parasitic forms of Blastomyces, Histoplasma and Sporothrix are yeasts, and this demonstrates the close relationship between the filamentous fungi (molds) and yeasts.

11. What must be done to make a ‘definitive identification’ of the primary pathogenic dimorphic fungi?
The definitive identification of the primary pathogenic dimorphic fungi can be made by getting the mold form to convert to the parasitic in vivo form in culture.
This is very time-consuming and a difficult undertaking.
The importance of this step depends on the organism.
The anthroconidia of the mold form of c. immitis are very distinctive, and only a few other organisms look like it.
In contrast, the mold form of Histoplasma capsulatum can easily be confused with the mold forms of nonpathogenic fungi, and thus it is usually necessary to convert it to the yeast form for definitive identification.

12. What’s an alternative method to identify primary pathogenic fungi?
An alternative method to identify primary pathogens is to demonstrate reactivity with antibodies that are specific to the antigens of the pathogenic fungus.
Such antibodies are commercially available and widely used.
More recently, nucleic acid probes have become available for the identification of some filamentous fungi and these are now used at UCSD to aid in the identification of Coccidioides immitus and Histoplasma capsulatum.

13. What are some features of Aspergillus?
Aspergillus species as examples of opportunistic fungi.
Unlike the primary pathogens, a large group of fungi are opportunistic pathogens that are capable of causing disease only in very special circumstances.
These fungi, which are monomorphic (growing as molds both in the patient and on culture media), usually cause disease in neutropenic patients or in patients whose PMN’s do not function normally.
People with normal numbers of functional PMNs are resistant to infection.
The organisms are identified on the basis of morphology alone.
One very important member of this group is Aspergillus fumigatus. This fungus has septate hyphae and a complex spore-forming apparatus consisting of a conidiophore that originates from the hyphae and supports the fruiting head (i.e., the spore-bearing structure). The mature colonies are usually gray-green in color.
Other species of Aspergillus differ in color and in the structure of the fruiting head. The other major opportunistic molds are the mucorales (Mucor and Rhizopus –discussed in Spring).

14. What are the three species of Aspergillus that are medically important?
There are many species of Aspergillus, but three are medically important, either because of their pathogenicity or because they are frequent laboratory contaminants.
They are:
Aspergillus fumigatus
Aspergillus flavus
Aspergillus niger

15. Candida albicans vs. Cryptococcus neoformans

16. Cryptococcus
vs. Candida

17. What is this? Give some features of this fungus.
Coccidioides immitis
Top photo shows “Spherule”
Bottom shows “Antroconidia”
Causes both pyogenic and granulomatous reactions.
Examine the spherules for endospores, which arise from internal division of the spherule.
Also note the septate hyphae and alternating anthroconidia.

18. Aspergillus fumigatus, flavus, & niger (2 lightings)

19. What is this?
Aspergillus fumigatus

20. What is this?
Aspergillus Niger

21. What is this?
Aspergillus Flavus