Laboratory work and demonstrations
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LABORATORY #5
Medical Mycology
Objectives: 1. To acquaint you with those few fungi which are of major clinical importance in the US. 2. To learn some simple techniques used to examine fungi in culture and clinical specimens.
Laboratory work and demonstrations
Do Not open any of the fungal cultures.
Cutaneous mycoses
1. Demonstrations
a. Fungal Cultures grown on Sabouraud's Dextrose Agar for 3 weeks at 30oC,
1. Epidermophyton floccosum
2. Microsporum canis
3. Microsporum gypseum
4. Trichophyton mentagrophytes
5. Trichophyton rubrum
6. Trichophyton tonsurans
b. Slides of wet preparations of each fungus culture above.
Subcutaneous Mycoses Demonstrations
1. Fungal Cultures - Mycelial forms grown on Sabouraud's Dextrose Agar
at 30oC on Blood Agar.
a. Fonsecaea pedrosoi
b. Sporothrix schenckii, mycelial form
c. S. schenckii, yeast form
2. Slides of wet preparations of F. pedrosoi, S. schenckii mycelium,
and S. schenckii yeast
Systemic mycoses
1. Demonstrations
a. Fungal cultures: Mold forms on Sabouraud's Agar. Yeast forms on
blood agar. Do not open any of the fungal cultures.
1. Histoplsama capsulatum--mycelium & yeast forms
2. Blastomyces dermatitidis--mycelium & yeast forms
3. Coccidioides immitis--mycelium form only
4. Cryptococcus neoformans--yeast form only
5. Candida albicans--yeast form only
6. Aspergillus fumigatus
7. Zygomycetes (rhizopus or mucor)
b. Slides of wet mount preparations of the above fungi.
c. Slides of tissues containing B. dermatitidis, C. immitis,
C. albicans, A. fumigatus, and H. capsulatum.
Work to be performed by each student
a. Prepare and examine wet mount of sputum containing B. dermatitidis.
b. Prepare and examine India ink slide of spinal fluid containing
C. neoformans
Introduction
Medical Mycology encompasses all fungi causing disease in man and animal.
Classically, the fungal (mycotic) diseases, or mycoses, have been
categorized as superficial, cutaneous, subcutaneous, and systemic,
depending upon the tissue or organ(s) involved. In classifying the mycoses,
as above, one must be well aware that there is considerable overlap.
For example, some of the systemic mycoses may exhibit cutaneous and/or
subcutaneous manifestations in addition to internal organ involvement.
Therefore, one of the first rules in Medical Mycology is to be aware of
variations in the host and the infecting fungal agent. Because of these
variations, overlap of clinical features, etc., the diagnosis of fungal
diseases is dependent upon the isolation and identification of the
etiologic agent, or in some cases, the demonstration of the morphologically
unique fungus in tissue.
Generally it is not the role of the physician to isolate and identify the
fungi from clinical specimens; however, he should be assured that the
proper specimen is collected and that the mycology laboratory personnel
are competent to isolate and identify the etiologic agents. Also, because
of the nature of some of the mycoses, the physician who is mycologically
astute may by simple examination of a clinical specimen obtain a diagnosis
within minutes, rather than waiting several days or weeks for a culture to
grow.
The two objectives of this laboratory exercise are (1) to acquaint you
with those few fungi which are of major clinical importance in the United
States and (2) to learn some simple techniques used to examine fungi in
culture and clinical specimens. With this knowledge you should be able to
recognize most common infecting fungi and to reach mycological diagnoses
more readily, since you may be able to isolate and identify the organism
yourself or at least may determine that the specimen is competently
processed by the laboratory personnel. Also, a working knowledge of Medical
Mycology should greatly increase your ability to interpret and evaluate
laboratory findings.
Because of time constraints, all fungi involved in human infections cannot
be examined in our single laboratory exercise. Consequently, you will study
the general morphological characteristics of only a few of the most
representative organisms. To obtain more detailed information on the
mycoses, you should refer to:
1. Manual of Clinical Microbiology, 5th Edition, American Society for
Microbiology, Chapters 57-65, 1991.
2. Kwon-Chung KJ, Bennett JE, Medical Mycology, 3rd Edition, Philadelphia,
1992, Lea & Febiger.
3. Rippon JW, Medical Mycology: The Pathogenic Fungi and the Pathogenic
Actinomycetes, 3rd Edition. W.B. Saunders Co., Philadelphia, 1988.
4. The information in the laboratory exercise will be complemented by the
Scope Monograph on Human Mycoses. Illustrations of the various fungi
in the "monograph" will be used as a reference for what you observe in
the laboratory.
A. SUPERFICIAL MYCOSES
In general, the superficial mycoses are mild, chronic, and of no great
medical consequence except for their cosmetic effect. Perhaps the most
common example of a superficial mycosis is tinea versicolor, which is a
relatively innocuous superficial skin infection, but may cause disease in
the compromised patient. The etiologic agent of tinea versicolor is
Malassezia furfur.
B. CUTANEOUS MYCOSES
The cutaneous mycoses are sometimes referred to as the dematomycoses,
ring worm or tineas. Current usage entails putting the suffix -osis to
the generic name, thus giving a more specific designation to the disease
produced by each fungus. The cutaneous mycoses would then be classified as:
1. Epidermophytosis - Genus: Epidermophyton--only one species
2. Microsporosis - Genus: Microsporum--numerous species
3. Trichophytosis - Genus: Trichophyton--numerous species
The cutaneous mycoses are infections of the epidermal tissue of human
beings and animals. The fungi of the three genera above are able to
penetrate and parasitize all the fully keratinized tissue of the body
(hair, skin, and nail); however, there is some tissue predilection by each
species--e.g.,
1. Genus Epidermophyton: skin (& rarely nails) Click Here to see an image
2. Genus Microsporum: skin & hair Click Here to see an image
3. Genus Trichophyton: skin & hair & nails Click Here to see an image
The fungi causing cutaneous diseases (dermatophytes) are with rare
exception, the only fungi causing communicable disease--i.e., are
transmitted by direct contact. Depending on the route of trans-mission,
the infection may be termed "anthropophilic" (man-to-man spread),
"zoophilic" (animal-to-animal or to-man spread), or "geophilic"
(soil-to-man spread).
A preliminary diagnosis of a dermatomycosis can be made by directly
examining the infected tissue (skin, hair, or nails) for the presence
of fungal elements. This is accomplished with a wet KOH preparation.
Infected tissue will contain hyphae of infecting fungus. Note: This
procedure will only indicate that a fungus is present, not which fungus
is present. To identify the infectious agent you must culture the tissue
or hair on media, such as Sabouraud's Dextrose Agar. The dermatophytes
are slow growing and should be incubated at room temperature (25-30oC)
for up to six (6) weeks.
The three genera of dermatophytes are morphologically distinguishable by
gross colony morphology, pigment production and morphology of their conidia
(spores). Representative species of each genus will be studied in the
laboratory to illustrate these differ- ential characteristics. Species
determination requires a more detailed study of the gross morphological
characteristics, spore characteristics as well as some biochemical tests.
Time will not allow for this detailed study in the laboratory.
1. Genus Epidermophyton (epidermophytosis) Gross colony: Tan to olive green in color, with slight aerial mycelium. Microscopic: Macroconidia abundant; conidia are oval to club shape, thin-walled,7-12 x 20-40 um, produced directly from hyphaeor in cluster. No microconidia are formed. |
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2. Genus Microsporum (microsporosis) Gross colony: In general, indis- tinguishable from Trichophytons. Microscopic: Usually an abundance of large (5-10 x 5-20 um) conidia (Macroconidia).Large conidia are pointed to round. There are usually few small condia (microconida). When present, they are 4-6 x 6-8 um in size and are smooth walled. |
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3. Genus Trichophyton (trichophytonosis) Gross colony: Variable characteristics, depending on species. Microscopic: Usually, there are few or rare, large conidia (macroconidia). When present, they are thin walled, smooth walled and are rounded at the end. Microconidia are usually abundant and are borne on the conidiophores in various ways , depending upon the species. |
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Student Work 1. Observe gross characteristics of each demonstration cutaneous fungus culture. 2. Microscopically observe the morphological characteristics of each fungus. Use low and high power objectives only. DO NOT USE OIL IMMERSION. 3. Note the following: a. Color of colony b. Shape of colony c. Texture of colony--fluffy, cottony, granular, etc. d. Pigmentation of reverse side of colony 4. Correlate the colony types and microscopic characteristics with the three genera. C. SUBCUTANEOUS MYCOSES The subcutaneous mycoses include a very heterogenous group of infections that are characterized by the formation of a lesion at the inoculation site. These infections are caused by the implantation of the fungus into the skin and thus are the result of traumatic inoculations. Generally, the fungus grows slowly at the inoculation site, causing a gradual spreading of the lesions. In some cases the organisms may reach the lymphatics but rarely becomes disseminated. Two examples of subcutaneous mycoses are (1) chromoblastomycosis (chromomycosis) and (2) sporotrichosis. These diseases are caused by "dematiaceous" fungi, that is, these fungi are darkly pigmented. (1) Chromoblastomycosis is caused by several different species of fungi included in three genera: Cladosporium, Fonsecaea, and Philaphora. This is a complex group of organisms taxonomically and only one genus will be examined in the laboratory.
Click Here to see another image
(2) Sporotrichosis is caused by the dimorphic fungus Sporothrix schenckii. This organism is dimorphic, growing in its mycelial (hyphal) form in soil, etc. and in the yeast form in tissue. Infection occurs when the mycelial form is inoculated into the tissue (or when inhaled), is subsequently converted to the yeast form, and continues to grow in the susceptible host tissue. Abscesses and granulomatous lesions are characteristic of this disease. The organism can be recovered from exudates or tissue. You will observe cultures and microscopic preparations of the mycelial and yeast forms of this organism.Student Work 1. Observe colony characteristics on the agar plates 2. Observe the microscopic characteristics of fungi in wet preparation and compare with the illustrations in this manual and monograph. D. SYSTEMIC MYCOSES The systemic mycoses are those fungal diseases involving the internal organs of humans and animals. Prior to the last decade only four or five species of fungi were considered to cause systemic disease; however, with the increased use of antibiotics, immunosuppressants, and the increase in chronic diseases (e.g., AIDS), the altered host is more susceptible to infections by fungi considered previously to be saprophytes and of no medical consequence. These latter fungi are now regarded as "opportunists," causing "opportunistic" disease. They are becoming an increasing problem in modern medicine and must be recognized as such by the knowledgeable physician. In this laboratory period you will study four of the fungi causing classical systemic mycoses in the United States. These fungi are: 1) Histoplasma capsulatum 2) Blastomyces dermatitidis 3) Coccidioides immitis 4) Cryptococcus neoformans In addition, you will examine three of the fungi most commonly causing opportunistic fungal infections in the United States: 5) Candida albicans 6) Aspergillus fumigatus 7) Zygomycetes Please note that these are presently the most common opportunists, certainly not the only opportunists. It cannot be overly emphasized that any fungus can cause infection or disease in a compromised human host. 1. Histoplasma capsulatum: This is a dimorphic fungus, growing as a mold in nature and on artificial media at room temperature (25-30oC) and as a yeast in tissue or on enriched media at 37oC.
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a. Mold Form-- --Gross: On Sabouraud's Dextrose Agar this fungus grows slowly, requiring 5-10 days to become visible. The mycelium is usually fluffy and white to tan in color. --Microscopic: Septate hyphae; smooth, round conidia (4-6 um); large conidia (macroconidia 7-15 um) which are thick walled, round, and tuberculated.
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b. Yeast Form-- --Gross: White to buff color colonies on blood agar; creamy consistency. --Microscopic: Small oval
budding yeast (2-5 um).
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This is a dimorphic fungus, growing as a mold at 25-30
oC, but as a yeast form in tissue or on enriched media at 37oC.|
a. Mold Form-- --Gross: On Sabouraud's Dextrose Agar the fungus appears in 5-10 days as a white fluffy colony. --Microscopic: Septate hyphae (3-5 um in diameter) bearing small conidia (4-6 um) on variable length conidiophores. (Note: These conidia are similar to those of other fungi; therefore, this fungus cannot be identified in its mold form).
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b. Yeast Form-- --Gross: Colonies on blood agar or other enriched media are white to tan, soft and "yeasty." --Microscopic: Round, thick-walled budding cell has a characteristically
wide septum (neck) at the bud base.
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This is a dimorphic fungus, growing as a mold at 25-30
oC and in a spherule form in tissue and in special media at 37oC. Note: The spherule is not a yeast and does not bud.|
a. Mold Form-- --Gross: White, moist looking colonies appear on Sabouraud's Agar in 4-8 days when incubated at 25-30oC. With age, the colony becomes fluffy and tan in the center. --Microscopic: Septate, branching hyphae which break up into arthroconidia. The arthroconidia are thick-walled, rectangular, barrel-shaped cells, with empty spaces between each cell.
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b. Spherule Form-- --Gross: Can grow only in special media. Usually not grown in the laboratory. --Microscopic: In tissue, or clinical specimens the spherules are 20-100 um in diameter. Mature spherules are filled with endospores.
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--Gross: Pasty, soft, colony which grows to visible size within 24-48 hrs on Sabouraud's Agar at 25-37oC. Colony may be white, mucoid and "runny" or may be "flesh" colored to light pink. This yeast grows on most microbiological media. --Microscopic: Small (5-8 um) oval budding yeast. Bud is separated from mother cell by a narrow, pinched off septum. Characteristically, the yeast is surrounded by a capsule varying greatly in size from 2-3 um wide at 30-40 um. Note: This characteristic capsule can be best demonstrated by using a background stain.
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--Gross: Grows well on most solid media in 24-48 hrs. The colony is usually white, glistening with rhizoid like projections representing pseudo- hyphal formation. --Microscopic: Round or oval yeast-like cells. Thick-walled chlamydospores may be produced on the branching pseudohyphae. Note: This fungus cannot be identified solely on basis of morphology.
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This is a mold commonly found in soil and decaying vegetation and is an opportunist, occupying residual pulmonary cavities as a fungus ball or invading tissue of compromised hosts. A. fumigatus is not the only species of the genus Aspergillus that is opportunistic; however, it is very frequently one.
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--Gross: Colony grows to observable size within 1-3 days on Sabouraud's Agar incubated at 25-30oC. The colony color varies from light green to dark green, growing darker with age. --Microscopic: Branching, septate hyphae. Conidiophores enlarge at the distal end forming vesicles which are covered at the tip with a single layer of Phialides bearing chains of conidia.
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Click Here to see an image Click Here to see an image Click Here to see an image
Student Work 1. Observe the gross characteristics of each fungal culture. Do not open any of the fungal cultures. 2. Observe the wet mount slides microscopically and sketch the morphological features of each fungus. 3. Examine microscopically the tissue slides and note the morphology of the fungi. Examine only methenamine silver stained slides of H. capsulatum and Rhizopus or Mucor. 4. Place one drop of sputum from B. dermatitidis on a clean glass slide. Cover with a clean cover slip and examine for fungi. Use low and high power objectives only.** 5. Place one small drop of India ink on a clean glass slide and then add a small drop of spinal fluid. Cover with a clean cover slip and observe for fungi. DO NOT USE OIL IMMERSION IN STUDYING FUNGAL MORPHOLOGY. **Reduce light through your microscope by partially closing the iris diaphragm.
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