06- Mycology & Mycobacteriology (Exam #6) Flashcards
How are fungi different from bacteria?
- Fungi are eukaryotic; they possess a true nucleus, with a nuclear membrane and mitochondria.
- Bacteria are prokaryotic, lacking these structures.
How are fungi different from plants?
- Like plants, fungi are eukaryotic.
- Unlike plants, fungi lack chlorophyll and must absorb nutrients from the environment.
- Fungal cell walls are made of chitin, whereas those of plants contain cellulose.
Define:
Saprobe/Saprophytic
- In contrast to classic pathogens, these fungi are found in the environment living on nonliving material.
- Isolation of these organisms would only be important in an immuncompromised patient
Fungal Diseases
It is helpful to organize the fungal diseases by the depth of the skin that they infect.
Describe
Processing Specimens for Fungal Culture
- Because of the additional hazard of airborne conidia, a class 2 biological safety cabinet should be used to reduce exposure of personnel to fungal elements.
- All specimens for mycology should be transported and processed as soon as possible. Because many pathogenic fungi grow slowly, any delay in processing compromises specimen quality and decreases the probability of isolating the causative agent as a result of overgrowth by contaminants.
- Although almost any tissue or body fluid can be submitted for fungal culture, the most common specimens are respiratory secretions, hair, skin, nails, tissue, blood, bone marrow, and CSF.
Describe
Direct Microscopic Examination of Specimens
-
10-20% KOH
Specimen is placed on a slide with a drop of KOH and a coverslip. The slide is heated and allowed to cool for ~ 15 mins
KOH breaks down keratin and skin layers to reveal fungi if present -
KOH with Calcofluor White
Calcofluor white binds to chitin and will fluoresce apple green -
India Ink
Added to CSF, to quickly detect capsulated yeast (Cryptococcus species)
Which microscopic method is seen in the picture?
KOH
Which microscopic method is seen in the picture?
India Ink
Which microscopic method is seen in the picture?
Calcofluor white
Describe
Primary Plating Media
-
Potato dextrose agar (PDA) is the recommended plating medium for the cultivation, enumeration, and identification of yeasts and molds from products, as well as from clinical specimens. The potato infusion encourages the growth of and sporulation by fungi.
Some labs use a similar agar = Sabouraud Dextrose Agar -
Potato dextrose with chloramphenicol (PDACH)
Same as above, but with chloramphenicol to inhibit bacteria - Mycosel/ Mycobiotic Agar are selective media used to isolate pathogenic fungi, dermatophytes, and systemic pathogens. The cycloheximide suppresses the growth of saprophytic fungi, whereas the chloramphenicol inhibits bacterial contaminants.
Describe
Primary Plating Media
Describe
Incubation of Fungal Cultures
- Fungal Culture: 30 degrees Celcius for 4 weeks (28 days)
- Fungal Screen: 30 degrees Celcius for 10 days
What test methods are used to ID yeasts?
- Germ Tube Test
- Urease Test
- Chrom Agar
- Carbohydrate Assimilation Tests
Describe
The Germ Tube Test
- Purpose: Rapid identification test for Candida albicans
- Principle: Candida albicans and Candida dubliniensis produces tube-like projections with parallel walls and no constrictions at the jucture of the parent cell.
-
Procedure:
1. Using an isolated colony make a light suspension of the organisms 1 mL of bovine albumin
2. Incubate for 2 hours
3. Prepare a wet preparation of the suspension
4. Examine on 10X and confirm on 40X
What is the purpose of the urease test?
Rapid screening test for urea positive yeast including Cryptococcus neoformans. Also positive for Rhodoturula
Principle: The test relies upon the release of ammonia during urea hydrolysis to change the pH of the medium to alkaline, converting the light orange phenol red indicator to pink.
Procedure:
Pipet 200 um of urea broth into a well on a microtiter plate
Using a wooden applicator stick, heavily inoculate the urea broth with the test isolate
Incubate in ambiently at 35-37 degrees C for 2-4 hours
What is the purpose of the Carbohydrate Assimilation Test (API 20C)?
Assimilation tests identify which carbohydrates a yeast can use aerobically as a sole carbon source.
How to ID Mold
Macroscopic:
1. Describe forward and reverse colony color
Microscopic:
1. Describe hyphae as hyaline or dematiaceous
2. Describe hyphae as septate or pauciseptate
3. Describe reproductive structures
How to ID Mold
Describe the appearance of mold.
Most molds have a fuzzy or woolly appearance
What is the name of the structures that give mold it’s fuzzy or wooly like appearance?
Mycelia.
Mycelia are made up of many long strands of tubelike structures called hyphae, which are either aerial or vegetative.
Aerial mycelia extend above the surface of the colony and are responsible for the fuzzy appearance. In addition, aerial mycelia support the reproductive structures that produce conidia. Conidia, in many cases, are used to identify different fungal genera. The vegetative mycelia extend downward into the medium to absorb nutrients.
How to ID Mold (Macroscopic)
Step 1: Describe the
Base = reverse side (light or dark)
Lid = forward side (colony color)
Mold ID (Microscopic)
How to ID Mold: Microscopic
Step 2:
What procedures are used to identify the structures of mold?
How to ID Mold: Microscopic
Step 2 Continued:
Reading the lactophenol cotton blue describe the hyphae as Hylaine or Phaeoid.
- Hyaline (moniliaceous) hyphae are nonpigmented or lightly pigmented
- Phaeoid (dematiaceous) hyphae are darkly pigmented because of the presence of melanin in the cell wall.
How to ID Mold: Microscopic
Step 3:
Describe the different types of hyphae.
- Septate hyphae show frequent cross-walls occurring perpendicular to the outer walls of the hyphae
- Pauciseptate show sparsely septate hyphae have few cross-walls at irregular intervals.
How to ID Mold: Microscopic
Step 4:
Identify reproductive structures.
- Fungi can reproduce asexually (imperfect) or sexually (perfect). Asexual reproduction results in the formation of conidia (singular, conidium) following mitosis. Asexual reproduction is carried out by specialized fruiting structures known as conidiogenous cells. These structures form conidia, which contain all the genetic material necessary to create a new fungal colony.
- In the clinical laboratory, most mold identifications are based on the structures formed as a result of asexual reproduction. Sexual reproduction requires the joining of two compatible nuclei, followed by meiosis . A fungus that reproduces sexually is known as a teleomorph. Occasionally, these fungi will also reproduce asexually. When this occurs, the asexual form is termed the anamorph. If more than one anamorph is present for the same teleomorph, the anamorphic strains are termed synanamorphs.
How to ID Mold (Microscopic)
Step 4:
Identify reproductive structures continued:
- Two common conidiogenous cells are the phialides and annellides.
- Phialides are vaselike structures
- Annellides are ringed structures
- Both form their conidia blastically (budding) like many yeasts; the parent cell enlarges and a septum forms to separate the conidial cell.
General Mycology
How do yeast reproduce?
- Single vegetative cell
- Reproduce by budding or fission
Yeasts reproduce by budding or fission. Budding involves maturation of the bud to an independent structure called what?
Blastoconidium (aka daughter cell)
This process involves lysis of the yeast cell wall so that a blastoconidium can form. As this structure enlarges, the nucleus of the parent cell undergoes mitosis. Once the new nucleus is passed into the daughter cell, a septum forms and the daughter cell breaks free. During fission, two cells of equal size are formed. These cells continue to grow from the tips of the cell and divide only after a medial fission is formed.
The term spore should be reserved for referring to what type of reproduction?
Meiosis (i.e., sexual reproduction), such as ascospores, oospores, or zygospores,
The term conidia should be reserved for referring to what type of reproduction?
Mitosis (i.e, asexual reproduction)
Asexual reproduction is carried out by specialized fruiting structures known as what?
Conidiogenous cells.
These structures form conidia, which contain all the genetic material necessary to create a new fungal colony.
Two common conidiogenous cells are the phialides and annellides. Phialides are vaselike structures that produce phialoconidia (Fig. 27.6), whereas annellides are ringed structures that produce annelloconidia. Both form their conidia blastically (budding) like many yeasts; the parent cell enlarges and a septum forms to separate the conidial cell.
Name another form of asexual reproduction that are formed by fragmentation of fertile hyphae.
Arthroconidia
This type of conidiation is characteristic of the mold form of Coccidioides immitis and Geotrichum species, among others.
Chlamydoconidia (aka chlamydospores) are formed from preexistent cells in the
hyphae, which become thickened and often enlarged. This type of conidiation is associated with which yeast?
Candida albicans
What type of hyphae are darkly pigmented due to the presence of melanin?
Phaeoid or dematiaceous
- Surface & reverse side of the plate are both dark
Look at the picture
Septate or aseptate hyphae?
Septate hyphae
Septate hyphae show frequent cross-walls occurring perpendicular to the outer walls of the hyphae,
Look at the picture
Septate or aseptate hyphae?
Aseptate hyphae
The term aseptate, which means absence of septations, has historically been used to describe the hyphae of the Zygomycetes.
Name the rootlike structures that might be seen in some of the Zygomycetes, and their presence and placement can assist with identification.
Rhizoids
What is mycoses?
Infections caused by fungi
Identify the structures in the picture
Which picture displays true hyphae? Pseudohyphae?
- Pseudohyphae and hyphae are commonly called the “filamentous” morphologies and are elongated in form, and are attached end to end.
- Pseudohyphal cells generally have constrictions at the septal junctions.
- True Hyphal cells generally possess true septa lacking constrictions and can create a germ tube.
A positive germ tube is a presumptive ID for what yeast?
Candida albicans
Candida dubliniensis is also positive but is isolated infrequently.
The procedure for inoculating a germ tube requires what type of suspension and is incubated for how many hours?
A light suspension incubated for 2 hours
Which Candida spp is most commonly isolated from humans, regardless of site?
Candida albicans
A common cause of thrush (an infection of the mouth or tongue).
Colonies from what Candida spp frequently form filamentous edges (“feet”) around the edges?
Candida albicans
Which Candida spp is:
* Frequently isolated from blood & urine
* Grows much more slowly than other Candida spp
* Positive for rapid trehalose assimilation
Candida glabrata
Which Candida spp is:
* An emerging pathogen associated w/ outbreaks healthcare facilities
* Causes serious invasive infection
* Multidrug resistant
Candida auris
Both C. albicans & C. dubliniensis are germ tube positive & green on ChromAgar, how can incubation temperature be used to differentiate between the two?
C. albicans can grow at 42 deg C.
C. dubliniensis can not.
What is cornmeal agar used for?
The patterns of growth on cornmeal agar are helpful in making a presumptive identification. The different types of morphology include blastoconidia, chlamydoconidia, pseudohyphae, and arthroconidia.
How is urease used to help ID yeast?
Almost all clinically encountered Candida spp. are urease negative, whereas all Cryptococcus and Rhodotorula organisms are urease positive.
Identify the colonies based on their color on Candida CHROMagar.
Which species of yeast tend to be mucoid due to capsule formation?
Cryptococcus spp.
The capsule of Cryptococcus that surrounds the budding yeast in CSF can be detected with the aid of what stain?
India ink
The cryptococcal capsular polysaccharide antigen can be detected in either serum or body fluid (usually CSF), this is commonly referred to as?
CAD (Cryptococcal Antigen Detection)
Cryptococcus only produce what type of conidia?
Cryptococcus spp. are noted for producing blastoconidia only, without producing true hyphae or pseudohyphae on cornmeal agar.
What is Cryptoccocus neoformans associated with?
Bird droppings (pigeon & chicken)
What is Cryptoccocus gattii associated with?
Eucalyptus trees
What yeast is the cause of tinea versicolor?
Malassezia furfur
It is also thought to be a cause of dandruff.
Describe the appearance of Malassezia furfur in the KOH
KOH preparations reveal budding yeasts, approximately 4 to 8 µm, along with septate, sometimes branched, hyphal elements. This microscopic appearance has gained M. furfur the nickname the spaghetti and meatballs fungus.
Because of M. furfur’s special nutritional requirements, routine fungal cultures are negative for growth. What does it require?
M. furfur requires lipids for growth and typical yeastlike colonies may be observed only after the culture medium has been overlaid with olive oil.
Identify
This organism is noted for their bright salmon pink color. They resemble the cryptococci because they bear a capsule and are urease positive. Some species are also nitrate positive. They are not common agents of disease but have been known to cause opportunistic infections.
Rhodotorula spp.
This yeast-like fungus can cause superficial mycoses where white piedra occurs producing white-to-tan gelatinous, pearly nodules surrounding the hair shaft.
Trichosporon spp.
Originally classified as protozoa, this fungus in immunocompetent individuals, is asymptomatic; however, in immunocompromised patients (AIDS), serious life-threatening pneumonia can develop.
Pneumocytis jirovecii
- The life cycle of Pneumocystis sp. has three stages— the trophozoite, which is 1 to 5 µm in size and is irregularly shaped; the precyst, 5 to 8 µm; and the cyst, which is a thick-walled sphere of about 8 µm containing up to eight intracystic bodies.
- Transmission of the organism is known to occur through the respiratory route, with the cyst being the infective stage. The spores or intracystic bodies are released from the cyst in the lung, and these trophic forms multiply asexually by binary fission on the surface of the epithelial cells (pneumocyte) lining the lung. Sexual reproduction by trophozoites also occurs, first producing a precyst and then the cyst containing spores or intracystic bodies.
Explain
Laboratory Diagnosis of Pneumocystis
- Infected patients typically present w/ respiratory symptoms
- Respiratory samples show typical exudate and organisms are easily seen with histologic stains such as Giemsa & Gomri methanamine silver.
- Calcofluor white can be used to screen specimens for Pneumocystis and other fungi.
Define
Dimorphic fungi
Dimorphic fungi include a mold phase and a yeast or spherule phase.
Which state is observed in vivo or when a dimporhic fungi is grown at 37 deg C. with increased concentration of CO2?
The yeast (or tissue) state.
Which state is observed when a dimorphic fungi is grown at room temp, in ambient conditions?
The mold state.
List the thermally dimorphic fungal species associated with human disease:
- Blastomyces dermatitidis
- Coccidioides immitis
- Coccidioides posadasii
- Emmonsia spp.
- Histoplasma capsulatum
- Paracoccidioides brasiliensis
- Sporothrix schenckii
- Talaromyces (Penicillium) marneffei
Define
Polymorphic fungi
- Polymorphic fungi** have both yeast and mold forms in the same culture.**
- This characteristic occurs despite growth conditions and is best observed in Exophiala spp., in which the yeast phase is typically observed initially, followed by the mold phase as the colony ages.
Thermally Dimorphic Fungi
- Found in the Ohio & Mississippi River valleys
- In soil contaminated by chicken and in caves containing bat droppings (guano)
- Primary infection is pulmonary
Histoplasma capsulatum
Thermally Dimorphic Fungi
- Found in Ohio & Mississippi river valleys & areas bordering the Great Lakes
- Found in soil, acquired through inhalation
Blastomyces dermatitidis
Thermally Dimorphic Fungi
Which fungi is probably the most virulent of all human mycotic agents?
Coccidioides spp
- Infectious arthroconidia are present in soil
- Aquired through inhalation (arthroconidia in lab also highly infectious)
After inhalation, the barrel-shaped arthroconidia round up as they convert to spherules