Exercise 11
Accomplished by:
› Isolating organisms on appropriate culture media
› Observing their morphological characters
Macroscopic
Microscopic
• Colonial Morphology may be of little value due to the natural variation among isolates that are culture media dependent
• (meaning: even fungi of the same species may differ in appearance depending on the media used to culture them)
Accurate identification of filamentous fungi is based on the microscopic organization of sporulating parts of a colony
› Each species has a characteristic morphology and arrangement of its spores and fruiting bodies.
Remember: Spores and fruiting bodies are more useful in distinguishing fungi—not their colony appearance grossly.
Cultures of:
› T. mentagrophyte
› M. gypseum
› E. floccosum
› Candida albicans
Cultures of:
› Penicillium sp.
› Aspergillus sp.
Sabouraud’s Dextrose Medium with 2% agar
2 microculture dishes
Inoculating needle
Glass slides, coverslips
Lactophenol Cotton Blue (LPCB)
Nail Polish
Microscopes
Tease Mount Slide Culture / van Tieghem cell
*Lactophenol Cotton Blue (LPCB) – this dye is used in both preparations
* Van Tieghem cell– a device mounted on a microscope slide
that is used to observe the development of a fungus' mycelium
Used for quick evaluation of fungal structures
Has three components:
› Lactic acid preserves fungal structures
› Cotton blue an acid dye; stains chitin of the cell walls of the fungi
› Phenol kills any live organisms suspended in the stain
Traditionally used by most laboratories
Primary Purpose:
› Demonstrate conidia
› Other reproductive structures, or
› Other morphological forms which might help in the identification of the organism
1. Using an inoculating needle, pick a small portion of each fungus growth and place on separate slides containing a drop of lactophenol cotton blue.
2. Tease with inoculating needle. Emulsify the growth of the yeast and yeast-like fungi.
3. Put a cover slip and examine under low and high power objectives
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
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Hypha are the microscopic form or tubular form of the mold
Mycelia are masses of intertwined hypha that are already visible to the eye.
Septate Non-Septate / Coenocytic /Aseptate
Vegetative
- hyphae embedded into the substrate
Reproductive / Aerial
- hyphae that sprouts vertically; gives rise to sexual spores
Hyaline
(Non-Pigmented)
Dematiaceous
(Pigmented)
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
Zygospores Ascospores Basidiospores
Container: zygosporangium Container: Ascus Container: Basidium
Sporangiospores
› The spores are contained in a sporangium (an enclosed sac)
› Typical of zygomycota
Conidia
› The spores are devoid of containing sac
› Present in the rest of the phyla
Blastoconidia
Poroconidia
Phialoconidia
Annelloconidia
Macroconidia / Microconidia
Chlamydoconidia Anthraconidia
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
Fluffy
Velvety
Powdery
Dry
Moist
Etc.
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
4. Observe, record, and draw the following important morphological features of the various fungi*
a) Nature and type of Mycelium = (+/-) crosswalls
b) Characteristic Arrangement, size, shape, and type of spores produced, and some special characteristics
c) Nature of Growth (fluffy, velvety, powdery, dry, moist)
d) Color of Growth on surface and reverse side
5. Label all drawings accordingly
6. Observe and Characterize the different fungal colonies
DERMATOPHYTE SKIN HAIR NAIL
Trichophyton
Microsporum
Epidermophyton
Recall:
T. mentagrophyte
› Gross / On S.D.A.:
Mycelium: spiral
Shape: Generally Flat
Color of Growth: Surface: White to Cream
Reverse: Yellow-brown to Reddish-brown
Nature of Growth: Powdery to Granular Surface
Other Characteristics: Some with central folding, or raised central tufts or pleomorphic suede-like to downy areas.
Source: Internet
T. mentagrophyte
› Microscopic:
Microconidia
often in dense clusters
Numerous single celled
Hyaline, smooth & thin walled
predominantly spherical to subspherical in shape; occasional clavate to pyriform
Chlamydoconidia
Spiral Hyphae
Macroconidia (smooth, thin-walled, clavate shaped, multicelled)
Source: Internet
Trichophyton mentagrophyteHyphae
Source: MicroLab Demo Slides
Macroconidia
Microconidia
Trichophyton mentagrophyte
Source: Internet
Macroconidia
Hyphae
Microconidia
Trichophyton mentagrophyte
Source: Internet
Spiral Hyphae
Chlamydoconidia
M. gypseum› Gross / On S.D.A.: Mycelium: fluffy white tuft
Shape: usually flat, spreading
Color of Growth: Surface: deep cream to tawny-buff to pale cinnamon red
Reverse: yellow-brown (often with central darker brown spot), in some strains, reddish brown
Nature of Growth: suede-like to granular
Other Characteristics: Many cultures develop a central white downy umbo (dome); some also have a narrow white peripheral border.
Source: Internet
M. gypseum
› Microscopic:
Microconidia
numerous but not diagnostic
Spindle shaped
Macroconidia
Ellipsoidal / Club shaped
Symmetrical
Thin-walled
Verrucose
4-6 celled
Source: Internet
• Terminal ends = slightly rounded• Proximal Ends (point of
attachment to hyphae) = truncate
Microsporum gypseum
Source: MicroLab Demo Slides
Hyphae
Microconidia??
Macroconidia
Microsporum gypseum
Source: Internet
Hyphae
4-6 celled
Macroconidia
E. floccosum› Gross / On S.D.A.: Mycelium: (Older cultures)
pleomorphic tufts
Shape: raised and folded in the center; flat periphery, with submerged fringe of growth
Color of Growth: Surface: greenish-brown or khaki coloured
Reverse: deep yellowish-brown
Nature of Growth: suede-like
Other Characteristics: Slow-growing
Source: Internet
E. floccosum
› Microscopic:
Microconidia
absent
Chlamydoconidia
Numerous
Formed in older cultures
Macroconidia
Smooth; club shaped
Thin-walled
Often in pairs, singles, or in clusters growing directly from hyphae
Source: Internet
Epidermophyton floccosum
Source: MicroLab Demo Slides
Chlamydoconidia?
Epidermophyton floccosum
Source: Internet
Macroconidia
Hyphae
C. albicans
› Gross / On S.D.A.:
Color of Growth: white to cream coloured
Nature of Growth: glabrous
Source: Internet
C. albicans
› Microscopic:
Spherical to subspherical budding yeast-like cells or Blastoconidia
Source: Internet
Candida albicans
Source: MicroLab Demo Slides
Blastoconidia
Candida albicans
Source: Internet
Blastoconidia
Considered the best methods for preserving and observing the actual structure of a fungus.
Unsurpassed as a routine means of studying fine points of the microscopic morphology of fungi
Demonstrates important microscopic structures and morphologic details in undisturbed state
used for microcultures of fungi
may be used to demonstrate important microscopic structures like spores
especially useful for filamentous fungi
1. Inoculate the microculture plates separately with each of the given fungus
2. Hold a microscope coverslip with a pair of forceps and put it on top of the agar inside the plates
3. Check the culture periodically for growth and sporulation. In most cases, room temperature incubation is best. Observe the fungus each day without lifting the lid of the petri dish. When the fungus has grown onto the square of the S.D.A. and out onto the small part of the microscope slide and coverslip, proceed with the next step.
The amount of incubation time required for this to happen varies with each fungus but usually:
• 5-10 days for contaminants
• 1-3 weeks for pathogens.
4. Remove the microscope slide from the petri dish. Take a new, clean microscope slide and place a drop of LPCB on it near the center. With a forcep, carefully lift up the coverslip with fungus growing on it and very slowly lower it onto the drop of LPCBon the new microscope slide. The LPCB will flow under the coverslip. (If it leaks out of the coverslip, the LPCB was too much).
To preserve the slide even for years, carefully seal the coverslip to the microscope slide with nail polish. Label the slide.
5. To make a second slide, pick up the first microscope slide which still contains the small block of S.D.A. with the fungus growing on it. Hold the slide upside down, with an inoculating needle or spatula, flick off the piece of S.D.A. in a beaker of disinfectant. Note that some fungus will still be growing on the microscope slide. Add 1-2 drops of LPCB to the center of the fungus colony (where S.D.A. square was) and place a new microscope coverslip onto it. Seal the coverslipwith nail polish and label the slide
6. Examine the slide for reproductive structures and notice the undisturbed morphology.
SLIDE CULTURE PREPARATION
Penicillium sp.
Penicillium sp.
› Gross / On S.D.A.: (25oC)
Color of Growth: Surface: grayish pink to brown (with
age); produces a diffusible brownish-red to wine-red pigment
Reverse: ???
Nature of Growth: suede-like to downy
Other Characteristics: fast growing
Penicillium sp.
› Gross / On B.H.I. (37oC)
Color of Growth: Surface: tan coloured
Reverse: ???
Nature of Growth: rough, glabrous. Yeast-like
Penicillium sp.
› Microscopic: (25oC) S.D.A
Conidiophores
Hyaline
Smooth walled
Some with terminal verticilsof 3-5 metulae, each bearing 3-7 phialides
Conidia
Globose to subglobose
Smooth walled
Produced in basipetalsuccession from the phialides
Penicillium sp.
› Microscopic: (37oC) B.H.I.
Yeast
Spherical to ellipsoidal
Divide by fission rather than budding
Hyphal Elements
Numerous
Short
Penicillium sp.
Source: MicroLab Demo Slides
Conidia
Conidiophore
Phialides
Penicillium sp.
Source: Internet
SLIDE CULTURE PREPARATION
Aspergillus sp.
Aspergillus sp.
› Gross / On S.D.A.:
Color of Growth: Surface: (depending on species)
white, yellow, yellow-brown, brown to black, or shades of green
Reverse: ???
Other Characteristics: fast growing; thick walled hülle cells
Aspergillus sp.
› Microscopic:
Conidiophores
dense, erect
Conidial Head
(UNISERIATE) a vesicle covered with either a single palisade-like layer of phialides
(BISERIATE) a layer of subtending cells (metulae) which bear whorles of phialides
UNISERIATE
vesicle
phialides
conidia
BISERIATE
vesicle
metulae
phialides
conidia
Aspergillus sp.
› Microscopic:
Conidia
One celled
Smooth/Rough walled
Hyaline/Pigmented
Basocatenate = describe such chains of conidia where the youngest conidium is at the basal or proximal end of the chain:
Divergent chains (radiate)
Compact columns (columnar)
Aspergillus sp.
Source: MicroLab Demo Slides
Conidia
Conidiophore
Septate Hyphae
Conidial Head
Phialides
1.a. Advantages of Slide Culture› Allows fungi to be studied with little disturbance
› No need to remove fungi from culture plate and trasnsfer to the slide since it is already cultured in the slide
1. b. Disadvantages of Slide Culture› Rapidly exhausts oxygen and nutrient supply; quick
to turn acidic
› Hard to maintain sterility in prolonged periods
› Alllows only a small amount of sample to be cultured
2.a. Advantages of Tease Mount Preparation
› Can be done quickly
› Sufficient (usually) to ID common fungi encountered in the laboratory
2. b. Disadvantages of Tease Mount Preparation
› May disrupt the characteristic arrangement of the conidia, or even the entire fungal structure upon the placement of coverslip with increased pressure
› Less arranged fungi vs. Slide Culture
3. a. Describe the scotch tape lactophenol mount as a means to demonstrate morphologic features of filamentous fungi.
› The clear tape is pressed down a colony surface, allowing observation of organism as it has grown in culture. The addition of LPCB kills and preserves the fungi—providing adequate staining of the fungal elements. The filamentous growth of the organism can be observed. Depending on species, structures such as sporangia, spores, sporangiospores, etc. can also be seen.
Based on the slides, list 4 characteristic structures / arrangements which could be used as guides to identification of the given fungi.
› Penicillum sp. Small, ovoid structures dividing by transverse fission Flat, powdery to velvety, tan to reddish yellow colonies Conidiosphore bearing short broad metullae At 37C colonies are soft and yeast-like, round or ovoid cells with
central septum are seen
› Aspergillus sp. Mycelia Fruiting heads Septate, monomorphic fungi Forms branches at acute angles
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