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 funginot their colony appearance grossly.

Cultures of:
T. mentagrophyte M. gypseum

Cultures of:
Penicillium sp. Aspergillus sp.

E. floccosum
Candida albicans

Sabourauds 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.

2.

Using an inoculating needle, pick a small portion of each fungus growth and place on separate slides containing a drop of lactophenol cotton blue. 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. 6.

Label all drawings accordingly 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. 6.

Label all drawings accordingly 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. 6.

Label all drawings accordingly 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. 6.

Label all drawings accordingly Observe and Characterize the different fungal colonies

Zygospores

Ascospores

Basidiospores

Container: zygosporangium

Container: Ascus

Container: Basidium

Sporangiospores
The spores are

Conidia
The spores are devoid

contained in a sporangium (an enclosed sac) Typical of zygomycota

of containing sac Present in the rest of the phyla

Blastoconidia

Phialoconidia

Poroconidia

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. 6.

Label all drawings accordingly 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. 6.

Label all drawings accordingly 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. 6.

Label all drawings accordingly 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. 6.

Label all drawings accordingly Observe and Characterize the different fungal colonies

Recall: DERMATOPHYTE Trichophyton Microsporum Epidermophyton SKIN HAIR NAIL

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 mentagrophyte
Hyphae Macroconidia

Microconidia Source: MicroLab Demo Slides

Trichophyton mentagrophyte
Hyphae Macroconidia

Microconidia Source: Internet

Trichophyton mentagrophyte
Spiral Hyphae Chlamydoconidia

Source: Internet

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 Terminal ends = slightly rounded Thin-walled Proximal Ends (point of Verrucose attachment to hyphae) = truncate 4-6 celled

Source: Internet

Microsporum gypseum
Hyphae Macroconidia

Microconidia?? Source: MicroLab Demo Slides

Microsporum gypseum
4-6 celled Macroconidia Hyphae

Source: Internet

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

Chlamydoconidia? Source: MicroLab Demo Slides

Epidermophyton floccosum
Macroconidia

Hyphae Source: Internet

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

Blastoconidia Source: MicroLab Demo Slides

Candida albicans

Blastoconidia Source: Internet

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 Hold a microscope coverslip with a pair of forceps and put it on top of the agar inside the plates

2.

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 coverslip with 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 brownishred 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 verticils of 3-5 metulae, each bearing 3-7 phialides Conidia Globose to subglobose Smooth walled Produced in basipetal succession 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.
Conidia

Phialides

Conidiophore Source: MicroLab Demo Slides

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 hlle cells

Aspergillus sp.
Microscopic:
UNISERIATE

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
BISERIATE

conidia phialides vesicle

conidia phialides metulae vesicle

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.
Phialides
Conidia

Conidial Head

Septate Hyphae
Conidiophore Source: MicroLab Demo Slides

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 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

1. b. Disadvantages of Slide Culture

2.a. Advantages of Tease Mount Preparation

Can be done quickly Sufficient (usually) to ID common fungi encountered in the laboratory 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

2. b. Disadvantages of Tease Mount Preparation

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 fungiproviding 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