exercise 11 - microscopic morphology of fungal culture

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

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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)

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

Fluffy

Velvety

Powdery

Dry

Moist

Etc.

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


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


Chlamydoconidia?

Epidermophyton floccosum

Source: Internet

Macroconidia

Hyphae

C. albicans


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

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.


Conidia

Conidiophore

Phialides

Penicillium sp.

Source: Internet

SLIDE CULTURE PREPARATION

Aspergillus sp.

Aspergillus sp.


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.


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

exercise 11 - microscopic morphology of fungal culture

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