Exercise 3

Morphology of FilamentousFungi

Coenocytic Fungi

Mucor

Macroscopic features:

Grows rapidly at mesophilic conditions (25-30 C) Fluffy appearance resembling cotton candy Usually white to grayish
features:

Microscopic

Non-septated with broad hyphae Conspicuous spores, sporangium and sporangiophores Presence of oidia or arthrospores in some species Sporangiophores short, erect, branched or unbranched, hyaline and tapered at the apex

Mucor

Columella hyaline, round cylindrical or pear-shaped Absence of apophyses (swelling of the somatic cell underneath the sporangium), rhizoids and stolon Sporangium spherical, gray to black filled with sporangiospores Sporangiospores round (4-8 m in diameter) or slightly elongated, smooth in texture Sporangiospores freely spread

Mucor

Rhizopus
Macroscopic

features: Grows rapidly and reaches maturation ~4days Texture similar to cotton Grows well at 37 C black bread mold

Microscopic

features: Non-septated with broad hyphae (6-15m in diameter) Presence of rhizoids and stolon

Rhizopus

Sporangiophores unbranched and brown arising at the node where rhizoids are also formed Solitary or in clusters Sporangium found at the tip of sporangiophore, large, usually brown to black Sporangiospores unicellular, round to ovoid, hyaline or pigmented (brown) and smooth and striated in texture Columella hemispherical with cup-shaped apophyses

Rhizopus

Septated Fungi

Aspergillus
Macroscopic

features: Colonies downy with powdery texture Usually white to grayish Downy to powdery

Microscopic

features: Septated hyphae hyaline; forms branching mycelium Conidiophore or stalk septate arise from foot cell

Aspergillus

Conidiophores swells Conidia in chains *Phialides fully or partially-covers vesicle Conidium circular forming radial chains (2-5 m in diameter) Vesicles bearing sterigmata from which the conidia are cut-off

*Phialides are cylindrical, with a small collarette, solitary or produced

as a component of a complex branching system

Aspergillus

Fusarium
Macroscopic

features:

Grows rapidly Colonies woolly to cottony; usually at first then turn pinkish or lavender when maturity is reached Presence of sclerotium (dormant mass of hyphae during conditions unfavorable to the species) may be observed Sporodochium (cushion-like mat of hyphae with conidiophores on the surface) may be present

Fusarium

Microscopic features: Several-celled Branched or unbranched conidiophores form macroconidia (thick walled, smooth, elongated to sickle shape around 3-8 x 11-70 m) Microconidia (2-4 x4-8 m) form from long or shortsimple condiophores; smooth, hyaline, ovoid to cylindrical Conidia born isolated or in chains Sometimes presence of chlamydospores

Fusarium

Penicillium
Macroscopic

features: Fast growing Colonies flat, filamentous, velvety, with woolly to cottony texture; initially white then turn bluegreen, green with gray specks, yellowish or pinkish after some time features: Septated; forms branching mycelia Branched or unbranched conidiophores Brush-like spore heads

Microscopic

Penicillium
Sterigmata or phialides borne in clusters and essentially in one plane Phialides form brush-like clusters referred to as "penicilli" Metulae (secondary branches sprout from conidiophore) and conidia are visible Conidia unicellular, round and unbranching at the tips of the phialides (~ 2.5-5m in diameter)

Penicillium

Trichoderma
Macroscopic

features:

Fast growing Colonies woolly and become compact at maturity White when viewed from the front Blue-green or yellow-green patches (may form concentric rings) appear usually when conidia are formed

Microscopic

features: Septated; hyaline hyphae Visible conidia, phialides and conidiophores

Trichoderma
Conidiophores hyaline, branched sometimes forming a pyramidal arrangement the final branch being a sterigma which cuts off spherical to slightly ovate Phialides flask-shape, hyaline and swollen at the bottom; may be isolated or in clusters Conidium unicellular, round(3 m in diameter in average); smooth or rough texture and most probably green Conidia forms slime ball

Trichoderma

Additional pictures of fruiting bodies/structural parts of molds

Additional pictures of fruiting bodies/structural parts of molds

Additional pictures of fruiting bodies/structural parts of molds

Additional pictures of fruiting bodies/structural parts of molds

Trichoderma

Additional pictures of fruiting bodies/structural parts of molds

Sclerotium

Additional pictures of fruiting bodies/structural parts of molds

Media used
Potato Dextrose Agar (PDA) - Ideal for the culture of yeast and agar to stimulate sporulation and enhance growth of poorly sporulating mycelia - The potato component is the basic source of nutrient of the growing molds. Molds are capable of breaking down the starch of PDA through their enzyme, amylase. - Dextrose, which is a carbohydrate, aids in the stimulation of growth. - By adding tartaric acid, the pH will be lowered which will inhibit the growth of bacteria

Media used

PDA (cont.) - Influences the rapid growth of:

Trichoderma Rhizopus
Malt agar but also thrive greatly in PDA

*Mucor and Trichoderma grow well in

PDA (additional)

Potato Dextrose Agar (PDA) is a general purpose medium for molds and yeasts that can be complemented with antibiotics or acid to inhibit bacterial growth. It can be used for growing clinically important molds and yeasts. The nutritionally rich base (potato infusion) nurtures mold sporulation and pigment production in some dermatophytes.

PDA (additional)
Components:

Potato Infusion and Dextrose: nurtures abundant fungal growth Agar: solidifying agent Tartaric Acid (10%): lowers pH of PDA to 3.5 +/- 0.1, preventing growth of bacteria

Media used

Czapek Dox Agar (CDA)

Medium used for the general cultivation of fungi Contains sodium nitrate as the sole source of nitrogen and sucrose as the source of carbon Uses dipotassium phosphate as buffer Influences the rapid growth of:

Penicillium Aspergillus

CDA (additional)

Czapek Dox Agar is a semi-synthetic medium used for the cultivation of fungi, containing sodium nitrate as the sole source of nitrogen. This medium is prepared according to the formula developed by Thom and Church (1), which has a defined chemical composition. Czapek Dox Agar is recommended by APHA (2) for isolation of Aspergillus, Penicillium, Paecilomyces and some other fungi with similar physiological requirements.

CDA (additional)
Sucrose serves as the sole source of carbon while sodium nitrate serves as the sole source of nitrogen. Dipotassium phosphate buffers the medium. Magnesium sulphate, potassium chloride, ferrous sulphate serves as sources of essential ions.

(retrieved from: http://himedialabs.com/TD/M075.pdf)

Mounting Media
Lactophenol

Cotton Blue

Components: *Lactic acid- preserves fungal structures *Phenol- kills the microorganisms *Cotton blue- stains the fungi ---*Glycerine/glycerol- prevents spore dispersion

Slide Culture Technique

It is a rapid method of preparing fungal colonies for examination and identification. Permits fungi to be studied virtually in situ with as little disturbance as possible. Fungi are identified mostly by close examination of their morphology and the characteristics they possess. In slide cultures, fungi are grown on thin film of agar (agar block) with coverslip then incubated..

Slide Culture Technique

(cont.) Through this, chances of damaging the features used for identification especially the spore-bearing structures are low since removal of a portion of the fungus is no longer necessary

Slide Culture Technique

These

are important because: V-rod used to remove of the slide easily without interrupting the fragile growth of the molds since the moisture in between the surface of the petri dish and the glass slide itself might create enough surface tension between the two. Moistened filter paper additional source of moisture to prevent drying out of growing fungi upon prolonged incubation

Answers to questions:
1.

What are the advantages of using slide culture technique for studying molds?

The slide culture technique enables one to observe the actual morphology of the mold. It is also a method of preserving the specimen. This technique promotes conditions essential for sporulation. Since the mold is grown on the coverslip straightly, inoculation from a colony is no longer needed so damaging of the fruiting bodies and other structural parts will be avoided. Isolated spore-bearing structures will also be easier to locate because of lesser number compared to a whole colony.

Answers to questions:
2.

Does the measurement of mold colony diameter give you accurate data on mold growth? Explain. No. Not all molds exhibit the same growth rate. Some molds grow faster than the others. For example, Mucor has a rapid mycelial growth rate and is observed to reach maturity within a shorter period of time. The media used should also be considered since the components affect the growth rate. Specifically, there are molds that grow faster in PDA while there are some which prefer CDA (i.e. Aspergillus and Rhizopus)

References:
http://www.doctorfungus.org http://www.misac.org.uk/PDFs/MiSAC_s uitable_and_unsuitable_microorganisms.pdf http://nelabservices.com/techsheets/Pota to_Dextrose_Agar.pdf http://himedialabs.com/TD/M075.pdf http://thunderhouse4yuri.blogspot.com/2011/11/slide-culturetechnique.html