Fun With Microbiology (What's Buggin' You?)

Cladosporium speciesRevisited (Kind Of) -A Black Mould -Hyphomycetes

This post is a bit of a revisitation to the fungus Cladosporium as I previously included the genera in a previous post on Cladophialophoraspecies. You can compare the two more thoroughly by checking out my previous post by clicking here.

Ecology:Cladosporium species are cosmopolitan saprophytic fungi found in soil, on plant debris and leaf surfaces.

Pathogenicity: Cladosporiumspecies are generally considered to be non-pathogenic although they may be considered as possible opportunists in the severely debilitated host. Potentially pathogenic species previously included under the Cladosporiumgenera have recently been reassigned to the genera Cladophialophora (eg. C.bantianum, C.carrionii). Cladosporium may be encountered in the laboratory as a culture contaminant and must be distinguish from the pathogenic Cladophialophora.

Macroscopic Morphology: The rate of growth is dependent on the particular Cladosporium species and can vary from slow to moderately rapid. The isolate discussed in this post expanded in size rather slowly though it matured to produce copious amounts of conidia rather quickly (~7 days). The colony was velvety to suede-like in texture. Other sources describe Cladosporium’s texture as ranging from powdery to woolly. The colony may become slightly heaped and develop gentle folds as it ages. Colour ranges from greyish-green to olivaceous-green to brownish-black. The reverse is a dark brown to black in colour.

Cladosporium species on SAB, 15 Days at 30˚C (Nikon)

Cladosporium species on SAB, 25 Days at 30˚C (Nikon)

Microscopic Morphology: Cladosporium produces erect, dark, septate hyphae. Conidiophores are also darkly pigmented, may be septate and show tree-like branching. Fragile chains of dematiaceous blastoconidia are produced and exhibit a dark hila or scar at their point of attachment to the conidiophore or other conidia. The 1-4 celled conidia are round to oval (3 -6 µm X 4 - 12 µm) and may be smooth-walled to verrucose in surface texture. Cells on the conidiophore which bear the chains of conidia are sometimes septate and appear in the shape of a ‘shield’. These cells are also conidia but are referred to as shield cells. Chains of conidia easily disarticulate (break up) and were frustratingly difficult to document using both adhesive tape andslide culture techniques.

Cladosporium species are not thermotollerant and some species may not grow at 37˚C.

Cladosporium species produce darkly pigmented, septate, branching hyphae,

(LPCB, DMD-108, 400+10X)

Cladosporium species - Conidiophores are also darkly pigmented, may be septate and also show tree-like branching. (LPCB, DMD-108, 400+10X)

Cladosporium species - The Lactophenol cotton blue stain has taken more deeply in this preparation. Septate hyphae are clearly visible. (LPCB, DMD-108, 400X)

Cladosporium species -1-4 celled conidia are round to oval (3- 6 µm X 4-12 µm) and may be smooth-walled to verrucose in surface texture.

(LPCB, DMD-108, 1000X)

Cladosporium species -the structures are clearly evident here: septate hyphae, darkly pigmented conidiophores and oval conidia in chains.

(LPCB, DMD-108, 1000X)

Cladosporium species -as above. Younger conidiophores & conidia stain more intensely with the LPCB while the mature structures have developed the pigmentation.

(LPCB, DMD-108, 1000X)

Cladosporium species -the structures easily dis-articulate (break up) and it was difficult to keep them intact regardless of using the adhesive tape technique or slide culture technique.

Short chains of oval, pinmented, conidia are seen.

(LPCB, DMD-108, 1000X)

Cladosporium species - a closer look at the conidiophores bearing round to oval pigmented conidia.

(LPCB, DMD-108, 1000+10X)

Cladosporium species -a single conidia (conidiophore?) seen at the end of a septate hyphae.

(LPCB, DMD-108, 1000X)

Cladosporium species - a structure visible in some of the previous photos is more clearly seen in this picture. Here you can clearly see what is referred to as a "shield cell", because of its resemblance to a warrior's shield. These are conidiophores as you can clearly see chains of conidia extending from them. (LPCB, DMD-108, 1000+10X)

Cladosporium species - `Shield` cell conidiophore and conidia

(LPCB, DMD-108, 1000+10X)

Cladosporium species - another view, as above.

(LPCB, DMD-108, 1000+10X)

Cladosporium species - `Shield`cells and chains of conidia

(LPCB. DMD-108, 1000X)

Cladosporium species - Septate hyphae with conidiophore bearing conidia at its apex. Insert is simply a change of focus. (LPCB, DMD-108, 1000X)

Cladosporium species - okay, one final photo, just for the heck of it!

(LPCB, DMD-108, 1000+10X)

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Malassezia furfur complex(Yeast)

Note: While reading a gram stain taken as the swab of the ear canal, I noticed a number of yeast cells which appeared to show broad-based budding. A Sabouraud-Dextrose media plate (SAB) was added to the routine culture to better isolate any yeast present. When no yeast grew after 24 hours incubation, my suspicion of Malassezia furfurcomplex was confirmed. Culture of the yeast is not necessary for confirmation M.furfurcomplex as its appearance (broad based mono-polar budding), location of isolation (lipid-rich ear canal), and lack of growth on basic mycological media is generally considered sufficient evidence. Never having tried the traditional olive oil overlay technique, I thought I’d shot on this specimen just for the fun of it. The photos that follow are the result.

History: Malasseziayeast was first identified by the French scientist Louis-Charles Malassez in the late 19^th century. Currently, the Malassezia furfur complex is thought to be comprised of 14 Malasseziaspecies, eight of which have been associated with humans. Of the eight, M.furfur, M.sympodialis, M.globosa and M.restricta are the most common. Another Malassezia species, not part of the complex, is Malassezia pachydermatis. M.pachydermatis is not lipophilic and while it can on occasion be isolated from human skin, it is most often associated with the ears of canines (dogs).

Ecology: Malassezia furfuris a lipophilic yeast (need lipids/fatty acids) to grow, therefore habitats are limited to where exogenous source of lipids are available. Malasszia furfur can be found as a saprobe, living on the excretions of normal skin flora on over 90% of healthy adults. Usually acquired at an early age, it prefers the oilier parts of the skin such as the scalp and ear canals.

Pathogenicity: While Malassezia furfur complex can be found as part of the normal human skin flora, members are implicated in a variety of diseases. Hyperhydrotic individuals (excessive sweating) may develop a disorder known as pityriasis versicolor[i](tinea versicolor) and Pityriasis folliculitis[ii]. It has been implicated in seborrheic hyperkeratosis[iii], and more recently as a causative agent of seborrhoeic dermatitis[iv]. So, under certain, poorly understood conditions, Malassezia furfur complex may cause or accentuate various skin conditions.

Catheter associated infections are commonly seen in neonates and adults receiving prolonged intravenous lipid supplements. Immunocompromised individuals and those with other underlying conditions may find themselves at increased risk to Malassezia furfur complex infection.

Colonial Morphology: Malassezia grows fairly rapidly, maturing in about 5 days at 30 -35˚C. It has a rather narrow temperature growth range as it grows poorly at 25˚C and some species will not grow above 37˚C. Colonies are cream to yellowish-brown in colour. They appear smooth and pasty, often becoming brittle and wrinkled as they age. The Margin can be entire or lobed. As already mentioned, lipid supplement is required for growth and in culture this can be as simple as adding an olive-oil overlay to the Sabouraud-Dextrose surface. Specialized media, specifically for the isolation and growth of Malassezia species is commercially available. Malassezia is resistant to cycolohexmide and therefore will grow on selective media such as Mycosel™ & Dermasel™. Urea test is positive.

Malassezia furfur growing on a SAB plate which was overlaid with a thin layer of olive oil. The olive oil is rich in long chain fatty acids which in nature is supplied by the host environment. The SAB agar is prepared using water which repels oil. However carefully you spread the oil, it will separate into individual droplets. The M.furfur will grow and produce colonies around or near the oil droplets where it can obtain the nutrient. (other techniques are possible)

(Mycosel™ Agar, 30˚C, 72 hrs, Nikon)

Malassezia furfur inoculated onto SAB media with olive oil supplement (left) and without olive oil supplement (right). As is evident, growth only occurs where the olive oil supplies the long-chain fatty acids. The slight haze (growth?) may be explained by the yeast continuing to grow for a short period of time on the reserve of fatty acids carried over from the original source. Once depleted, growth stops. The first photograph was taken of the yeast growing on Mycosel™ agar which shows that M.furfur is resistant to cycloheximide. In comparison between these two photos, there appears to be better growth on the Mycosel than the SAB. This may simply be due to the inoculation load, that is a heavier inoculum placed on one than the other.

(SAB, 30˚C, 72 hrs, Nikon)

Microscopic Morphology: On initial examination of material taken from the patient (skin swab or scrapings), both yeast and hyphal forms may be present. This appearance is often referred to as “spaghetti & meatballs” when both round & linear elements are seen mixed together. Hyphal elements are usually absent on culture but rudimentary forms may occasionally be seen.

The yeast-like cells (1.5 µm – 4.5 µm X 3 µm – 7 µm) are actually phialides and may show small collarettes, though they may be difficult to discern with the light microscope. The cells are referred to as being unipolar, with one end round and the other somewhat blunt, where bud-like structures form singly on a broad base. (Relative size of the budding base differs between M.furfur complex species).

Note: While I cannot say which species of Malassezia furfur complex this is, it has to be one of the eight species associated with disease in humans. From this point on, I will refer to this yeast as M.furfur for simplicity, however the reader must realize I am referring to M.furfurcomplex.

M.furfur complex as seen in the original gram of a child's ear swab. Arrows point to the individual yeast cells amongst some cellular debris. The 'B' points to one yeast cell which is exhibiting broad based budding. "Broad" in that the daughter cell may appear to be attached to the parent cell by a connection, perhaps by up to half the width of the cell. Other yeast such as Candida albicans may exhibit a narrow, almost point like area of attachment before the daughter cell is 'pinched off'.

(Direct gram Stain, 1000X, DMD-108)

M.furfur direct gram stain as above but at a higher magnification. Without the arrows, can you pick out the yeast cells? (Direct gram stain, 1000+10X, DMD-108)

M.furfur seen in a potassium hydroxide suspension. The appearance is that of typical yeast.

(KOH, 400X, Nikon)

M.furfur -arrows point to cells with broad-based budding

(KOH, 400+10X, DMD-108)

M.furfur - Broad based budding evident between daughter and parent yeast cell.

(LPCB, 400X, Nikon)

M.furfur - Broad based budding again seen

(Gram from culture, 1000X, Nikon)

M.furfur -daughter cell about to split from mother cell.

(Gram from culture, 1000X [cropped], Nikon)

Note:I applied a drop of olive oil to a Sabouraud-Dextrose agar plate (SAB) and spread it as evenly as possible over the surface. This is most effectively accomplished using a 'hockey stick'(Canadian, eh?), which is just a plastic or glass rod bent in the shape of an ''L". The moisture in the agar media repels the olive oil to some degree as it can be seen beading up in the plate photos. Sufficient oil remains distributed to allow growth and growth may be seen greatest around the globules of oil. An alternative method is to soak some sterile filter-paper discs or strips in olive oil and lay them on the agar surface. Growth will be observed closest to the impregnated disc edge where the lipid is available. As an afterthought, I was going to try this for photographic purposes but regretfully, I had already discarded the isolate. Perhaps I’ll try this next time.

[i]Pityriasis versicolor–a condition characterized by a rash on the trunk and proximal extremities, usually caused by M.globosa of the M.furfur complex, and occasionally by M.furfur itself. May be more prominent in hot, humid environments. Results in pale, dark-tan or pink patchy skin.

[ii] Pityriasis folliculitis– Proliferation of Malassezia yeast within hair follicles resulting in acne like eruptions particularly on the trunk.

[iii]Seborrheic hyperkeratosis– a pigmented waxy or scaly raised growth which may resemble skin cancer, however it is benign. It occurs more frequently in older adults.

[iv]Seborrhoeic dermatitis– an inflammatory skin disorder affecting the scalp, face and torso resulting in red flaky, scaly, itchy skin, particularly in areas rich in sebaceous glands.

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Scopulariopsis brumptii(Mould/Fungus)

Compare this species to Scopularis brevicaulis.

Ecology: A cosmopolitan fungus (found just about everywhere), particularly in soil and in household dust.

Pathogenicity:Scopulariopsis brumptii is rarely implicated in disease; however, as with many other ‘non-pathogenic’ fungi, they may be considered opportunists and may be increasingly found as the cause of pulmonary (lung) infections in severely immunocompromised patients. Encountered in the laboratory as a possible contaminant, they must be distinguished from more pathogenic fungi.

Macroscopic Morphology: S.brumptii is a moderately rapid grower, usually maturing within about 5 days. Surface growth may initially be whitish, then darkening from a light grey to a sepia-grey (reddish overtones) or fuscous (darker brown-grey). One source described the colour as that of cigarette ash. The grey overtones distinguish it from the cinnamon, tan or buff brown colour of Scopulariopsis brevicaulis. The texture is described as velvety to powdery.

Scopulariopsis brumptii - Sabouraud-Dextrose Agar

(SAB, 12 Days, 30^oC, Nikon)

Microscopic Morphology: S.brumptii produces septate hyphae that are hyaline when young but darkens as it matures. Conidiogenous (conidia producing) cells develop from the hyphae and can appear singly or in brush-like arrangements. These conidiogenous cells are more specifically referred to as annellides as they produce conidia (annelloconidia) in succession, each leaving a ring-like collar on the annellide when the annelloconidia is released. The annellides (5 µm – 10 µm X 2.5 µm to 3.5 µm) are somewhat flask-shaped, with a swollen base which tapers near the apex, however they are generally more cylindrical that those of S.brevicaulis. The annelloconidia (4.0 µm – 5.25 µm X 3.5 µm – 4.5 µm) in size and although they can be smooth, the texture is usually described as finely roughened. They are usually pyriform (pear-shaped), dark brown in colour and have a flattened or truncated base where once attached to the annellide. The annellations (scar/ring) produced by S.brumptii may not be very conspicuous. Annelloconidia are produced in loose chains from the apices of the annellides.

Scopulariopsis brumptii - darkly pigmented annelloconidia.

(KOH, 400X, Nikon)

Scopulariopsis brumptii -hyaline hyphae become pigmented as are the ellipsoidal annelloconidia.

(KOH, 1000X, Nikon)

Scopulariopsis brumptii - conidiogenous cells develop from the hyphae either singly or in brush-like arrangements. These annellides produce the oval or ellipsoidal, darkly pigmented annelloconidia.

(KOH, 1000+10X, DMD-108)

Scopulariopsis brumptii - anannellide is seen in the center of the photo extending from the hyphae. The "three-fingered" brush-like arrangement at the apex has anelloconidia still attached and some which have been released. (KOH, 1000+10X, DMD-108)

Scopulariopsis brumptii- appear to be two single annellides, with annelloconidia being produced at the apex. (KOH, 1000X, Nikon)

Scopulariopsis brumptii -the hyphae is septate and has produced a single annellide, bearing a single annelloconidium, at the center of the photo. (KOH, 1000X, Nikon)

Scopulariopsis brumptii -two brush-like arrangements of the annellides of S.brumptii are seen here in the center-left of the photo. Numerous annelloconidia, both attached and free are seen.

(LPCB, 1000X, DMD-108)

Scopulariopsis brumptii - two bunches of annellides (the right one more brush-like than the left) are seen in the center of the photo. Pigmented annelloconidia are seen at the apex.

(LPCB, 1000X, DMD-108)

Scopulariopsis brumptii -More brush-like annellides showing their arrangement and attatchment of annelloconidia. (LPCB, 1000X, DMD-108)

Scopulariopsis brumptii - another branching annellide, thinned out a bit so the arrangement of the annellides are more easily seen. The ring or collar is clearly visible on the annelloconidium seen at the lower middle of the photo. It appears as a scar or flattened base where the annelloconidium was once attached to the annellide. (LPCB, 1000X, Nikon)

Scopulariopsis brumptii -okay, too many photos, but what else am I going to do with them?

Hyphae, annellide & annelloconidia extending from the tips.

(LPCB, 1000X, DMD-108)

Scopulariopsis brumptii -ditto. Lots of annelloconidia produced by this brush-like structure.

(LPCB, 1000+10X, DMD-108)

Scopulariopsis brumptii -two single annellides seen extending from the hyphae traversing the center of the photo. Each has an annelloconidium still attached to the apex. Look closely at the darkly pigmented mature annelloconidia and you will see the collarette (annellide ring) on many.

(LPCB, 1000+10X, DMD-108)

Scopulariopsis brumptii -a poorer example of the photo explanation above. Two single annellides producing annelloconidia. Many more single annellides are present in S.brumptii cultures than in S.brevicaulis cultures where the arrangements are primarily brush-like.

(LPCB, 1000X, DMD-108)

Scopulariopsis brumptii -again, as above, single, rather than brush-like arrangements might be seen more frequently in S.brumptii, than in S.brevicaulis.

(LPCB, 1000X, DMD-108)

Scopulariopsis brumptii -ditto - more single, rather than brush-like annellides may be seen in S.brumptii cultures. (LPCB, 1000X, DMD-108)

Scopulariopsis brumptii -and a single annellide with an attached annelloconidium seen in the center of the photo. A single free annelloconidium is seen in the upper left-third of the photo - the annellide ring or collarette visible at the bottom. (LPCB, 1000+10X, DMD-108)

Scopulariopsis brumptii - a crowded photo showing at least two brush-like arrangements of annellides within a 'sea' of pigmented annelloconidia.

(LPCB, 1000X, DMD-108)

Scopulariopsis brumptii -oh, what the heck! -here's one more. A brush-like arrangement of annellides and darkly pigmented, oval to ellipsoidal annelloconidia.

(LPCB, 1000+10X, DMD-108)

Note: Scopulariopsisspecies may show variable sensitivity to cycloheximide and therefore may or may not grow on Mycosel/Dermasil® agar.

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Trichosporon species: (Basidiomycetous yeasts)

This post contains three species from the genus Trichosporon. I chose to deal with these together because of their common elements and that relevant features could be illustrated with a minimal number of photographs for each. Included here will be Trichosporon mucoides, Trichosporon asahii and Trichosporon inkin. I’ll add others if and when I come across them in the laboratory.

Generic Description of Trichosporon:

Ecology: Ubiquitous – found in soil and on plant material as well as humans and animals. It may be present as normal flora on the skin, on nails and in the mouth of humans.

Pathogenicity: Five species of Trichosporon are generally considered to be of clinical significance, these being T.mucoides, T.inkin, T.asahii, T.asteroides and T.cutaneum. Recent molecular studies have altered the taxonomy and with it the species names associated with various ailments. The species T.beigelii appears to be obsolete and may have been composed of the species listed above. Localized systemic as well as disseminated infections are increasingly being found in immunocompromised patients such as those with acute leukemia. Most frequently implicated are T.asahii or T.mucoides. Other species may be involved in superficial mycoses. All of these Trichosporon species have been implicated in a superficial, asymptomatic cosmetic condition called white piedra. It is characterized by the presence of relatively soft, white nodules located along the shafts of hair. T asahii is considered most closely linked to white piedra, although some authorities believe T ovoides is the main agent of white piedra of the scalp while T.inkin prefers the groin area and pubic hairs.

Note: Trichosporonspecies initially appear as yeast cells which develop arthroconidia as they mature. Adhesive tape mounts are of little value and slide cultures fare only slightly better. The method I employed here in observing Trichophyton was to inoculate a Corn Meal Agar (CMA) plate by scratching the yeast into the agar surface and covering it with a cover slip. (The coverslip prevents you from driving your objective into the culture when viewing) After appropriate incubation, the slide holder can be removed from a microscope and the arthroconida observed under low power by placing the open agar plate on the microscope stage and carefully lowering the objective into position.

CAUTION: Make sure you apply a coverslip to the media at the time of inoculation. After appropriate incubation, this allows the culture to be viewed undisturbed. Also, DO NOT use this technique for moulds (filamentous fungi) as the spores will become airborne, enter your nose and cause your head to fall off!!

Microscope with the slide holder removed, incubated plate in place, and objective carefully lowered to view the growth under (and around the edge of) the cover slip. The plate can be carefully moved around by a very steady hand (do this before coffee break!). Objective turret should be raised when placing or removing the plate and when changing objectives. Stick to the lower powers.

* * *

All descriptions below are from Trichosporon species grown at 30˚C on Sabouraud Dextrose Agar (SAB) or Corn Meal Agar (CMA) as indicated. Corn Meal Agar is used as it is less nutritious than the SAB. With a less favorable nutrition source, the organism may be somewhat more stressed and therefore develop spores, be they blasto or arthro, as a mechanism for dissemination and survival.

Some elements in the photos may appear larger or smaller at the same magnification. This is most likely due to my enlarging, then cropping a photo to emphasize a particular feature.

Trichosporon mucoides:

Macroscopic Morphology: On SAB agar at 30˚C colonies appeared yeast-like, moderately expanding with a shiny, moist or mucoid appearance (hence the name). They developed a light cream-like colour.

Trichosporon mucoides, SAB, 8 Days at 30˚C. Note the mucoid texture, hence the name. (Nikon)

Microscopic Morphology: Initially the cells appear as budding yeast cells which expand to produce slightly barrel-shaped arthroconidia. Terminal or lateral blastoconidia are present when mature.

Trichosporon mucoides colony as seen on Corn Meal Agar (CMA) viewed through the microscope as described above.It should be evident that when viewed as described, the cells are unstained. The creamy yeast like forms expand to produce hyphae & pseudo-hyphae (chains of individual cells which mimic hyphae) which grow out from the center of inoculation. (100X, Nikon)

Trichosporon mucoides -edge of the colony growing outwards from the center. The hyphae can be seen to branch. (CMA, 250X, Nikon)

Trichosporon mucoides -a closer look at the hyphae. Branching is evident.

(CMA, 250X, Nikon)

Trichosporon mucoides -hyphae can be seen developing into individual arthrospores along with blastospores along the sides. (CMA, 250X, Nikon)

Trichosporon mucoides -as above (CMA, 250X, Nikon)

Trichosporon mucoides -ditto. (CMA, 250X, Nikon)

Trichosporon mucoides -Chains of arthroconidia (AC) have developed from the hyphae. Lateral blastoconidia (BC) are present, as the name implies, on the sides of the arthconidial chains or hyphae. (CMA, 250X, Nikon)

Note: See also differentiation table at the end of this post.

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Trichosporon asahii:

Macroscopic Morphology: White to light cream coloured colonies which develop a farinose covering (covered with a whitish, mealy dust) and has a fissured marginal zone.

Trichosporon asahii - SAB, 72 hours at 30˚C. Culture almost looks mixed with lighter and darker colonies. Colonies appear quite dry in contrast to the Trichosporon mucoides described above.

(Nikon)

Trichosporon asahii - SAB, 30˚C, one week. Again, note the dry wrinkled apperance. The white mealy growth in the heaviest area (Lower right quadrant) is the farinose texture described in the text above. (Nikon)

Different techniques can be used to view all the Trichosporon species. Here I used both the plate method viewed on a microscope withe the slide holder removes, and the adhesive tape technique. Try whatever technique best captures the features that describe the species.

Microscopic Morphology: Again, initial culture appears as yeast cells which grow to develop barrel-shaped arthroconidia. Budding cells and lateral conidia are absent.

Trichosporon asahii - Initial growth on the CMA plate as viewed through the microscope set-up described above. Fine hyphae can be seen extending out comprising the colony.

(CMA, 100X, Nikon)

Trichosporon asahii -as above but photo taken at edge of the outgrowth.

(CMA, 250X, Nikon)

Trichosporon asahii -Taking a step back, these are the T. asahii yeast cells as they appear taken from the SAB colony plate. It is this form that makes Trichosporon a yeast-like fungus. Trichosporon grows as a yeast, both at room temperature and at 37˚C (not to be confused with a true dimorphic fungus). These yeast grow out forming hyphae or pseudohyphae, fragmenting into arthrospores.

(1000+10X, LPCB, DMD-108)

Trichosporon asahii -edge of a colony as viewed using the adhesive tape method. Not the best method to use because of the yeast-like texture of the colony. Free cells are seen to the left with the edge of the colony to the right.

(100X, LPCB, DMD-108)

Trichosporon asahii -the edge of the colony is to the left in this photo. Extending out from the colony are hyphae and chains of arthroconidia (near top of photo)

(400X, LPCB, DMD-108)

Trichosporon asahii -with a closer view, the difference between the smooth-walled hyphae and the chains of arthroconidia is evident. Here they lie side by side. In the upper right of the photo, the transition of the hyphae as it fragments into arthroconidia is quite evident.

(1000X, LPCB, DMD-108)

Trichosporon asahii -another view, as above.

(1000X, LPCB, DMD-108)

Trichosporon asahii -as above. Regardless of the technique used to observe the fungus, note that there are no lateral blastoconida on T.asahii that we saw on the T.mucoides isolate.

(1000X, LPCB, 1000X)

Trichosporon asahii - Barrel-shaped arthroconidia, but no lateral blastoconidia.

(1000X, LPCB, 1000X)

Trichosporon asahii -a hyphal element running from right to left through the center of the photo can be seen disarticulating (fragmenting) into barrel-shaped arthroconidia.

(1000X, LPCB, DMD-108)

Note: See also differentiation table at the end of this post.

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Trichosporon inkin:

Macroscopic Morphology: Colonies are initially yeast-like, off-white to cream coloured, smooth, moist and soft in texture. As the colonies mature they become finely cerebriform (wrinkled), farinose (covered with a whitish, mealy powder) or crumb-like. The center of the colony may become heaped up and the aging colony may adhere to and even crack the agar.

Trichosporon inkin -SAB, 30˚C, 72 hours. Off-white, soft cream-like texture

(Nikon)

Microscopic Morphology: Initially the cells are yeast-like however on incubation true hyphae as well as pseudohyphae develop. Long, cylindrical arthroconidia (2 – 4 µm X 3 – 9 µm) develop as the colony matures. Blastoconidia may form singly or in short chains, however lateral conidia are absent. Appressoria (a speciallized cell which assists the fungus in infection) may be present and best visualized in slide cultures.

Trichosporon inkin -rather than using the LPCB stain as with the T.asahii (above), here you can see the yeast cells as they appear suspended in ~10% Potassium Hydroxide solution (KOH).

(I have this noted as 400X, KOH, Nikon, but the magnification may be higher -ooops!)

Trichosporon inkin -as with the other Trichosporon species presented above, this is the T.inkin colony producing hyphae and pseudo-hyphae which extend from the point of inoculation.

(CMA, 100X, Nikon)

Trichosporon inkin -a blastoconidium can be seen developing at the end of a hyphae (upper right). Lateral blastoconidia, as seen with T.mucoides, are not produced.

(CMA, 250X, Nikon)

Trichosporon inkin -blastoconida at the apex of the hyphae.

(CMA, 250X, Nikon)

Trichosporon inkin -disarticulation (fragmentation) of the hyphae into arthroconida is seen here.

(CMA, 250X, Nikon)

Trichosporon inkin -branching hyphae & pseudo-hyphae fragmenting into arthroconidia.

(CMA, 250X, Nikon)

Trichosporon inkin -another view as described above.

(CMA, 250X, Nikon)

Trichosporon inkin - and yet another (ditto)

(CMA, 250X, Nikon)

Below is a table of physiological features which will help distinguish between the species presented here.

Differentiation: Trichosporondiffers from Cryptococcus species in that it produces arthroconidia while Cryptococcusdoes not. Trichosporon can be differentiated from Geotrichum in that Geotrichumdoes not produce blastoconidia and is urea negative.

Inoculation onto Malt Extract Broth at room temperature encourages the production of blastoconidia in Trichosporon species. Corn meal agar may do the same.

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Chrysonilia sitophila -Hyphomycetes (formerly Monilia sitophila)

Ecology:

Chrysoniliaspecies are a cosmopolitan (widespread) saprobe (lives of dead or decaying plant matter). Chrysonilia may be found within the home as ‘red bread mould’, named for the colour of growth found on contaminated bread. Chrysoniliais the asexual state (anamorph) of Neurospora, its sexual or (teleomorph) state. Neurospora’s ascospores are stimulated by applied heat and therefore Neurosporaand the Chrysonilia anamorph may be the first organisms to repopulate areas devastated by grass or forest fires. For the same reason, it is notorious for populating heat sterilized soils such as those found in greenhouses. Chrysonilia’steleomorph, Neurospora, is frequently used in the study of genetics and basic eukaryotic cell biology because of its desirable growth characteristics.

Chrysonilia sitophila was formerly called Monilia sitophila; however the genus Monilianow only encompasses plant pathogens.

Pathogenicity:

Chrysonilia is considered to be a contaminant and is not considered to be very pathogenic. It has been implicated in peritonitis, eye infections as well as occupational asthma.

Macroscopic Morphology:

· Colonies exhibit extremely rapid growth, maturing within 72 hours.

· Colonies can range in colour from white to a pale pink, a salmon colour to light orange.

· Texture is very cottony which quickly fills the petrie dish, often referred to as a “lid-lifter” as it crawls up the sides of the dish and presses against the lid.

Chrysonilia sitophila - 24 Hours growth on SAB agar plate incubated at 30˚C (Nikon)

Chrysonilia sitophila - This composite shows how quickly this fungus grows. Within one day the centrally inoculated plate has been filled with the mycelium. By 4 hours it has already completely filled the standard petrie dish and the areal hyphae are pushing against the lid. By day three, the slight pinkish to salmon to light-orange colour, characteristic of this fungus is evident. After a week the growth may have collapsed back on itself and be even be trying to crawl out the sides of the petrie dish. This organism is notorious for contaminating laboratories with the multitude of spores (conidia) produced. (Nikon)

Microscopic Morphology:

· Chrysonilia produces smooth-walled, hyaline, septate hyphae.

Sources differ on their description of the conidia produced. The majority of sources I consulted only mention rectangular ‘arthroconida’, produced as the hyphae disarticulate or break-up. Yet another source mentions blastoconidia in addition to the arthroconidia. By definition these should emerge as a bud which is then cleaved off at maturity, often leaving a mark or scar at the point of attachment. I have pointed these out where I believe they match the description.

· Simple, poorly differentiated conidiophores can be single or branched.

· Conidiophores produce branching chains of oval conidia (5-10 µm X 10-15 µm)

· Mature hyphae break up forming thick-walled rectangular arthroconidia connected by disjunctors.

Chrysonilia sitophila -a first look at low magnification.

(100X, LPCB, DMD-108)

Chrysonilia sitophila -septate hyphae are seen with poorly differentiated conidiophores producing chains of rather round blastoconidia. Rectangular arthroconidia are also evident, produced as the hyphae fragment or disarticulate.

(400X, LPCB, DMD-108)

Chrysonilia sitophila - another view as above.

(400X, LPCB, DMD-108)

Chrysonilia sitophila -and another. The rather round Blastoconidia (Bc) are being produced in addition to the rectangular Arthroconidia (Ac). Are the so called 'Blastoconidia' referring to immature or developing arthrospores in one source? The blastoconidum shown clearly does not appear to be the product of a fragmenting hyphal element. The hyphae are septate (S), along the lines where they will disarticulate into separate arthroconidia,

(400X, LPCB, DMD-108)

Chrysonilia sitophila -Branching hyphae & conidiophores.

(400X, LPCB, DMD-108)

Chrysonilia sitophila -ditto

(400X, LPCB, DMD-108)

Chrysonilia sitophila -whether these are considered to be blastoconidia or immature arthroconidia, chains are clearly shown to be developing from a branch extending from a main hyphal element.

(400X, LPCB, 400X)

Chrysonilia sitophila -arthroconidia can be seen joined to each other via a disjunctor cell (Dj) which appears as a small bridge separating one cell from the next. Various texts show the disjunctor as being more pronounced than seen here.

(1000X, LPCB, DMD-108)

The photograph show above, as several of the previous, are taken from an adhesive tape preparation. I have found at times that an adhesive tape preparation preserves features better than a slide culture and sometimes the opposite. Use whatever technique works best for you. Overall, I like slide cultures as it generally produces a much clearer photograph. In tape preps such as this, you can often detect the unevenness of tape surface and distribution of the adhesive. Here the background is full of bubbles from trapped air and the adhesive.

Chrysonilia sitophila -hyphae disarticulating into arthrospores (AC).

(1000X, LPCB, 1000X)

Chrysonilia sitophila -once again, it appears to me that there is a distinct difference between the blastoconidia (BC) which often appear to develop from the side of the hyphae or rudimentary conidiophore and those of the already rather rectangular arthrospores at the apex of a fragmenting hyphal element. (Dj = Disjunctor)

(1000+10X, LPCB, DMD-108)

Chrysonilia sitophila - One last photo again showing what I call a round blastoconidium (BC) on a very basic conidiophore (Cp). To my eye, these look distinctly different from the rectangular arthrospores forming by the disarticulation of the hyphae which they originate.

(1000+10X, LPCB, DMD-108)

Physiological Tests:

· +Growth at 37˚C.

· +Growth on Cycloheximide Agar

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Ascaris lumbricoides(Intestinal Nematode) “Roundworm”

Geographic Distribution:

Ascaris lumbricoides can be found throughout the world but is more commonly found in moist temperate and tropical regions. Ascaris is increasingly prevalent where poor sanitation exists. In cultures where human excrement (night soil) is used as fertilizer, infection may again be greater.

Pathogenicity:

Infection occurs with the ingestion of fully embryonated eggs (ova). Infection with Ascaris lumbricoides is termed Ascariasis and it affects more of the world’s population than any other parasitic disease. Ingestion of small numbers of eggs may be asymptomatic for the host however larger numbers may cause Ascaris pneumonitis[i]or Loeffler’s[ii]syndrome. Those infected with Ascaris lumbricoides may develop asthmatic attacks, shortness of breath, wheezing or a persistent cough. A heavy infection in the small intestine may result in a bowel obstruction, especially in children. Symptoms may include fever and generalized malaise possibly with abdominal distension, associated tenderness and possible vomiting.

Life Cycle:

Ingested embryonated eggs travel to the duodenum (small intestine) where they hatch and begin an obligatory migration throughout the body before they return to the duodenum to mature into adulthood. Hatched larvae start the migration by penetrating the duodenum wall to enter the blood or lymphatic system which eventually carries the larvae to the liver and the heart to eventually enter pulmonary circulation. In the lungs, the larvae break free of the capillaries to enter the aveoli where they continue to grow. After about 3 weeks, they begin to migrate through respiratory passages to reach the esophagus where they are swallowed and once again reach the small intestine.

At about two to three months post ingestion of the eggs, the now mature worms begin to lay their own eggs. It has been estimated that mature female worms may produce an average of 200,000 eggs daily. Ascaris lumbricoides females are oviparous[iii]. When passed, the eggs require about 2 to 3 weeks outside of the host to develop into the infective embryonated stage. While the eggs are susceptible to excessive heat and drying, they can remain viable in moist soils for long periods of time.

Ascaris Worm:

The adult Ascaris lumbricoides worms area creamy-white in colour, occasionally with a pinkish cast. Female worms range from 20 to 35 cm in length. Males are generally shorter and usually do not exceed 30 cm. Female worms are also generally thicker (3-6 mm) than the more slender males (2-4 mm). Males can be distinguished from females by their incurved tail. Females have a straight tail. Adult worms are believed to live up to a year. Ascaris worms do not attach themselves to the intestinal wall but rather maintain their position through constant movement.

Ascaris lumbricoides: Adult male worm show with its distinctive curved tail. This particular worm was about 24 cm or 10 in in length, (Nikon-Macroscopic)

Ascaris Eggs (Ova):

Diagnosis of ascariasis is by the demonstration of the characteristic eggs in the feces.

Fertile eggsare broadly oval in shape and typically yellow-brown in colour, stained by the bile in freshly passed stools. They measure 45 to 75 µm in length by 35 to 50 µm in breadth. The outer albuminoid coat is coarsely mammillated covers a smooth shell which is difficult to distinguish in laboratory preparations. Some confusion in diagnosis may occur if the Ascarisegg has lost its mammillated coat (decorticated) as it may somewhat resemble Hookworm or Trichostrongylus species ova. Ascaris eggs contain only one cell when passed in the feces.

Infertile eggsare elongate, about 85 – 95 µm by 43 – 47 µm in size. Their mammillated layer may vary from being coarsely irregular to a relatively smooth layer, almost devoid of mammillations. The internal contents of infertile eggs appear as a mass of disorganized, refractile granules.

Note: All the following photos are take from the re-suspended sediment after fecal concentration.

Ascaris lumbricoides egg seen in the center of the photograph.

(Nikon, 250X)

Ascaris lumbricoides egg has a distinctive appearance. This egg shows the rough looking surface due to the mammillated albuminoid coat.

(DMD-108, 400X)

Ascaris lumbricoides: another photo of the egg with its rough mammillated coat.

(Nikon, 400X)

Ascaris lumbricoides: a photo of the egg with an alternate focus inserted for comparison. As the egg is three-dimensional, adjusting the focus can aid in visualizing the egg's structure.

(Nikon, 400X)

Ascaris lumbricoides: as above.

(DMD-108, 400X)

Ascaris lumbricoides:Two A.lumbricoides eggs seen in the same field.

(Nikon, 400X)

Ascaris lumbricoides: Typical appearance of the Ascaris egg. The presence of these eggs in a fecal specimen is diagnostic for an Ascaris infection.

(DMD-108, 400+10X)

Ascaris lumbricoides: The thick albuminoid mammillated layer is quite evident on this fertile Ascaris egg. (DMD-108, 400+10X)

Ascaris lumbricoides: As above with a slightly altered focus.

(DMD-108, 400+10X)

Ascaris lumbricoides: Another photo of the Ascaris egg in a fecal concentrate.

(DMD-108, 400+10X)

Ascaris lumbricoides: As above but with an alternate focus of this fertile Ascaris egg. I post these alternate focus photos in an attempt to demonstrate the three-dimensional nature of the egg and their complexity. (DMD-108, 400+10X)

Ascaris lumbricoides: Yet another example, 'cause more is better!.

(DMD-108, 400+10X)

Ascaris lumbricoides: At a higher magnification. The large single cell interior is clearly visible.

(Nikon, 1000X)

Ascaris lumbricoides ova: Okay, more is better, right? Another photo showing the thick outer albuminoid mammillated layer. The inner wall often cannot be visualized unless the egg loses this outer layer (decorticated). The single celled contents are clearly visible.

(Nikon, 1000X)

Ascaris lumbricoides: Too many photos? I have to do something with them! Another photo of the Ascaris egg however it appears as if the interior cell has divided into four or more individual cells.

(Nikon, 1000X)

Ascaris lumbricoides: An Ascaris egg with the inset enlargement of the Ascaris egg. There appears to have been a number of divisions of the original cell contained within the egg.

(Nikon, 250X)

Too many photos? Just a few more to go..

While the Ascaris lumbricoides ova can be found in iron-hematoxylin stained preparations, the eggs usually stain so intensely that they appear as silhouettes and very little structural information can be discerned. They may be missed by an inexperienced eye. A thinner part of the stained slide may offer the best chance of recognizing and finding these eggs. The sedimentation concentration wet preparation technique is preferable in visualizing these eggs when present.

Note: The following photos are taken from Iron-Hematoxylin stained fecal preparations.

Ascaris lumbricoides:Two Ascaris eggs are seen in this photo which appear as two large dark masses on either side of the picture. The dark blob in the lower center is just an artifact present in the stool and clearly does not have the outline of the Ascaris egg.

(Nikon, 250X)

Ascaris lumbricoides: As with the concentrates, if a good microscopic field is found in the stained preparation, the Ascaris egg exhibits different textures as one focuses through it.

(Nikon, 1000X)

Ascaris lumbricoides: Same photo as above but with altered focusing. The mammillated layer appears as a scalloped edge surrounding the single celled contents of the egg.

(Nikon, 1000X)

Ascaris lumbricoides: In most iron-hematoxylin stained preparations the Ascaris ova will be heavily stained and often appear in silhouette with very little structure revealed.

(Nikon, 1000X)

Ascaris lumbricoides: As above. If a thin field can be found, the stained ova may show some detail.

(Nikon, 1000X)

Ascaris lumbricoides: Okay, last one. This egg doesn`t look particularly healthy. The `scalloped`edge of the mammillated coat is somewhat visible along the upper edge.

(Nikon, 1000X)

Diagnostic Problems:

Both fertile and infertile Ascaris eggs concentrate well however well using sedimentation techniques, however, if the laboratory only uses a flotation method for concentrating, infertile eggs may be missed by this technique.

[i]Pneumonitis refers to an inflammation of the lungs which can be cause by a variety of agents, from allergic, chemical or an infection. Ascaris pneumonitis is an inflammation caused by the presence of Ascaris lumbricoides migration through the lungs.

[ii]Löffler's syndrome or Loeffler's syndrome is a disease in which eosinophils accumulate in the lung in response to a parasitic infection.

[iii]Oviparous –egg development and maturation occurs outside of the female worm.

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Lots of photos for this one....probably too many, but didn't know what else to do with them!

Hymenolepis nana (Cestode) –Parasite

Also known as the “dwarf tapeworm”

Geographic Distribution:

Hymenolepis nanais a cosmopolitan parasite as it has worldwide distribution.

Associated Disease:

Hymenolepiasis, or Dwarf Tapeworm Infection. H.nana is often carried by the common house mouse. While it is more frequently isolated from children, adults are also quite susceptible. The location of the worm in the infected host is the small intestine. Light infections may be asymptomatic however a large worm burden may cause abdominal pain, diarrhoea, headaches, dizziness and anorexia.

Life Cycle:

Infection usually occurs following the ingestion of H.nana eggs (ova) which make their way to the small intestine where they subsequently hatch. The released sixed-hooked oncospheres, bury into the intestinal villi where, after a few days, they develop into cysticercoid larvae. The cysticercoid larvae are quite small, containing a single scolex. When mature, the larvae break out of the intestinal villi to enter the lumen of the small intestine. From the ingestion of eggs to the emergence of mature worms may take between two to three weeks.

Eggs of H.nanamay also develop into infective cysticercoids in various intermediate hosts, particularly grain beetles. Accidental ingestion with contaminated grain products allows the larva to grow into adult worms in mice and most probably, in humans as well.

Autoinfection is also possible. In this case, eggs passed by the adult tapeworm hatch within the intestine, develop through the cysticercoid stage and mature within the intestine as adult tapeworms.

Egg (Ova) Morphology:

H.nana eggs are spherical to sub-spherical in shape and have a thin hyaline (clear) shell. They measure between 30 – 47 µm in diameter. The six-hooklet oncosphere is surrounded by a membrane with two polar thickenings, from which arise four to eight filaments that extend into the space between the embryo and the outer shell. The related Hymenolepis diminuta has no polar filaments and this is one feature that aids in their differentiation.

Hymenolepis nana egg (ova): a first look at the fairly low power of 250 times magnification. Here an egg is seen amongst other fecal debris in a concentrated fecal specimen. Care must be taken so as not to over look the parasite as there may be other structures that may mimic or obscure the egg in the concentrate.

(Fecal concentrate, 250X, DMD-108)

Hymenolepis nana egg: A closer view of the H.nana ova in a fecal concentrate.

(400X, Nikon)

Hymenolepis nana egg: Typical appearance of H.nana egg.

(400X, Nikon)

Hymenolepis nana egg: More detail revealed.

(400+10X, DMD-108)

H.nana eggs are spherical to sub-spherical in shape. They may be described as 'broadly oval'.

Hymenolepis nana egg: Another view - inset showing details.

(400X, Nikon)

Hymenolepis nana egg: Four hooklets are visible in the lower left of the inner oncosphere surrounded by a membrane.

(400X, Nikon)

Hymenolepis nana egg: Another view with the magnified inset showing the position of one of two polar thickenings from which 4 to 8 polar filaments (PF) arise.

(400X, Nikon)

Hymenolepis nana egg: Yet another view.

(500X, Nikon)

Hymenolepis nana egg: Ditto

Hymenolepis nana egg: Now were getting to higher magnifications. Eggs (Ova) measure between 30 – 47 µm in diameter.

(1000X, Nikon)

Hymenolepis nana egg: Learn to recognize the ova regarless of the orientation and the shape it may be in. (500X, Nikon)

Hymenolepis nana egg: Cell wall appears to be damaged on the left side of this photo. Cell may not be viable, (500X, Nikon)

Hymenolepis nana egg: Oncosphere with a few of the six hooklets are visible in this photo.

(1000X, Nikon)

Hymenolepis nana egg: The pointed structures within the oncosphere (upper part of the inner cellular structure) are the hooklets. Thin, hyaline cellular wall is evident as well.

(1000X, Nikon)

Hymenolepis nana egg: Ditto

(1000X, Nikon)

Hymenolepis nana egg: At least four of the six hooklets are seen within the oncosphere which is surrounded by a membrane. This is contained withing the cell surrounded by the hyaline cell wall.

(1000X, Nikon)

You should be able to recognize the Hymenolipis ova in a fecal concentrate after having viewed all the preceding photos. While the characteristic structures are most clearly viewed in a freshly passed or concentrated fecal specimen, you should be able to recognize the egg in a stained smear as well. A number of photos of iron-hematoxylin stained permanent smears follow.

Hymenolepis nana egg: Here in the same field, one can see that the uptake of the stain and the appearance of the egg can differ significantly.

(500X, Nikon)

Hymenolepis nana egg: At first glance, the egg may even resemble a large amoeba cyst such as Entamoeba coli. Focusing through the cell will not reveal the 8 nuclei expected in E.coli. The size difference also should eliminate the cyst. (1000X, Nikon)

Hymenolepis nana egg: Ditto

(500X, Nikon)

Hymenolepis nana egg: Oncosphere visible, surrounded by a clearing with the outer cell wall barely visible.

(1000X, Nikon)

Hymenolepis nana egg: Oncosphere is visible but cell wall is not.

(1000X, Nikon)

Hymenolepis nana egg: Iron-Hematoxylin stained showing little detail in the oncosphere but cell wall is visible. (1000X, DMD-108)

Hymenolepis nana egg: Another variation of the egg's appearance in an Iron-hematoxylin stained smear.

(1000X, DMD-108)

Hymenolepis nana egg: Dehydration process during staining has distorted the cell wall

(1000+10X, DMD-108)

Hymenolepis nana egg: Egg with outer cell wall and inner ocosphere. Shadows of several hooklets can be seen within, (1000+10X, DMD-108)

Hymenolepis nana egg: Last one.

(1000+10X, DMD-108)

Adult Worm Morphology:
H.nana adult tapeworms are quite small, measuring 2.5 – 4.0 cm in length. The tiny, knob-like scolex has four suckers and a rostellum bearing a ring of 20 - 30 hooklets. Proglottids (segments) are wider than they are long.

Sorry, I have no worm to take a photo of, however they can be found elsewhere on the web.

Diagnosis:

Diagnosis is made by demonstrating the characteristic eggs in the fecal sample. Unstained, concentrated specimens are preferable as the details are more evident. Also, thin-shelled eggs may collapse on permanent stained smears, making them difficult to identify. Specimens preserved in PVA (polyvinyl alcohol) do not exhibit morphological characteristic nearly as well as those preserved in formalin fixed specimens. The adult worm or proglottid segments are rarely seen in the stool.

Confusion may occur with the related Hymenolepis diminuta, however H.dimunataeggs of the ‘Rat Tapeworm’ are much larger (70 – 85 by 60 - 80 µm in diameter) than those of H.nana. They also do not possess the polar filaments as previously mentioned.

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Aureobasidium pullans (Hyphomycetes) –Black yeasts

Happy New Years, 2015 - Another post with far too many photos....

Ecology:

Aureobasidium pullans’ preferred habitat is on the aerial portions of plants, particularly the leaves. It may reside there as a saprobe (lives on dead organic matter) but may be a phytopathogen on susceptible species of plants. It is a cosmopolitan fungus (found just about everywhere) but prefers temperate zones. It may be isolated from humid indoor environments such as, foodstuffs, textiles, shower curtains and soil. A.pullans may be found as a laboratory contaminant.

Pathogenicity:

A.pullansappears to be opportunistic, with systemic infection often the result of traumatic implantation. It has been implicated in peritonitis and pulmonary infections. It may rarely be the cause of keratitis or cutaneous infections.

Macroscopic Morphology:

Most sources describe the rate of growth as “rapid”. Structures in the photographs below do develop rapidly (3-5 days), however the colony itself expands at a moderate rate. Initially the colony appears white, cream or pinkish in colour but then adds shades of brown, grey and black as it ages (due to the development of chlamydoconidia). The colony may have a white or slightly greyish fringe along the expanding edge. The texture is moist or creamy, glistening under reflected light. The reverse is pale in colour but becomes dark as the colony matures.

Aureobasidium pullans on SAB media after 3 weeks incubation at 30^oC (Nikon)

Aureobasidium pullans on SAB -progression of growth at 30^oC. Most sources state that Aureobasidium pullans is a rapid grower. The colony may mature fairly rapidly but expansion of the colony is more moderate. (Nikon)

Microscopic Morphology:

Young colonies appear yeast-like, consisting of unicellular, budding cells. As the colony ages, two types of vegetative hyphae (3 – 12 µm dia.) appear to be produced. The first are described as thin walled, hyaline (clear) hyphae which produce blastoconidia (also hyaline)synchronously in tufts (ie. simultaneously, from poorly differentiated conidiogenous cells along the length.)

Blastoconidia (3 – 6 X 6 – 12 µm) are described as oval to ellipsoidal but can vary in size and shape

The second type of hyphae appears to have a thicker wall and is dematiaceous (darkly pigmented) which develop into brown coloured arthroconidia and chlamydoconidia. Sources seem to be unclear as to whether these two hyphal forms are truly different or simply different stages of development. I found that both forms appear to be present as the colony matured.

Sources also state that endoconidia may be present within intercalary cells but were not observed in the isolate presented here. Perhaps the development of endoconidia is media dependent.

Two techniques seem to be necessary to best view the structures of Aureobasidium pullans. I found that just using the adhesive tape technique or the slide culture technique to view the structures, failed to capture where the blastoconidia were being produced. The microscopic fields were abundantly full of blastoconidia, however they were all free and how they originated was not at all obvious. The Dalmau plate method, described below was also used. I used this technique on a previous post to view various Trichosporon species.

The Dalmau plate method can be employed to view the blastoconidia 'in-situ'. What is shown below is a Corn Meal Agar (CMA) plate inoculated with Aureobasidium by simply scratching it into the surface and then covering it with a coverslip. The coverslip simply aids in focusing and prevents the objective to be contaminated by inadvertently lowering it into the inoculated agar. After appropriate incubation, the petrie dish can be placed on a microscope stage (remove plate lid & stage slide holder) and viewed under low power. The hyphae growing out from the center of inoculation are virtually undisturbed and should now show the blastoconidia growing synchronously in tufts from poorly differentiated conidiogenous cells along the length, as already described in the previous paragraph.

Aureobasidium pullans on CMA after 72 hours incubation at 30^oC. (Nikon)

Here is the technique described above, which I used for the next five photographs. Fungi, primarily being aerobic organisms can be seen growing out from the coverslip where the oxygen tension is lower. As the colony expands on this less nutritious Corn Meal Agar plate, the blastoconida can be seen having been produced in tufts along the length of the hyphae. When focusing the low power objective (100X or 250X) on the edge of the growth, the inserted photo is what appears (purple arrow). The following 4 photos were taken from this plate.

Aureobasidium pullans on CMA -hyaline hyphae bearing blastoconidia growing out from central inoculation point. (100X, Nikon)

Aureobasidium pullans on CMA -at slightly higher magnification, the somewhat oval blastoconidia are evident. (250X, Nikon)

Aureobasidium pullans on CMA -at still higher magnification, the somewhat oval blastoconidia are seen growing singly and in tufts along the length of the septate, hyaline hypha.

(400X, Nikon)

Aureobasidium pullans on CMA -after additional incubation (~1 week), tufts of blastoconidia can be seen along the length of the hypha.

(250X, Nikon)

The following photos are taken from slide cultures of Aureobasidium pullans after the stated incubation times. The adhesive tape techique can be used but as the fungus has a yeast-like texture, pressure may just "squash" the structures rather than preserve them by adhering to the tape.

Aureobasidium pullans - I just found this to be a cute photo. A small piece of agar adhered to the glass cover slip when removed. Hyphae can be seen growing out from the dematiaceous center

(100X, LPCB, DMD-108)

Aureobasidium pullans -the growth at the edge of a slide culture adhering to the cover slip. A mass of blue stained blastoconidia can be seen from which the hyphae are extending towards the top of the photo. Some hyphae are already becoming darkly pigmented.

(250X, LPCB, DMD-108)

Aureobasidium pullans -at higher magnification, a large mass of blue stained yeast-like cells are seen in the upper portion of the photograph. Sources speak of "yeast-like cells" and "blastoconidia" but fail to clarify if these are in fact, the same. I fail to see distinctions that would make these different.

Also seen in this photograph is a hyaline, septate hypha which already appears to be developing into arthroconidia at the far left end. (400X, LPCB, DMD-108)

Aureobasidium pullans - As above, hyphae being produced and reaching out from central mass of yeast-like cells. A few dematiaceous (darkly pigmented) hyphae also are present towards center-right of the photo. (400X, LPCB, DMD-108)

Aureobasidium pullans - as above.

(400X, LPCB, DMD-08)

Aureobasidium pullans - again, as with the previous descriptions but here at the top of the screen there appears to be two type of 'single' cells, with the smaller lighter blue as the yeast-like cells and the darker, larger and somewhat oval cells still clinging to the hyphae being the blastoconidia.

(400X, LPCB, DMD-108)

Aureobasidium pullans - hyphae breaking up into individual arthrospores.

(400X, LPCB, DMD-108)

Aureobasidium pullans - indivdualconidia remain at the bottom of the photograph while hyphae are becoming darkly pigmented. Development of arthroconidia and chlamydoconidia is evident along the hyphae. (400X, LPCB, DMD-108)

Aureobasidium pullans - the organism appears to take on some bizarre shapes with the darkly pigmented chlamydoconidia and more box-car shaped arthroconidia now developing at about 72 hours if incubation.

(400X, LPCB, DMD-108)

Aureobasidium pullans - loose oval-shaped blastoconidia with dematiaceous hyphae and formation of chlamydoconidia

(400X, LPCB. DMD-108)

Aureobasidium pullans - blue stained blastoconidia with dematiaceous chains of chlamydoconidia and arthroconidia.

(400X, LPCB, DMD-108)

Aureobasidium pullans - ditto

(400X, LPCB, DMD-108)

Aureobasidium pullans - Individual dematiaceous chlamydoconidia and blue-stained, hyaline hyphae extending out towards right side of photo. Individual blastoconidia seen scattered throughout.

(400X, LPCB, DMD-108)

Aureobasidium pullans

(1000X, LPCB, DMD-108)

Free blastoconidia
Dematiaceous, boxcar-shaped, arthroconidia
Dematiaceous, round, intercalary chlamydoconidia
Hyaline hyphae developing as arthroconidia

Aureobasidium pullans - a few photos as described above.

(1000X, LPCB, DMD-108)

Aureobasidium pullans - As above

(1000X, LPCB, DMD-108)

Aureobasidium pullans - As above

(1000X, LPCB, DMD-108)

Aureobasidium pullans - As above

(1000X, LPCB, DMD-108)

Aureobasidium pullans - As above

(1000+10X, LPCB, DMD-108)

Aureobasidium pullans - okay, only a few more photos. Here, the terminal chlamydoconidia appears to be germinating (arrow), releasing new growth of hyaline cells (hypha). A few blue-stained blastoconidia remain. This is from a slide culture after 4 days of incubation.

(1000X, LPCB, DMD-108)

Aureobasidium pullans - Hyaline hyphae stained blue showing some internal structure or inclusions. Endoconidia, (conidia found within intercalary hyphal cells) do not seem to be present.

(400+10X, LPCB, DMD-108)

Some sources describe the presence of intercalary endoconidia being produced within the hypha by Aureobasidium pullans. I did not find evidence of these on the isolate presented here. Perhaps the production is media related or perhaps strain dependant.

Aureobasidium pullans - Again, hyaline hypha stained blue showing some internal structure or inclusions. These do not appear to be endoconidia.

(1000+10X, LPCB, DMD-108)

Aureobasidium pullans

(1000X, LPCB, DMD-108)

Physiology:

Aureobasidium pullans:

· Grows best at about 25^o C and may be inhibited at 35^oC.

· Tolerates up to 10% NaCl

· Is inhibited by cycloheximide

· Urea Positive

· Nitrate Positive

Note:Blastoconidia formation may best be visualized using the Dalmau plate method as for demonstrating chlamydoconidia in Candida albicans

A.pullans may most frequently be confused with Hormonema dematiodes and possibly Wangiella (Exophiala) dermatiditis or Hortaea werneckii when young and yeast-like.

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Ochroconis species(Hyphomycete) Fungus

Ecology:

Ochroconisspecies are primarily soil saprobes (live on decaying vegetative matter), found in the soil worldwide. As of 2014, there are thirteen recognized species of Ochroconis.

Pathology:

Ochroconisspecies have been recovered from central nervous system (CNS) infections as well as pulmonary (lung) infections, from both immunocompromised and immuocompetent hosts. In particular, Ochroconis gallopava is considered to be a neurotropic opportunist and proposals have been made to place this fungus into a new genus, Verruconis.

Ochroconisspecies are considered to be mesophilic (preferring moderate temperatures) however they can cause disease in several species of cold-blooded animals, particularly fish such as coho salmon and rainbow trout. Ochroconisspecies are known to cause encephalitis in chickens, turkeys and other fowl.

Macroscopic Morphology:

The rate of growth is rather slow growing as measured by the expanding colony but will mature to produce conidia usually within 5 days.

The texture is described as velvety to felt-like or floccose.

The colony colour is usually a reddish-brown to chocolate brown to a dark olive-grey. The reverse is a dark brown to black.

A red to brown pigment may diffuse into the medium.

Ochroconis on Sabouraud Dextrose Agar (SAB) incubated at 30˚C for 3 weeks. (Nikon)

Ochroconis- same organism as above but with different background and lighting to show variations in texture and pigment. SAB, 30˚C, 3 weeks. (Nikon)

Ochroconis on SAB - colony center rises off and above the agar surface resembling and inverted shallow bowl. Looked like a small hollow mountain!

Below, right - shows the Reverse of the Ochroconis presented here. The lighter section which appears in the center of the larger colony is the area that has "cupped" and lifted off of the surface of the agar. (Nikon)

Microscopic Morphology:

Ochroconisproduces septate hyphae which are hyaline (clear) to pale brown in colour.

Conidiophores are also hyaline to pale brown. They arise erect and unbranched from the hyphae and usually have a knobby or bent appearance. The conidiophores have apical denticles in a sympodial arrangement from which the conidia have formed. Conidia (2.5 – 4.5 µm X 11 – 18 µm) are usually 2 to 4 celled, depending on the species. Conidia are cylindrical to club shaped and after detachment from the conidiophore (denticle), an inconspicuous frill may remain on both the denticle and the conidium base.

Ochroconis species - edge of growth of slide culture as initially viewed at low magnification. Hyphae radiating out from point of inoculation after 1 week of incubation.

(LPCB, 250X, DMD-108)

Ochroconis species - Conidia extending from hyphae now become evident at this higher magnification. (LPCB, 250X, Nikon)

Ochroconis species - as we once more increase magnification individual conidia attached to phialides can be seen in more detail. (LPCB, 400X, Nikon)

Ochroconis species - another view with conidia attached to their phialides. Brown pigmentation seen in lower right of photo. (LPCB, 400X, Nikon)

Ochroconis species - one more view. Remember, the fungus grows in three dimensions and even here, within the space between a microscope slide and a cover slip, hyphae and phialides extend forward, into the photo and some backwards, out of the photo. It is for this reason that the photos often appear to be out of focus as only those features that lie relatively flat along the focal plane appear clearly in the picture. (LPCB, 500X, Nikon)

Ochroconis species - a massive ammount of conidia with the most mature to the right where the brown pigment is most evident. (LPCB, 500X, Nikon)

Ochroconis species - edge of slide culture (as previous)

(LPCB, 500X, Nikon)

Ochroconis species - a hypha weaves its way from top center to bottom center of this photograph. Along its length you can see phialides with attached conidia. (LPCB, 500X, Nikon)

Ochroconis species -a two-celled conidium is seen attached to a phialide that extends from the hypha. The conidium shows a slight constriction near the center.

(LPCB, 1000X, Nikon)

Ochroconis species -Numerous phialides with attached conidia shown here. Phialides seen bearing multiple conidia. Dark pigmentation is also evident as the colony ages. Most conidia are two-celled, some showing a slight constriction near their center, others (center) showing the furthest end of the conidium being larger than that nearest the phialide from which it originated. The arrow points to a phialide which has lost it's conidium -a slight scare remains.

(LPCB, 1000X, DMD-108)

Ochroconis species -a mass of darkly pigmented, septate hyphae as well as a dark blue conidium seen near the left of the photo (LPCB, 1000X, DMD-108)

Ochroconis species -Hyphae with phialides bearing conidia. Arrows point to phialies with multiple (two) conidia, the one on the left showing the scar remaining after the conidium has detatched.

(LPCB, 1000X, DMD-108)

Ochroconis species -a three-celled conidium appears to be present along with the more numerous two-celled conidia. (LPCB, 1000+10X, DMD-108)

Ochroconis species -center of photo, conidium attached to a long phialide.

(LPCB, 1000X, DMD-108)

Ochroconis species -Nice photo of a phialide bearing two conidia attached to the parent hypha.

(LPCB, 100X, DMD-108)

Ochroconis species -yet another photo showing much the same. Phialides bearing multiple conidia with the arrows showing the ragged attachment points which remain after the conida have detatched.

(LPCB, 1000+10X, DMD-108)

Ochroconis species -septation within the hyphae are clearly visible as is the developing brown pigmentation. Tree Conidia still attached to their brownish pigmented phialides which extend from the hyphae. Near center, one phialide is seen with a detached conidium nearby.

(LPCB, 1000X, DMD-108)

Ochroconis species -Aging phialides & conidia. Brown phialide in center of photo has a thinner denticle at it's apex to which the conidium is attatched.

(LPCB, 1000X, DMD-108)

Ochroconis species -again, septations in hyphae are clearly evident. Conidium near center of photo appears to be supported by a bent denticle. (LPCB, 1000+10X, DMD-108)

Ochroconis species -conidium at apex attached to phialide by a short denticle.

(LPCB, 1000X, DMD-108)

Ochroconis species - Seen more clearly here, the two-celled conidium is attached to the phialide via a somewhat wavy denticle. The phialide, in turn is attached to the hypha from which it originated.

(LPCB, 1000+10X, DMD-108)

Ochroconis species - Here we are looking at the conidium 'head-on' so it appears spherical. The conidium is attatched to the hypha by this darkly pigment, and apparently degenerating, phialide-denticle structure. (LPCB, 1000+10X, DMD-108)

Ochroconis species - Almost done here. A phialide bearing two conidia.

(LPCB, 1000+10X, DMD-108)

Ochroconis species

(LPCB, 1000+10X, DMD-108)

Ochroconis species - Phialide bearing multiple conidia.

(LPCB, 1000+10X, DMD-108)

Physiology:

Growth is inhibited by cycloheximide.

I'm uncertain as to which specific species I have pictured in this blog as the conidia occasionally show more than 2 cells and can be constricted in the center. The majority of the conidia from the isolate presented here are two-celled and rather ellipsoidal or cylindrical in shape

Differentiation of the more common species:

Conidia usually 4-celled= Ochroconis tshawytschae

Conidia usually 2-celled = Ochroconis gallopava

Conidia distinctly clavate (club-shaped), with the upper cell wider than the basal cell = Ochroconis humicola

Conidia broadly ellipsoidal and constricted at the septum = Ochroconis constricta

Ochroconis constricta is not known to be pathogenic

A new genus Verruconis is proposed for the neurotropic opportunist Ochroconis gallopava.

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Acremonium species -Hypocreaceae Family (obsolete name Cephalosporium spp.)

Ecology:

Acremoniumspecies are yet another cosmopolitan fungus (ie. found just about everywhere) which can be isolated from soils as well as decaying plant material. There are about one hundred recognized species of Acremonium.

Pathogenicity:

Acremoniumspecies is associated with ‘white grain mycetoma’[i], an infection most commonly of the foot. Acremonium has also been implicated in meningitis, endocarditis, endophthalmitis and corneal ulcers. Onychomycosis or ‘tinea unguium’ (fungal infections of the nail) may also be caused by localized infection with Acremonium. Disseminated infections are rare and may result from traumatic injury and may be more likely in immunocompromised hosts.

Macroscopic Morphology:

Acremonium exhibits moderately rapid growth, rather flat colonies that may be slightly raised in the center. The colony has been described as glabrous to membrane-like initially, becoming powdery, cottony or even felt-like as it matures. The colony may be white to cream in appearance or even yellowish to coral or pinkish in colour. The reverse is pale, yellowish to pinkish in colour.

Acremonium species -Sabouraud Dextrose Agar (SAB) incubated at 30˚C for 18 days. (Nikon)

Acremonium species -Sabouraud Dextrose Agar (SAB) incubated at 30˚C for ~21 days. (Nikon)

Note -On the Challenges of Plate Photography:

This is the same species as above with a few more days of incubation. The reason I posted both these photos is to show the difficulty I have in expressing the true colour (and often texture) of the fungus even when grown on the same media and under identical conditions. The first photo was taken with the plate placed against a white background and the second against a black background.

The other challenge is lighting. I take these photos for my own entertainment and education - for "fun" as the title of the blog states. The acute care lab I work out of is interested only in a quick and accurate identification of fungi in clinical specimens. Documentation by photography is not a concern and we have no professional set up for taking photographs. I have rigged up my own apparatus for use as a camera stand for use within a biological safety cabinet (BSC) (see the post entitled 'Toys'). Photography of macroscopic plates is confined to the BSC as the lab could quickly become contaminated with spores if fungal plates were to be examined outside of the BSC. Many fungi produce vast quantity of spores which can become airborne with only the slightest breeze.

I have little control over the lighting. I often take a large number of photos using both white and black backgrounds. I simply use a sheet of white paper which I have run through a photocopier without a "target". In other words, I leave the cover of the photocopier open and "copy the air" which results in a sheet of paper covered with back toner on one side but white on the back. I can flip this paper over to the black or white side as I wish and as the background paper may be contaminated with spores, I can discard it safely when finished.

I use the fluorescent tube lighting of the BSC itself, I have an incandescent source of lighting I can bring into the cabinet, and I can use the flash unit on the camera itself in order to manipulate lighting. Light coloured fungi often show more detail when on a dark background and dematiacious fungi (darkly pigmented) stand out better on white backgrounds - but not always! I try to avoid cast shadows. I also find that the flash often produces such glare and reflection off of the petrie dish and agar surface that it obscures the true nature of the organism. By taking a large number of photos on various backgrounds and with various combinations of the lighting I have available, I can usually find a photograph that reflects the true characteristics of the fungus I am attempting to document.

Microscopic Morphology:

Acremoniumproduces septate hyphae from which erect, unbranched and tapering phialides extend. Most phialides (but not necessarily all) have a basal septum which delimits them from the hyphae proper. Conidia are oblong (2–3 X 4–8 µm) are usually one-celled, however bicellular conidia may occur. The conidia are produced and accumulate as balls (rarely as chains) at the apices of the phialides but they are fragile and easily disrupted.

Phialide: A specialized conidiogenous cell (conidiophore) that produces conidia in basipetal succession without increasing in length. (Mycology Online)

Acremoniumspecies - above is a little piece of agar which adhered to the slide culture microscope cover slip when removed from the agar block (See Post on Slide Cultures). From this little flake of agar you can see phialide extending outwards with little "balls" of conidia attached at their apices. (LPCB, DMD-108, 250X)

Acremoniumspecies - a collection of hypha run through the center of the photo from which you can see the phialide bearing conidia at their apices. This is like "micro-botany" with the fungi as tiny plants, the 'seeds' (conicia) at the tips of the stems. (LPCB, DMD-108, 400X)

Acremoniumspecies - As above but at a slightly higher magnification.

(LPCB, DMD-108, 400+10X)

Acremoniumspecies - the individual conidia can now be seen, gathered at the tapering tips of the phialides. (LPCB, DMD-108, 1000X)

Acremoniumspecies - not the greatest shot but try to picture the three 'balls' of conidia siting at the top of the phialides which are extending upwards, towards you, the viewer. The phialides are blurred (out of focus) as they are out of the focal plane of the camera, lying beneath the balls of conidia, where they attach to the hyphae. (LPCB, DMD-108, 1000X)

Acremoniumspecies - The same as above but viewed more from the side. The originating hyphae and tapering phialides are slightly out of the focal plane of the camera. The conidia that were produced at the apex of the phialide remains undisturbed as a ball around the top.

(LPCB, DMD-108, 1000_10X)

Acremoniumspecies - The previous photos show the phialides and conidia almost in three-dimensions. In the photos that follow, they are lying fairly flat, making it easier to see the features.

(LPCB, Nikon, 400X)

Acremoniumspecies - in the center of the photograph, there are two phialides, both with a collection of conidia at their apices, where they remain after being produced.

(LPCB, DMD-108, 1000X)

Acremoniumspecies - another view of the tapering phialides extending from their parent hyphae where elongated, ellipsoidal conidia lay gathered.

(LPCB, DMD-108, 1000X)

Acremoniumspecies - pretty much the same as in the above photo. The phialide at the very top of the photo appears to have produced only one conidium. Perhaps it is younger than the others.

(LPCB, DMD-108, 1000X)

Acremoniumspecies - another photo of tapering phialides and the ellipsoidal conidia gathered around the apices. (LPCB, DMD-108, 1000X)

Acremoniumspecies - insert -one tapering phialide with individual conidia gathered at the apex.

(LPCB, DMD-108, 1000X)

Acremoniumspecies - Two phialides, side by side producing large quantities of conidia.

(LPCB, DMD-108, 1000+10X)

Acremoniumspecies - tapering phialide with single ellipsoidal conidia at the apex. A slight collarette can be seen remaining around the apex. Conida already produced almost seem to have fallen down and accumulated around the base of the phialide and hypha.

(LPCB, DMD-108, 1000+10X)

Acremoniumspecies - as previously -another view of the tapering phialides and the balls of conidia adhering to the apex of the phialides where they were produced.

(LPCB, DMD-108, 1000+10X)

Acremoniumspecies - As previously.

(LPCB, DMD-108, 1000X)

Acremoniumspecies - a few barren phialides can be seen and the large mass of dispersed, primarily single celled conidia seen throughout.

(LPCB, DMD-108, 1000X)

Acremoniumspecies may be confused with Verticilliumand some isolates of Fusarium where macroconidia are not present. Rate of growth and colony morphology differs from that of Acremonium species and may provide initial clues for differentiation.

[i]Mycetoma, or maduromycosis, is a slow-growingbacterial or fungalinfectionfocusedin onearea of thebody,usuallythefoot. Approximatelyonemonth or moreaftertheinjury,a painless noduleformsundertheskinsurface. The nodules develop into a tumor which produces sinuses to drain fluid. Thefluidcontainstinygrains, which may be a clue as to the type of organism is causing the infection.

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Exophiala jeanselmei-Mitosporic fungi; Hyphomycetes

Note: With the advent of molecular testing, some sources are stating that Exophiala jeanselmei should more correctly be referred to as the Exophiala jeanselmei complex. See explanation at the bottom of this post.

Also: Compare Exophiala jeanselmei with Exophiala dermatitidis elsewhere in this blog by clicking here.

Ecology:

Exophiala species can be found worldwide and may be isolated from soil, decaying wood and fresh water sources.

Pathogenicity:

Exophiala jeanselmei may be an agent of mycetoma[i]and phaeohyphomycosis[ii]has also been implicated as an agent of back grain mycetoma and chromoblastomycosis[iii]. While occurring worldwide, some evidence suggests that infections, particularly neurotropic infections, may be more prevalent in East Asia.

Macroscopic Morphology:

Colonies are initially moist and yeast-like in texture; however they soon form a velvety surface due to the production of aerial mycelia. Some isolated can remain yeast-like without further development.

The colony can be brownish-black to greenish-black with the reverse being black in colour.

Exophiala jeanselmei grows slowly, maturing within about 14 days at 25ᵒC to 30ᵒC.

Exophiala jeanselmei on SAB after incubation for 3 weeks at 30ᵒC.

While this isolate matured rather quickly, compared to the "up to 14 days" stated by some sources, the colony expanded outward slowly. (Nikon)

Exophiala jeanselmei on SAB after incubation for ~3 weeks at 30ᵒC.

Heaped up, folded appearance. (Nikon)

Microscopic Morphology:

Young cultures with budding yeast-like cells may eventually develop septate hyphae. Hypha, initially hyaline (clear) become pale brown to olivaceous as the colony ages. The conidiogenous annellophores (structure producing annelloconidia) are slender, sometimes branched, and narrow towards the apex (tip). The smooth annelloconidia (2.5 – 6.0 µm X 1.2 – 2.5 µm) are oval or ellipsoidal in shape and if undisturbed, accumulate in clusters around the tip and upper sides of the annellide. Potato Dextrose Agar (PDA) or Corn Meal Agar (CMA) may enhance conidia production, however all the photos on this post were taken from isolates grown on Sabouraud Dextrose Agar (SAB) with copious conidia produced.

Exophiala jeanselmei - mycelial growth adhering to small piece of agar from a slide culture after 4 days of growth. (250X, LPCB, DMD-108)

Exophiala jeanselmei - small clusters or balls of conidia become evident, dispersed along the mycelia at this magnification. (250X, LPCB, Nikon)

Exophiala jeanselmei -Individual conida can now be made out at this magnification.

(400X, LPCB, DMD-108)

Exophiala jeanselmei -septate hyphae appear as if vacuolated. Conidiogenous structures (annellophores) do not appear to be highly differentiated from the hyphae themselves and can be intercalary (along the hyphae) or terminal (at the end of the hyphae).

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -as above, with typical accumulation of the oval to ellipsoidal annelloconidia around the top, and along the sides of the annellophore.

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -again, intercalary, and terminal production of annelloconidia.

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -and another.

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -Both clusters of conidia and individual conidia can be seen at the tips of the annellophores. (1000, LPCB, DMD-108)

Exophiala jeanselmei -Branched annellophore with (annello)conidia.

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei - and another...

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei - ditto, as above.

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -branched conidiophore at various stages of conidia production.

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -okay, too many photos of the same structures!!

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -the annellophore appears undifferentiated from the hyphae from which it arises. Nice photo showing the annelloconidia accumulated around the apex of the annellophore from which they were produced. Conidia are single-celled with the darkly staining band across the center of the conidia not being a septum. Also visible on many of the conidia is the remnants of a fringe, the scar that remains from where they were attached to the conidiophore. This represents a specific method of reproduction and fringe or scar (annellide) is why we specify the conidiophore as an annellophore, and the conidia as an annelloconidia. (1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -as the colony matures, the hypha become pigmented which gives the colony its distinctive colour. (1000X, LPCB, DMD-108)

Exophiala jeanselmei -as above, just another view,

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -an interesting shot showing the annellophore rising upwards from it's parent hyphae with the annelloconidia clustered about the apex. Almost three-dimensional.

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -copious amounts of conidia are produced.

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -as previously stated, the darkly staining band across the center of the annelloconidium is not a septum. The single-celled annelloconidia have the fringe or scar which remains at one end of the cell which is why the conidia are specifically referred to as annelloconidia.

(1000+10X, LPCB, DMD-108)

Exophiala jeanselmei -conidium germinating.

(1000X, LPCB, DMD-108)

Exophiala jeanselmei -annelloconidia (1000+10X, LPCB, DMD-108)

[i] Mycetoma is a chronic, progressively destructive morbid inflammatory disease usually of the foot but any part of the body can be affected. Infection is most probably acquired by traumatic inoculation of certain fungi or ‎bacteria into the subcutaneous tissue. (WHO)

[ii] Phaeohyphomycosis is a heterogeneous group of mycotic infection caused by dematiaceous fungi whose morphologic characteristics in tissue include hyphae, yeast-like cells, or a combination of these. (Wikipedia)

[iii] Chromoblastomycosis is a chronic fungal infection of the skin and the subcutaneous tissue caused by traumatic inoculation of a specific group of dematiaceous fungi. (Medscape)

Note: As with most fungi, speciation of the genus Exophiala is undergoing change brought about by molecular analysis. Some sources are now referring to the ‘Exophiala jeanselmei complex’.

Isolates from the United States that in the past had been identified as E. jeanselmei based on morphologic and physiologic characteristic have been shown by molecular methods to be mostly two newly named species closely related to E.jeanslemei, i.e. Exophiala oligosperma and Exophiala xenobiotica. A lower percentage were found to belong to several other species of Exophiala. The precise species in the complex can only be determined by molecular testing.

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Sporothrix schenckii Complex –Hyphomycetes (Dimorphic Fungus)

Note1:I first added a post entitled Sporothrix schenckii back in November of 2008 while bedbound, recovering from a serious injury. I had just discovering ‘blogging’ and toyed with the idea of posting a few film photos that I had tucked away in a drawer – just for the fun of it. The poor quality photo demanded it be upgraded once I decided to keep up my blog ‘Fun with Microbiology’. Finally, here is the upgraded blog post, perhaps more accurately entitled Sporothrix schenckii complex Revisited.

Note2: In recent years, gene sequencing studies have revealed that the species previously known as Sporothrix schenckii is actually composed of several species. The currently accepted species names of the species which comprise the complex are as follows: S.albicans (formerly Sporothrix pallida), S.brasiliensis, S.globosa, S.luriei, S.mexicana, and S.schenckii , ‘sensu strictu’. As many clinical laboratories may not have routine access to molecular technology for specific speciation, this blog simply describes Sporothrix schenckii complex with macroscopic, microscopic and physiological features found in most current textbooks.

Ecology:

Sporothrix is a cosmopolitan (found just about everywhere) fungus which is commonly isolated from soil and decomposing plant matter. Peat moss is a particularly well known source of Sporothrix. It may also be found on living plants such as rose bushes leading to what has been termed ‘rose handler’s disease’ where the fungus gains entry to the host through thorny pricks.

Pathogenicity:

Sporothrix schenckii is the agent responsible for sporotrichosis, a chronic infection that most frequently begins as a skin puncture with introduction of the fungus into the subcutaneous tissues. Eventually it will involve the lymph nodes and lymphatic channels that drain the infected area. Implantation of the fungus is usually by puncture by items contaminated with plant material harbouring the fungus such as wood splinters, sphagnum moss, hay or thorns as previously mentioned. Pulmonary (respiratory) infection may also develop in predisposed individuals after inhaling fungal spores. Rare cases of disseminated Sporothrix infection with a fatal outcome have been reported.

Laboratory acquired infections have also been reported.

Sporothrix schenckii is a thermally dimorphic fungus, meaning it can take on one of two forms depending on the temperature it finds itself in.

· At 25ᵒC exhibits the mould (filamentous) form, with a glabrous, moist texture. Initially white or cream coloured, the colony may acquire a black colour with aging

· At 37ᵒC exhibits a yeast-like form, with a creamy texture, cream to beige in colour.

Macroscopic Morphology:

The mould or filamentous phase in greater detail:

Growth is described by most sources s moderately rapid to rapid, with the colony becoming mature within 7 days.

The filamentous phase grown on SAB or PDA is generally cream coloured with some sources describing orange to orange-grey colouration. As the fungus matures, a salt & peppery brown or black colour develops with the colony retaining a narrow whitish border. Isolates may vary in their colour, some being dark/black from initial growth. Stock cultures kept for long periods may lose their dark colour completely.

The reverse of darkly pigmented colonies is usually dark in the center with a progressively lighter periphery.

Sporothrixinitially has a moist appearance but becomes wrinkled and leather to velvety in texture as it ages.

Sporothrix schenckii - SAB, 30ᵒC, 3 weeks incubation (Nikon)

The Yeast Phase:

The yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures). At 37ᵒC, Sporothrix exhibits a yeast-like form, with a creamy texture, cream to beige in colour which also may darken with aging.

(Yeast Phase Photo further below)

Microscopic Morphology:

Fillamentous Mould Phase:

Sporothrixproduces narrow (1 – 2 µm dia.) hyaline, septate and branching hyphae.

Sporothrixproduces two types of conidia:

· Slender, tapering conidiophores arise at right angles from undifferentiated hyphae. Hyaline conidia are produced at a small swelling at the conidiophores apex by sympodial growth resulting in a “rosette-like” appearance. These conidia (2 – 3 X 3 – 6 µm) are tear-drop shaped to round in appearance and unless disturbed, remain attached to the conidiophore in young cultures via thread-like denticles. (Rosette)

· Single thick-walled brown to black (dematiaceous) sessile conidia (2 – 4 µm dia.) can also be present, arising directly from the hyphae.

Sporothrix schenckii -Initial look at the growth attached to a cover slip from a slide culture.

(100X, LPCB, DMD-108)

Sporothrix schenckii - A closer look where detail begins to emerge.

(400X, LPCB, DMD-108)

Sporothrix schenckii - ditto

(400X, LPCB, DMD-108)

Sporothrix schenckii - At yet a higher magnification, fine structures emerge. Conidia are easily seen at the tips of conidiophores. At the center of this photo one such structure is seen in a 'rosette' arrangement, typical for this fungus.

(1000X, LPCB, DMD-108)

Sporothrix schenckii - hyphae bearing conidiophores with conidia being produced sympodially at the apex. (1000X, LPCB, DMD-108)

Sporothrix schenckii - appearing somewhat in 3-D, the hypha is seen running across the photo with conidiophores extending at right angles from the hypha. Conidia formation is is seen at different stages with the conidiophore at the right having accumulated numerous conidia.

(1000X, LPCB. DMD-108)

Sporothrix schenckii - again in "3-D" perspective, you can see a number of hyphae below, and out of the plane of focus of the camera, bearing conidiophores which appear to be rising upwards, towards the camera/viewer. Conidia are present at the apex.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -septate hyphae are show and two well defined conidiophores are shown with a 'rosette' of conidia at the apex.

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -another look at the conidiophores with conidia accumulated at the apex (tips). Fine thread-like denticles can be seen attaching the ellipsoid or tear drop shaped conidia to the conidiophore.

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -yet another view, as above.

(1000+10X. LPCB, DMD-108)

Sporothrix schenckii -what is meant as a "rosette" configuration of conidia. This is typical of Sporothrix schenckii and assists in its identification.

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii - I particularly like this photo. Like a shower of delicate flowers. What is seen are the conidia rosettes at the tips of unseen conidiophores and hyphae, below the plane of focus. (1000X, LPCB, DMD-108)

Sporothrix schenckii -a single hypha running through the photo, bearing conidia in typical rosette pattern. (1000X, LPCB, DMD-108)

Sporothrix schenckii -single, sessile and begining to show dark pigment (dematiaceous), are seen along the hypae. (1000X, LPCB, DMD-108)

Sporothrix schenckii -ditto. Black pigment much more pronounced in this photo.

(1000X, LPCB, DMD-108)

Sporothrix schenckii - again, as above.

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii - just another photo...

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -Sessile (attached directly to hypha) with many developing a dark pigmentation (dematiaceous) which gives the macroscopic colony its distinctive colour.

(1000X, LPCB, DMD-108)

Sporothrix schenckii - dematiaceous cells are more abundantly produced as the colony ages.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -sessile dematiaceous conidia are seen lining a hypha running through the photo, as well as some conidiophores bearing the tear-drop shaped conidia.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -another view of the sessile, dematiaceous conidia lining the hypha with a few conidiophores extending from the hypha showing the delicate rosette arrangement at the tips. (1000X, LPCB, DMD-108)

Sporothrix schenckii -and just another photo showing what was described in the last few photos.

(1000X, LPCB, DMD-108)

Sporothrix schenckii -a nice rosette formation in the center of the photo.

(1000+10X, LPCB, DMD-108)

The Yeast Phase:

As previously mentioned, the yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures). Yeast cells are round to ovoid of varying size (1 – 3 X 3 – 10 µm), producing single or multiple buds often resembling rabbit ears or ‘Mickey Mouse’ ears extending from the primary yeast cell.

Sporothrix schenckii -the very same organism added to the nutritionally rich Brain-Heart Infusion (BHI) agar and incubated at 37ᵒC for ten days. A typical pasty, yeast form develops which is why this organism is 'dimorphic'. (Nikon)

Sporothrix schenckii -yeast cells seen in a suspension in Lactophenol Cotton Blue.

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii - Sporothrix in the yeast phase may show single yeas like cells with some of the population budding with typical "rabbit ears" or "Mickey Mouse" ears. They appear as two elongated cells projecting from the same surface of the parent cell. One can be seen here, one-third in from the center left edge of the photo. (1000X, LPCB, DMD-108)

Sporothrix schenckii -"Mickey Mouse' ear configuration in the cell at the upper center.

(1000X, LPCB, Nikon)

Sporothrix schenckii -again, yeast cells with two attached daughter cells on the same side giving the apearance of "rabbit ears" or "Mickey Mouse" ears.

(1000+10X, LPCB, DMD-108)

Sporothrix schenckii -ditto (as above) -inset -floppy 'rabbit ears"

(1000X, LPCB, DMD-108)

Sporothrix schenckii - that's it, I'm done!

(1000+10X, LPCB, DMD-108)

Physiology:

Sporothrix is resistant to cycloheximide; however growth is inhibited at temperatures of 39 - 40ᵒC.

Return to the top (Most Recent Posts)

* * *

Emmonsia pasteuriana

Note: I've held back on uploading this particular post only because I'm not satisfied with the quality of the photographs I've taken. It is unlikely that I will come across another strain of this fungus in my career so I will post it at this time hoping any readers will understand.

Emmonsia species: Emmonsia currently consists of three species: E.parva, E.crescens &E.pasteuriana.

Ecology: Emmonsia is a cosmopolitan soil saprobe (found just about in all temperate climates and lives of decaying organic matter). It has been isolated from a variety of mammalian species, particularly small rodents.

Pathogenicity: Emmonsia parva and Emmonsia crescens are the etiologic agents of adiaspiromycosis. Usually this presents as an asymptomatic pulmonary infection in animals, and more recently in immunocompromised humans after the inhalation of the fungal spores. Lung biopsy may be necessary to diagnose the illness as the organisms may not be present in sputa or bronchial alveolar lavages. Dissemination of the infection may occur more readily in immunocompromised hosts. E.crescens is more commonly isolated from humans while E.parva is isolated more often from animals.

Reports of E.pasteurianahave increased in recent years, presenting as disseminated cutaneous (skin) mycosis in persons with underlying AIDS infections[i]. Unlike E.parvaand E.crescens, E.pasteuriana does not produce adiaconidia and therefore does not cause adiaspiromycosis. E. pasteurianaappears as yeast in infected tissue.

Emmonsia pasteuriana:

The following description is for an isolate resembling Emmonsia pasteuriana.

Macroscopic Morphology: E.pasteuriana exhibits slow to moderate growth at 30ᵒC, with a colony diameter of about 25mm after 10 days and 60mm after 21 days. The colony appeared velvety to powdery in texture with folded, wrinkled, or cerebriform surface contours. The colony spontaneously acquired splits in the surface as it aged (see photo) The colony was primarily white in colour and remained so while some sources state that it may develop a light brown colour as it ages. The colony produced no diffusible pigment. The reverse was tan or light brown in colour.

Emmonsia pasteuriana - Saboraud-Dextrose Agar. The splits in the colony were created by the growth and not by any prodding of my own. (SAB), 30ᵒC, 1 Month. (Nikon)

Emmonsia pasteuriana - Dermasel® agar, 30ᵒC, ~18 Days. (Nikon)

Microscopic Morphology: Emmonsia pasteuriana is a dimorphic fungus meaning it can exhibit the filamentous fungus form at one temperature and the yeast form at another.

Filamentous form:This isolate produce septate, hyaline (non-pigmented) hyphae of about 1.0 to 1.3 µm diameter. Thin-walled, slightly verruculose (minutely verrucose or warty), globose to sub-globose (round-ish) conidia (2 – 3 µm X 3 – 4 µm) are formed on slender (0.4 – 0.5 µm) pedicles (stalks) or at the apex of inflated cells. Initially a conidium may be found at the end of a delicate pedicle which may then develop further to form four to eight pedicles with conidia, establishing a ‘floret’. Additional sessile or broad-based verrucose (warty) conidia may also be present.

Emmonsia pasteuriana - hyphae with conidia visible throughout.

(400X, LPCB, DMD-108)

Emmonsia pasteuriana - Globose to sub-globose (round-ish) conidia. They are generally found at the end of a delicate pedicile (stem) which can barely be seen at this magnification. A few more photos to follow of much the same as this is this really was my first impression of this fungus and what clued me into what it might be.

(400X, LPCB, Nikon)

Emmonsia pasteuriana - ditto. Numerous conidia formed.

(400X, LPCB, Nikon)

Emmonsia pasteuriana - another as previously, but here in this photo there may be evidence of the conidia occuring in small bunches, each on its own pedicile branching off from a central delicate pedicile. (400X, LPCB, Nikon)

Emmonsia pasteuriana -small conidia occuring in bunches (as previously)

(400X, LPCB, Nikon)

Emmonsia pasteuriana - and another.

(400X, LPCB, Nikon)

Emmonsia pasteuriana -at a higher magnification, the rather round (globose) or 'round-ish' (sub-globose) conidia can be seen with a couple at the end of a pedicile (stem or stalk) and one (center-left) which may be growing directly from the hypha (sessile).

(1000X, LPCB, DMD-108)

Emmonsia pasteuriana -small clusters of conidia.

(1000X, LPCB, DMD-108)

Emmonsia pasteuriana - as above. It was my impression of the tiny, round conidia attached by a delicate pedicile which directed me to the identity.

(1000X, LPCB, DMD-108)

Emmonsia pasteuriana - a solitary conidium at the end of a pedicile is seen in this photo (inset)

(1000X, LPCB, DMD-108)

Emmonsia pasteuriana - Initially a conidium may be found at the end of a delicate pedicle (see above) which may then develop further to form four to eight pedicles with conidia, establishing a ‘floret’. Here we see more distinctly what is described as a 'floret' (inset).

(400+10X, LPCB, DMD-108)

Emmonsia pasteuriana -a very good example of the 'floret', usually composed of between 4 to 8 conidia at the end of the slender delicate and slender (0.4 – 0.5 µm) pedicles.

(1000X, LPCB, Nikon)

Emmonsia pasteuriana -another example of a 'floret' as above.

(1000X, LPCB, DMD-108)

Emmonsia pasteuriana -a string of florets along a delicate hyaline (non-pigmented) hypha of about of about 1.0 to 1.3 µm diameter running through the photo from lower left to upper right.

(1000X, LPCB, Nikon)

Emmonsia pasteuriana -a string of florets along delicate hyaline (non-pigmented) hyphae.

(400X, LPCB, Nikon)

Emmonsia pasteuriana - Additional sessile (directly from hypha) or broad-based verrucose (warty) conidia may also be produced.

(1000X. LPCB, Nikon)

Emmonsia pasteuriana - once again, single conida on slender stalks.

(400X, LPCB, DMD-108)

Emmonsia pasteuriana - two conidia can be seen at center left, attached to the delicate pedicile.

(400+10X, LPCB, DMD-108)

Emmonsia pasteuriana -sessile (directly from hypha) or broad-based verrucose (warty) conidia may be produced. (1000X, LPCB, DMD-108)

Emmonsia pasteuriana - Once again, thin-walled, slightly verruculose (minutely verrucose or warty), globose to sub-globose (round-ish) conidia (2 – 3 µm X 3 – 4 µm) are formed on slender (0.4 – 0.5 µm) pedicles (stalks) or at the apex of inflated cells. Here in this photo is a conidium at the apex of an inflated cell. (1000+10X, LPCB, DMD-108)

Emmonsia pasteuriana - more conidia at the born on delicate pediciles.

(1000X, LPCB, DMD-108)

Yeast form: The filamentous fungus form can be converted to the yeast form by incubating a freshly inoculated culture at 37ᵒC for 10 to 14 days. Conversion is enhanced by cultivation on the nutritionally richer Brain-Heat Infusion (BHI) agar. Smooth, butyrous colonies appear cream to beige in colour which may darken as they age. Colonies consist of globose to oval yeast colonies which may show narrow-based budding.

Emmonsia pasteuriana - isolate was inoculated onto Brain-Heart Infusion (BHI) agar and incubated at 37ᵒC for 14 days. Emmonsia pasteuriana converted to the yeast form which is the form that is directly recovered from skin lesions.

(400X, LPCB, DMD-108)

Emmonsia pasteuriana - a more convincing example of the yeast-like phase of E.pasteuriana.

(400+10X, LPCB, DMD-108)

Note: At various stages of development, Emmonsia species may resemble other fungi such as Blastomyces dermatitidis, Paracoccidioides brasiliensis, Histoplasma capsulatum and Chrysosporium species. It has been noted that some cross-reaction (false positives) may occur between Emmonsiaspecies and Blastomyces dermatitidiswith a both a DNA probe and direct immunofluorescent-antigen tests.

Adioconidia:When the hyphae and conidia are incubated at their maximum temperatures on enriched media, the hyphae become distorted and usually disintegrate while the conidia swell to become round, thick-walled adiaconidia (formerly called adiaospores). Production of adiaconidia is best achieved by incubation at increased temperatures: 37ᵒC for E.crescens (20 – 14 µm dia) and at 40ᵒC for E.parva (10 -25 µm dia). As previously mentioned, E.pasteurianacannotbe induced to produce adiaconidia at any temperature.

Physiology: Emmonsia pasteuriana is not inhibited by cycloheximide and therefore can be grown on Mycosel® or Dermasel® agar.

[i] A Dimorphic Fungus Causing Disseminated Infection in South Africa

Chris Kenyon M.D. et al.,

N.Engl. J. Med. 2013, 369 – 1416 - 1424

Epidermophyton floccosum(mould/dermatophyte)

Note: While I've had photos of Epidermophyton floccosum for some time now, I've never been satisfied with the quality of the microphotographs I've taken. I remain unsatisfied here. The photos for this and every other post contained in this blog were taken by myself on my own time, before or after regular work hours. Unfortunately I find myself so busy at times that my own projects take a distant back seat. Primary cultures sometimes may become contaminated or overgrown. Isolates may revert to a sterile form on repeated subculture, as in this case, before sufficient study. While I've obtained several isolates over the years, E.floccosum always seems to defeat my best efforts to obtain those "text book" quality photos. Time is running out...

Ecology:

Epidermophyton floccosum is a cosmopolitan (worldwide distribution)anthropophilic (man is the primary host & reservoir) dermatophyte [i]. A once though related species, Epidermophyton stockdaleae has been determined to be a synonym for Trhycophyton ajelloi which exhibits no known pathogenicity to humans or animals

Pathogenicity:

E.floccosumcauses tinea pedis (athlete’s foot), tinea cruris (groin infections or “jock-itch), and tinea corpis (body infections), and to a lesser extent onychomycosis (nail infections). Skin infections are also known as “ring-worm” though there is no ‘worm’ involved. Infection with E.floccosum may be transmitted in gym facilities where unprotected feet may share a common floor. E.floccosum rarely infects the scalp and does not infect hair or hair follicles.

Macroscopic Morphology:

E.floccosum exhibits moderate growth, becoming mature in about 10 – 14 days. Surface colonies (media influenced) have been described as mustard yellow or yellowish brown to olive-grey (khaki) in colour. Colonies can be powdery, velvety or felty in texture and acquire a folded appearance as growth progresses. After prolonged incubation, sterile floccose (hairy) white mycelia may cover the colony. The reverse has been described as ochre, mustard-yellow to yellow-brown and even orange in colour.

E.floccosum -colony heaped up at center on SAB after 2 weeks at 30ᵒC (Nikon)

E.floccosum -colony on SAB (Saboraud Dextrose Agar) after 2 weeks at 30ᵒC (Nikon)

E.floccosum -colony on SAB after 3 weeks at 30ᵒC (Nikon)

E.floccosum -another colony on SAB after 3 weeks at 30ᵒC (Nikon)

E.floccosum -yet another colony on SAB after 5 weeks at 30ᵒC.

Note: white floccose patches beginning to develop. (Nikon)

E.floccosum -colony on SAB, 30ᵒC after repeated subcultures has developed white floccose patches which are areas of sterile hyphae. (Nikon)

Microscopic Morphology:

E.floccosum has septate hyphae however microconidia are not produced which differentiates it from the other genera of dermatophytes. Macroconidia develop as lateral or terminal outgrowths from mature hyphae and initially lacks a basal septum. Rather thin walled macroconidia (10 -40 µm X 6 – 12 µm) contain 2 to 5 cells can occur singly or in characteristic clusters. As the culture ages, macroconidia may transform into chlamydoconidia (chlamydospores) so they are best observed earlier in growth. The macroconidia are smooth walled, and clavate (club shaped) with a blunt tip. This also differentiates it from Microsporumand Trichophyton. (Again, see endnote 1).

Note:

Stock cultures are best maintained on SAB media with 3 – 5% sodium chloride. This may reduce or prevent the isolate from becoming sterile.

E.floccosum - a first look at low power.

(100X, LPCB, DMD-108)

E.floccosum - Slightly higher magnification reveals the macroconidia more clearly.

(250X, LPCB, DMD-108)

E.floccosum - as above, numerous club shaped macroconidia are clearly seen.

(250X, LPCB, DMD-108)

E.floccosum - club shaped macroconidia with internal septations.

(1000X, LPCB, DMD-108)

E.floccosum - club shaped macroconidia with internal septations.

(1000X, LPCB, DMD-108)

E.floccosum - club shaped macroconidia with internal and basal septations.

(1000+10X, LPCB, DMD-108)

E.floccosum - again, club shaped macroconidia with internal septations. My isolates tended to produce single macroconidia over the grouped macroconidia where several macroconidia crowd each other growing out from the same area of the hypha.

(1000+10X, LPCB, DMD-108)

E.floccosum - a single macroconidium. Note that in this and other microphotographs in of E.floccosum, there are no microconidia.The lack of microconidia is one feature which distinguishes E.floccosum from other dermatophytes.

(1000+10X, LPCB, DMD-108)

E.floccosum - several macroconidia.

(1000X, LPCB, DMD-108)

E.floccosum - a single mature macroconidium.

(1000+10X, LPCB, DMD-108)

E.floccosum - a single mature macroconidium with a curious little kink in it's side.

(1000+10X, LPCB, DMD-108)

E.floccosum - macroconidium measures 35.18µm in length.

This was obviously an adhesive tape preparation which may trap air bubbles or even reveal uneven adhesive application which may detract from the photograph.

(1000+10X, LPCB, DMD-108)

E.floccosum - club shaped macroconidia. I have this photo recorded as taken at 400X which is confirmed by the micron bar in the upper right. However, the macroconidia seem extremely large for this magnification if compared to previous photos at 1000X. The same goes for the photo which follows. Curious...

(400X, LPCB, DMD-108)

E.floccosum - numerous club shaped macroconidia as above.

(400X, LPCB, DMD-108)

E.floccosum - this photo was taken from a culture that was just over three weeks old. Numerous roundish chlamydospores have developed. Again, compare the micron bar in the upper right to the previous photo which shows identical magnification yet the macroconidia vary greatly is size.

(400X, LPCB, DMD-108)

E.floccosum - macroconidia and chlamydospores present in this adhesive tape preparation

(1000X, LPCB, DMD-108)

E.floccosum - macroconidia on prolonged culture. Some sources say that arthroconidia may also develop, however, I have never observed them in my older E.floccosum cultures.

(500X, LPCB, Nikon)

E.floccosum - finger-like group of macroconidia.

(1000X, LPCB, Nikon)

[i]Dermatophyte– fungi which thrive on keratin for growth therefore they primarily infect skin, hair and nails depending on the genera and species. Epidermophyton, Microsporum and Trichophytonare dermatophytes. Epidermophyton had macroconidia that are clavate (club shaped) while Microsporum produces fusiform (spindle shaped) macroconidia and Trichophytonpossesses cylindrical or ‘cigar-shaped’ macroconidia. E.floccosumdoes not produce microconida which also serves to differentiate it from the other dermatophytes.

* * *

Chrysosporium species (Mould)

Note: Chrysosporiumis another species I’ve been holding onto in hopes of obtaining additional specimens (strains) and taking better photographs. I’m really not satisfied with what I’ve taken to date but here they are for what they’re worth.

Ecology:

Chrysosporiumis a ubiquitous, cosmopolitan fungus. It is a rather common saprobe (living on dead organic matter).

Pathology:

A number of species are keratinophiles and although they may be isolated from skin and nails, they are generally considered to be contaminants.

Rare reports of systemic infections in immunocompromised hosts have been published; however, their importance in the disease process remains uncertain. Skin infections of snakes, iguanas, crocodiles and dogs have been more commonly reported.

Colony Morphology:

Growth is described as slow to moderately rapid, reaching maturity within about one week.

Colony morphology may be quite variable between isolated species. Texture is powdery to cottony to woolly. The colony may remain compact or be spreading and may show further variation by being flat or raised. Pigmentation is usually white but may vary from pale yellow, pink, pale brown or weakly orange. The reverse is most often white but may be yellow, tan or brownish.

Chrysosporium species - on Saboraud Dextrose Agar (SAB), 14 days at 30ᵒC. (Nikon)

Chrysosporium species - after 21 days on SAB, at 30ᵒC. (Nikon)

Microscopic Morphology:

Chrysosporiumproduces septate, hyaline hyphae. Conidia (aleurioconidia) often appear to be minimally differentiated from the hyphae and may appear to form directly on the hyphae (sessile). Conidia more often form at the ends of simple or branched conidiophores of varying length. Conidiophores may be ramified, forming tree-like structures. Conidia are usually one-celled (2 – 9 X 3 – 13 µm). They appear as clavate (club shaped) with the apex (top) rounded while the base being broad and flat. Remnants of the attaching structure may, for a time, remain attached. Conidial walls are thin-walled and the exterior is usually smooth. Intercalary conidia are sometimes formed and may appear as a cylindrical or barrel shaped structure or may be seen as a bulge on only one side of the hyphae.

Chrysosporiumis the asexual form of Nannizziopsis vriesii and therefore ascocarps (large, sexual fruiting bodies) may occasionally be seen in fresh cultures.

Chrysosporium species - initial look at a slide culture at low power. Can't see much detail but I just liked the look of this photo.

(250X, LPCB*, DMD-108)

* Lactophenol Cotton Blue Stain

Chrysosporium species - another slide culture. Picture a coverslip on a block of agar about a square centimeter in size. All along the four edges of the coverslip, the fungus is growing. Gently removing the coverslip from the agar block has some of the fungus adhering to the glass coverslip. Placing the coverslip onto a microscope slide that has a drop of Lactophenol Cotton Blue Stain on it. The right side of the above photo shows the edge that was against the agar block from which the fungus grew.

This slide shows the extensive branching mycelia with free and bound conida throughout.

(250X, LPCB, DMD-108)

Chrysosporium species - hyphae and conidia still attached to the conidiophores and hyphae.

(250X, LPCB, DMD-108)

Chrysosporium species - Conidiophores may be 'ramified', forming tree-like structures.

(400X, LPCB, DMD-108)

Chrysosporium species - conidia frequently stain more intensely than do the conidiophores which bear them or the hypha themselves.

(100X, LPCB, DMD-108)

Chrysosporium species - conidia may form short chains or develop as intercalary conidia (within the hyphae). Here (arrow) appears to show such a short chain or possibly intercalary conidia.

(400X, LPCB, DMD-108)

Chrysosporium species - another example of what appears to be a chain of conidia or alternatively might be described as intercalary (within the hypha) conidia.

(400+10X, LPCB, DMD-108)

Chrysosporium species - intercalary conidia are sometimes formed and may appear as a cylindrical or barrel shaped structure or may be seen as a bulge on only one side of the hyphae (arrows).

(1000X, LPCB, DMD-108)

Chrysosporium species -typical appearance.

(400X, LPCB, DMD-108)

Chrysosporium species - as above.

(400X, LPCB, DMD-108)

Chrysosporium species - another example.

(400X, LPCB, DMD-108)

Chrysosporium species

(400X, LPCB, DMD-108)

Chrysosporium species - sometimes it is difficult to tell whether there is a chain of conidia, a true intercalary conidium or whether there are just overlapping conidiophores which give the appearance of chaining. (400+10X, LPCB, DMD-108)

Chrysosporium species

(400+10X, LPCB, DMD-108)

Chrysosporium species - conidia are rather thin walled (see center right of photo)

(1000X, LPCB, DMD-108)

Chrysosporium species - oversaturation of the blue could not be effectively corrected with photo editing programs - then again, I just like this shot!

(1000X, LPCB, DMD-108)

Chrysosporium species - conidia are usually one-celled and about (2 – 9 X 3 – 13 µm).

(1000X, LPCB, DMD-108)

Chrysosporium species - conidia are clavate (club shaped) and have a round apex (far end) and a broad, flat base where attached to the conidiophore. Sources state that remnants of the attachment point (conidiophore) may remain attached to free conidia, however, I have not observed this in the photos I've taken. The separation of the conidia from the conidiophore appears to be quite clean.

Here also you can see that the conidiophores are minimally differentiated from the hypha itself. There is no uniquely recognizable,or elaborate structure to the conidiophores Conidiophores appear to have variable lengths. Condia appear to arise directly from the hyphae (sessile) and also may be on longer, simple or branched conidiophores.

(1000X, LPCB, DMD-108)

Chrysosporium species - rather delicate looking conidiophores - like fine pedicile-like strands attaching the conidia to the hyphae. (1000X, LPCB, DMD-108)

Chrysosporium species - again, fine structured conidiophores attached to the tear-drop or clavate (club shaped) conidia. (400X, LPCB, DMD-108)

Chrysosporium species - conidia on conidiophores arising from all sides of the hyphal element.

(1000X, LPCB, Nikon)

Chrysosporium species - Last photo. Teardrop or clavate shaped conidia attached to septate hyphae via delicate, minimally differentiated conidiophores.

(1000X, LPCB, Nikon)

Physiology:

Chrysosporiumis resistant to cycloheximide and therefore may be isolated on selective media used for primary isolation of dermatophytes.

Chrysosporium is urea positive.

Notes: Chrysosporiumspecies may develop branches diverging at 45ᵒ angles whereas the branches formed by dermatophytes are borne at angles closer to 90ᵒ.

Chrysosporiummay produce conidia as short, terminal chains which are not seen in dermatophytes.

Young cultures of Chrysosporiummay be confused with Blastomyces dermatitidis however they are not thermally dimorphic as is Blastomyces.

Chrysosporiummay also be confused with Emmonsia parva, however Chrysosporium does not produce adiaconidia at 37ᵒC and some species fail to grow at 37ᵒC.

Chrysosporiumcan be distinguished from Sporotrichumspecies as the latter spreads rapidly to cover the entire media surface, fails to grow on cycloheximide, and usually forms abundant arthroconidia which may break from the hyphae to form clusters.

One source states that species which produce short terminal chains of conidia, grouped in small tree-like clusters, are now considered to belong to the genus Geomycesrather than Chrysosporium.

* * *

Fonsecaea pedrosoi/monophora

Note 1: Recent changes in genus Fonsecaea: Previously the genus Fonsecaea was considered to be comprised of F.pedrosoi and F. compacta. Recent revisions indicate that F.compacta is simply a morphological variant of F.pedrosoi and therefore considered the same organism. DNA analysis, however, has added a second species to the genus known as F.monophora. There are subtle morphological differences between the two; however, they are best differentiated by molecular means. The fungal disease was first described by Alexandrino Pedroso in 1911, hence the name.

Ecology:

While Fonsecaea can be found worldwide it is more commonly found in tropical and sub-tropical regions where it is found as a saprobe (lives on dead organic matter) in soils and rotting plant materials. Agricultural workers in Central and South America, India, Africa and Madagascar are more commonly exposed to these soils and exposure to Fonsecaea. It is usually acquired through traumatic implantation via a splinter or thorn. Cold blooded animals living near or around swamps may also be infected and carry the fungus.

Pathology:

Fonsecaea is the most common cause of chromoblastomycosis, a chronic subcutaneous infection which is characterized by verrucous lesions and the formation of brown sclerotic fission cells, described as “copper pennies”[i]within the tissue. Other dematiaceous fungi responsible for chromoblastomycosis are Phialophora verrucosa and Cladophialophora carrionii. Both F.pedrosoi and F.monophoraare recognized agents of human chromoblastomycosis; however, in F.pedrosoi a strict association with this disease is noted, while F.monophorais a more general opportunist. While prognosis is generally good, the infection itself is difficult to treat and long-term therapy is required. The presentation of the disease can initially be confused with squamous cell carcinoma. Systemic (internal) infections have rarely been described however F.pedrosoihas been implicated in a fatal brain infection acquired via haematogenous dissemination. Keratitis (corneal infection) and a case of paranasal sinusitis have also been reported.

Macroscopic Morphology:

Growth rate for F.pedrosoi/monphora is slow with the colony maturing in about 14 days on Sabouraud Dextrose medium (SAB) at 30ᵒC.

The colony surface may be dark green to olive brown to dark grey or jet-black depending on the strain and medium. It is covered with a fine, velvety or downy mycelium. Colonies start of relatively flat however they usually produce a raised convex protrusion at the center where initially inoculated. The colony becomes somewhat embedded in the agar surface and may break apart when probed. The colony reverse is black.

Note 2: I will refer to the organism throughout the remainder of this post as Fonsecaea pedrosoi for my own ease, however, the reader should keep in mind that the organism could be Fonsecaea pedrosoi or Fonsecaea monophora as discussed in Note 1.

Fonsecaea pedrosoi - Sabouraud Dextrose Agar (SAB), 10 Days, 30ᵒC (Nikon)

Fonsecaea pedrosoi - Sabouraud Dextrose Agar (SAB), ~3 Weeks, 30ᵒC (Nikon)

Microscopic Morphology:

Note 3: One source (Larone-See Sidebar) suggests that conidiation may be enhanced by growing the organism on Corn Meal Agar (CMA) or Potato Dextrose Agar (PDA). In fact the isolate presented here did not show any conidiation until grown on CMA. All microphotographs presented here are from growth on CMA.

Be aware that the scale of the micron bar within photos may vary.

Fonsecaea pedrosoi - grown on SAB media, this isolate failed to produce any 'fruiting structures'. It was only after growing the fungus on Corn Meal Agar (CMA) that conidiophores and conidia were observed. (400X, LPCB, DMD-108)

All further photos are taken from growth on CMA.

Fonsecaea produces dematiaceous[ii](dark/brown) septate and loosely branching hyphae. The conidia produced are pale brown or olivaceous in colour. They are sub-hyaline, smooth textured, thin walled and ovoid or clavate (club-like) in shape. The conidia (3.5 – 5.0 X 1.5 – 2.0 µm) are produced in short chains at the apex of the conidiophores.

Fonsecaea pedrosoi - view of mature fungus at lower magnification.

(400X, LPCB, DMD-108)

Fonsecaea pedrosoi -initial 'budding' growth of conidiophores/conidia appear along the hypha.

(1000X, LPCB, DMD-108)

Four types of conidial formation may be observed the same strain of Fonsecaea.

Fonsecaea type: Conidiophores are septate, erect, and compactly sympodial. The distal (far) end of the conidiophore develops swollen denticles that bear primary single-celled ovoid conidia. Denticles on the primary conidia support secondary single-celled conidia that may produce tertiary conidia, but long chains of conidia are not formed. Elongate conidia often form in verticils at fertile sites along the conidiophore, producing an asterisk-like (*) appearance.

Rhinocladiella type: Conidiophores are septate, erect, and sympodial; swollen denticles bear ovoid conidia at the tip and along the side of the conidiophore. Usually only primary conidia develop.

Cladosporium type: Conidiophores are erect and give rise to large primary shield-shaped conidia that in turn produce short, branching chains of oval conidia having small dark hila (scars of attachment)

Phialophora type: Phialides are vase shaped with terminal cup-like collarettes. Round to oval conidia accumulate at the apex of the phialide. This type of conidiation is often scant or lacking.

Fonsecaea pedrosoi - as the colony matured, the hyphae darkened with the production of melanin pigment. Initially only primary, or a single tier of conidia were seen to be produced which suggested that this organism might be Rhinocladiella species. Therefore, this is what I believe sources refer to as Rhinoladiella conidiation as described in the previous text.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi -again, only primary conidia (each conidia attached to the conidiophore) is seen suggesting the Rhinocladiella type conidiation.

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi -an interesting feature observed is the sympodial growth of the hyphae/conidiophore (arrow).

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi -Conidia have dispersed revealing the sympoidal growth pattern (arrows)

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi -a final photo showing this feature.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi -secondary conidia appear which are attached to the primary by a delicate denticle. Rhinocladiella is not known for producing other than primary conidia therefore the evidence began to suggest that this was, indeed, Fonsecaea.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - still mostly primary conidiation however a typical example of Fonsecaea.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - typical growth. Branching conidiophores present.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - from edge of slide culture on CMA.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - compactly sympodial conidiation of Fonsecaea.

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - another photo (sometimes I post a photo just 'cause it looks cool!)

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - more typical of mature Fonsecaea as you can see that there are several tiers to the conidiphore & conidia fruiting structure.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - conidiophores bearing conidia along the septate hyphae.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - typical complex fruiting structure -center left of photo.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - another typical example.

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - more examples. Conidiophores of varying lengths along the hyphae. Primary and more mature central hypha has developed dark pigmentation.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - a typical example of the conidiphore with compactly sympodial growth of conidia. (1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - Fonsecaea type conidiation: Conidiophores are septate, erect, and compactly sympodial. The distal (far) end of the conidiophore develops swollen denticles that bear primary single-celled ovoid conidia. Denticles on the primary conidia support secondary single-celled conidia that may produce tertiary conidia, but long chains of conidia are not formed.

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - Elongate conidia often form in verticils at fertile sites along the conidiophore.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - Elongate conidia often form in verticils at fertile sites along the conidiophore.

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - Elongate conidia often form in verticils at fertile sites along the conidiophore, producing an asterisk-like (*) appearance. (Below)

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - Elongate conidia often form in verticils at fertile sites along the conidiophore, producing an asterisk-like (*) appearance.

(500X, LPCB, Nikon)

Fonsecaea pedrosoi - Cladosporium type conidiation: conidiophores are erect and give rise to large primary shield-shaped conidia (inset) that in turn produce short, branching chains of oval conidia having small dark hila (scars of attachment). Conidia were easily disrupted and I have no photos for chaining of the conidia) (1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - remnants of conidial attachment on a septate conidiophore.

(1000X, LPCB, DMD-108)

Fonsecaea pedrosoi - Septate conidiophore with conidia shown. The conidia produced are pale brown or olivaceous in colour. They are sub-hyaline, smooth textured, thin walled and ovoid or clavate (club-like) in shape. The conidia (3.5 – 5.0 X 1.5 – 2.0 µm) are produced in short chains at the apex of the conidiophores.

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi - Phialophora type conidiation (?): Phialides are vase shaped with terminal cup-like collarettes (inset - arrows). Round to oval conidia accumulate at the apex of the phialide. This type of conidiation is often scant or lacking. The 'staggered' sympodial growth pattern appears evident at base of inset photo)

(1000+10X, LPCB, DMD-108)

Fonsecaea pedrosoi

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[i] In tissues, this fungus, as well as other etiologic agents of chromoblastomycosis appears as large (5 – 12 µm diameter), round, brownish and thick-walled bodies, hence the resemblance to the coin and common description of “copper pennies”. When the fungus is cultured on laboratory media at 25, 30, or 37ᵒC, the fungus is filamentous.

[ii] Dematiaceous fungi represent a large and heterogeneous group of filamentous moulds containing melanin in their cell walls. The term phaeohyphomycosis was proposed by Ajello and Georg in 1974 as “a collective name for a group of mycosis caused by diverse genera and species of dematiaceous fungi”

* * *

So the laboratory proficiency mycology specimens arrive in the lab and I eagerly have a first look at the direct, teased out, samples. One specimen is labeled as having been taken from the scalp so I suspect the sample may possibly contain a dermatophyte. Carefully scanning at low power, I search for any tell-tale clues which may provide a ‘head-start’ (no pun intended). Then, I see something unexpected. Hey! What’s this? What are you doing here? Where did you come from? That specimen from the scalp has brought along a hitch-hiker. So who are you anyways? Follicle Fred? Afro Al? Blow-Dried Billy-Bob? Hmmm, Hairy Harrison? (Hairy Harry to be less formal).....but wait….what do we have here? Eggs! That’s not Harry the hitch-hiker….it’s Harrietthe hitch-hiker!

I suppose it was an unintended addition, but our mould sample from the scalp, also contained a tick(?) of some sort. The specimen donor has more problems than just a possible dermatophyte!

‘Googling’ ticks brought up the ‘mug shots’ of a number of possible suspects, however, I’ll leave it up to some entomologist out there to positively identify my freeloader and replace the alias with a proper name.

Below are Hitch-hiking Harriet’s mug shots to add to the unsavory lot of ticks, lice and louses imprisoned in cyberspace. Sorry, she wouldn’t turn for me to get a profile photo. No smile either! One tough character!

Lactophenol Cotton Blue is the background stain as I was looking for moulds in this preparation.

Harriet, my unknown scalp tick which hitch-hiked along on a scalp mycology specimen.

(Didn't keep detailed notes on this one. X100, I believe -Nikon)

Harriet again. Female for sure -check out the 5 oval eggs in her abdomen

(?250X, Nikon)

Same 'Harriet' but image reversed due to the difference in the optics between the Nikon and the DMD-108 Digital Micro-imaging Device.

(100X, DMD-108)

And the last mug-shot at a higher magnification.

(?100+10X, DMD-108)

Links below redirect to the identical post within this blog.

While I like using both photographic devices (Nikon Coolpix microscope mounted camera) and the Leica DMD-108 Digital Micro-imaging Device, I feel that each platform has an advantage in different situations. I like to try both methods and then chose which gave the best representation of the subject I was attempting to document. Often it is in the eye of the viewer.

What? you mean you don't give a familiar name to your organisms?

Fun With Microbiology - as the title of the blog proclaims.

* * *

Saksenaea vasiformis complex- Zygomycete

Note: I had been debating with myself whether to post this organism at all as I have so few photos to share. The photos I am posting show only the most basic characteristic features that would clue you in that this is a Zygomycete. Still, it may be of interest to some….and, what else am I going to do with the photos?

Until recently, Saksenaea vasiformis was recognized as the sole species within this genus. Currently, three species are recognized[i]and can be differentiated by genetic, morphological and physiological characteristics. Now recognized are; S. vasiformis which produces mainly cylindrical sporagniospores with rounded ends, S. erythrospora which has large sporangiophores and sporangia, which produce biconcave ellipsoidal sporangiospores, and S. oblongiaspora, characterized by oblong sporangiospores and its failure to grow at 42ᵒC.

Ecology: Saksenaeaspecies can be found worldwide, having been isolated from soils in India, Brazil, Panama, Honduras, as well as the United States.

Pathology: Though found worldwide, Infections occur most commonly in tropical and sub-tropical climates. Saksenaeaspecies have been implicated in both human and animal disease. Unlike many of the emerging fungal diseases, it appears that Saksenaea infects immunocompetent hosts more readily than those with underlying, and usually predisposing conditions. The most common mode of infection is through some traumatic implantation of material containing the sporangiospores though the respiratory tract may be the route of infection with disseminated infections. Clinical symptoms and presentation can be quite varied –from a slow, localized invasion to rapidly spreading disseminated infection. The spectrum of infections described in the literature range from skin and soft tissue infection to bone (osteomyelitis) and rhino-orbito-cerebral involvement. Skin and soft tissue infections may present with necrotizing fasciitis or cellulitis. While infections remain localized, they may respond to a combination of necrotic tissue debridement and aggressive antifungal therapy, otherwise amputation may be the only recourse. Overall the mortality rate as a result of infection is about 40%[ii]. Disseminated infections have a significantly higher associated mortality rate of about 75%, while rhino-orbito-cerebral infections have an estimated mortality rate of 83% based on published reports. Thankfully, infections with Saksenaea species remain relatively uncommon.

Saksenaea species characteristics (in general) follow

Macroscopic Morphology:

A rapidly expanding fluffy, spider-like colony on Sabouraud Dextrose Agar incubated at 30ᵒC. The growth may fill the Petrie dish within 48 hours. It may be a “lid-lifter” and growth attempt to escape through poorly sealed plates after 72 plus hours. The colony appears off-white to greyish in colour.

Saksenaea species - 48 hours growth on SAB (or SDA) at 30ᵒC (Nikon)

Saksenaea species -as above, but an oblique view (Nikon)

Saksenaea species - 4 days growth on SAB (or SDA) at 30ᵒC (Nikon)

Microscopic Morphology:

Saksenaea produces broad hyaline, mostly aseptate, hyphae. Simple and unbranched sporangiophores (24 – 64 µm in length), develop a flask-shaped sporangium (50 – 150 µm in length). The base of the sporangium above the hemispherical collumella is rather broad or swollen in appearance but narrows down into a long neck towards the apex. Smooth walled sporangiospores (1.5 – 2.0 X 3 – 4 µm) vary in shape depending on the species as described above in ‘Notes’. When mature, the sporangiospores are released through the top of the sporangiophore. Dichotomously branching rhizoids develop at the base of the sporangiophore. If this Zygomycete has produced its typical fruiting structure (sporangium), the overall structure of Saksenaea is unique and easy to identify. But there lies the problem…

Problems:

It is rather difficult to induce Saksenaea to induce produce fruiting structures and subsequent sporulation. Let’s back up a bit…

If you get a rapidly growing, fluffy colony which produces broad (wide) hyphae, there is a good chance you have a Zygomycete. If you place the mould on relatively rich mycological media, such as Sabouraud Dextrose Agar or Potato Dextrose Agar, and don’t get fruiting structures produced, you may have either a Saksenaea species orApophysomyces species. Both are notoriously suborn when it comes to inducing sporulation on richer mycological media. Media such as Czapek agar may produce better results. If this fails and you have lots of patience, you can try the agar block – sterile water technique as outlined by Padhye & Ajello[iii]. While the experienced mycologist may be able to tell Apophysomyces from Saksenaea without inducing sporulation, it is valuable in further speciation when not resorting to other means of identification such as molecular.

Although I attempted the agar –sterile water technique described by Padhye & Ajello, I was not successful in inducing sporulation and therefore the distinctive flask-like sporangium was not produced, nor the spores within. Features not produced cannot be photographed and I regret that I am unable to share them with you. As I work in a clinical laboratory, we carry only basic media and time is limited –I regret that I could not continue to pursue this challenge.

Because the organism could not be induced to produce its fruiting structures and sporangiospores, it was impossible to use these morphological structures to determine the specific identification of this Saksenaea species.

Saksenaea species - so here is about all I could get. The photo above shows the sporangiophore with the hemispherical shaped collumella at the apex (top). From the inset photo you can see what the fungus would look like had it produced (or retained) the flask-like sporangium.

(400+10X, LPCB, DMD-108)

Important: For the first time ever, I have posted a photo on this blog site which I have not personally taken. I felt that I could not post the photos I had without showing what the mature and fully intact Saksenaea looked like. The inset photo is shown here by the kind permission of Dr. David Ellis of the University of Adelaide's Mycology Website. My photos are free to share, however I request that this photo not be shared as the inset photo is the property of Dr. Ellis.

Saksenaea species - line drawing of structures discussed

Saksenaea species - again we see the hemispherically shaped collumella at the apex of the sporangiophore (arrows). Sporangium and sporangiospores are absent.

(400X, LPCB, DMD-108)

Saksenaea species - as above

(400X, LPCB, DMD-108)

Saksenaea species - ditto

(400+10X, LPCB, DMD-108)

Saksenaea species - a clue that this is a zygomycete is the broad, almost aseptate hyphae that the mould produces. The measurement seen within the large hyphae running through the photo reads 24.22 µm -that is wide!
(400X, LPCB, DMD-108)

Saksenaea species - a close look at the hyphae in the previous photo. Note that there is the septation at the center of the photo. (1000+10X, LPCB, DMD-108)

Saksenaea species - for the most part, the growth that I obtained, whether by slide culture or adhesive tape technique, appeared as above - just a tangled myceleum with no reproductive fruiting structures. (400X, LPCB, DMD-108)

So there you have it - the best I could do with the time and resources I had at hand.

[i]Molecular phylogeny and proposal of two new species of the emerging pathogenic fungus Saksenaea

Alvarez, E, Garcia-Hermoso, D, Sutton, DA, Cano, JF, Stchigel, AM. Hoinard, D, Fothergill, AW, Rinaldi, MG, Dromer, F, Guarro, J. Microbiol 2010;48 (12):4410-16

[ii]Mucormycosis caused by unusual mucormycetes, non-Rhizopus, -Mucor, and Lichtheimia species

Gomes, MX, Lewis, RE, Kontoyiannis, DP, Clin Microbiol Rev. 2011; 24(2):411-445

[iii] Simple Method of Inducing Sporulation in Apophysomyces elegans and Saksenaea vasiformis

Arvind A. Padhye and Libero Ajello; Journ Clin Micro, Sept 1988; 1861 - 63

* * *

Stachybotrys species

Ecology:

Stachybotrys is a common cosmopolitan saprobe (lives on decaying vegetative material) which can be found in soil. It is frequently found in the indoor environment, particularly in damp areas. Anyone who has removed water damaged wallpaper or lifted soggy cardboard boxes and found black discolouration on the material, may very well have encountered Stachybotrysmould. It seems to particularly like to feast on the glues used in wallpaper and paper tape adhesives. That powdery black residue on water damaged books may also yield Stachybotrys. Stachybotrysmay contain up to fifty species though I suspect this is under review using molecular methods.

Pathogenicity:

Stachybotrys has long been considered as non-pathogenic to humans or animals. More recently, evidence has been gathering that, although not a source of topical or systematic infection, the mould may be responsible for toxicosis following inhalation of the spores. It has been suspected as a cause of acute idiopathic pulmonary haemorrhage in infants, but its complete relation to human diseases is not yet fully understood.

The fungus has also been implicated in what has been termed ‘sick building syndrome’. Buildings which have an overall moisture problem, or those that have had been damaged by flood waters may be prone to extensive Stachybotrys(and other) fungal invasion. Wall coverings, ceiling tiles, and gypsum (sheet rock) wall boards and jute floorcoverings may all be contaminated. Unless extensive renovations are undertaken, occupants may developfatigue, headaches, chest tightness, mucous membrane irritation and pulmonary disease as a result of the fungal infestation.

Animals and horses in particular, may develop, what has been termed stachybotryotoxicosis after ingestion of feed (hay, etc.) heavily contaminated with Stachybotrys chartarum. It is characterized as an irritation of the mouth, throat, and nose which may lead to dermal necrosis and possible shock. Stachybotrys chartarum is known to produce a variety of toxins; macrocytic trichothecenes and related trichoverroids: roridin E and L-2; satratoxins F, G, and H; isosatratoxins F, G, and H; verrucarins B and J; and the trichoverroids, trichoverrols A and B and trichoverrins A and B.

According to the Center for Disease Control and Prevention, "The term 'toxic mould' is not accurate. While certain moulds are toxigenic, meaning they can produce toxins (specifically mycotoxins), the moulds themselves are not toxic, or poisonous.

Macroscopic Morphology:

Note: Sadly I have no photos of Stachybotrys colonies to share with the reader. A colleague provided swabs from blackened areas on damp washroom wallboard during a renovation. Days later, a quick adhesive tape mount of preliminary growth revealed the Stachybotrys posted below. Unfortunately it was quickly overgrown by other moulds present in the same sample and at a particularly busy time in the lab, I could not devote more time to its isolation. My hope was to obtain the mould from some other source at a more convenient time, which sadly, never came. 'Google Images' brings up a number of images of both the colonies on mycological media as well as 'in-situ' -on walls etc. Permissions could not be obtained to link to the photo which I felt best represented this mould's growth.

Stachybotrys exhibits rapid growth, maturing in three to four days. It has a powdery to cottony texture.

The surface colouration is initially white but quickly becomes black but may also exhibit pink or orange on the surface depending on the growth medium and species.

The reverse is also dark brown or black

Microscopic Morphology:

Stachybotrys has hyphae which are septate and hyaline when young but may darken with maturity.

Conidiophores may also be hyaline or may develop an olivaceous or dark pigmentation. The conidiophores may be septate, simple or branched. Conidiophores may show a rough-wall texture, particularly at the upper part, near the phialides.

Phialides (9-14 µm in length) may be hyaline, olivaceous to black in colour. They are ellipsoidal in shape and form in groups of 3 to 10 at the apex of the conidiophore
Phialides produce conidia singly and successively into a slime droplet that covers the phialides.

The conidia are 7 to 12 by 4 to 6 µm, black, unicellular, ellipsoidal in shape and smooth to rough-walled. Conidia are borne in slimy masses at the apices of the phialides.

Stachybotrys species -phialides are 9-14 µm in length and are borne in slimy masses at the apices of the phialides.

(1000X, LPCB, DMD-108)

Stachybotrys species -phialides are ellipsoidal in shape and form in groups of 3 to 10 at the apex of the conidiophore. The phialides are 9-14 µm in length.

(1000X, LPCB, DMD-108)

Stachybotrys species -conidiophores may show a rough-wall texture, particularly at the upper part, near the phialides.

(1000X, LPCB, DMD-108)

Stachybotrys species - conidiophores may be septate, simple or branched. Here both septation and branching are clearly visible.

(1000X, LPCB, DMD-108)

Stachybotrys species -conidia are 7 to 12 by 4 to 6 µm, black, unicellular, ellipsoidal in shape and smooth to rough-walled. In this photo the slimy mass of conidia have been disrupted.

(1000X, LPCB, DMD-108)

Stachybotrys species -This photo again shows a disrupted mass of ellipsoidal conidia. The dark pigmentation obliterated fine detail. Rough texture on the upper portion of the phialide is evident.

(1000X, LPCB, DMD-108)

Stachybotrys species -conidiophores may also be hyaline or may develop an olivaceous or dark pigmentation. (1000X, LPCB, DMD-108)

Stachybotrys species -hyphae are septate and hyaline when young but may darken with maturity.

(1000X, LPCB, DMD-108)

Notes:

Stachybotrys is highly cellulolytic which is why it enjoys growing on the various substrates already mentioned. It grows quite readily in materials low in nitrogen.

Stachybotrys differs from Memnoniella by not producing conidia in chains

While both the above genera are environmental contaminants, they are seldom isolated in the clinical laboratory.

Stachybotrysgrows well at 37ᵒC

One method of isolating or recovering Stachybotrys is to streak suspected conidia from the source onto dampened filter paper (eg. Whatman). Incubate the paper at 25 to 30ᵒC for about a week and look for dark patches. Transfer any growth to Sabouraud Dextrose Agar or Corn Meal Agar. Some sources claim that the fungus grows better on less nutritious media and this feature may assist in isolating it from more fastidious, yet fast growing fungi.

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Common and Alternative Stains for Viewing Moulds &Yeast

While cleaning out some files, I came across a number of microphotographs which might be of interest to the reader. Most of my posts show the organism in question after it has already been isolated and not how it may first clue the microscopist/technologist that an organism is present in a specimen. A few photos of specimens containing fungi follow below.

I worked on some of these specimens personally while others were brought to my attention by colleagues in the microbiology or histology laboratories. For those I did not work on personally, I have few specifics such as the final identification.

Yeast cell producing pseudohyphae in a sputum specimen. Numerous white blood cells present.

(1000X, Gram Stain, Nikon)

Numerous yeast cells, many budding, along with other bacteria in this sputum sample. A large epithelial cell is seen in the lower left.

(1000X, Gram Stain, Nikon)

Several yeast cells seen along the center of this sputum specimen photo. This sample also grew Streptococcus pneumoniae (pneumococcus) and Haemophilus influezae. White blood cells (wbc) and epithelial cells also present as one might expect.

(1000X, Gram Stain, Nikon)

Several individual yeast cells and the long pseudohyphae present seen in a vaginal swab specimen. Epithelial cells and commensal bacteria also present.

(1000X, Gram Stain, Nikon)

Yeast pseudohyphae in a kidney aspirate.

(1000X, Gram Stain, Nikon)

Another view (as above) of the yeast peudohyphae in the kidney aspirate, criss-crossing the photo.

(1000X, Gram Stain, Nikon)

Rather elongate yeast cell(s) in a blood culture. Identification was not noted.

(500X, Gram Stain, Nikon)

Candida parapsilosis in a blood culture.

(1000X, Gram Stain, Nikon)

Another photo as above - budding Candida parapsilosis in a blood culture.

(1000X, Gram Stain, Nikon)

This bizzare looking structure was from a direct gram stain of a blood culture and grew Candida albicans. It appears as if the yeast cells became clumped in this material (?) and were producing pseudohyphae seen as the 'branches' protruding outwards.

(250X, Gram Stain, DMD-108)

Another view as above. Pseudohyphae protruding from material clumped together in a blood culture.

(400X, Gram Stain, DMD-108)

Fungal conidium germinating in an ear. Two true hyphae can be seen extending from the conidium. Single epithelial cell seen in upper center.

(400+10X, Gram Stain, DMD-108)

Another photo of the ear swab (as above), here showing the dichotomous branching of the fungal hyphae. Septations within the hyphae are visible.

(400X, Gram Stain, DMD-108)

...And yet one more as above.

(400X, Gram Stain, DMD-108)

Fungus present in the gram stain from a nasal aspirate. This isolate was identified subsequently identified as Aspergillus flavus.

(1000X, Gram Stain, DMD-108)

Another photo of the nasal aspirate showing the fungal hyphae. Gram's crystal violet stain is strongly retained in this preparation resulting in the intense dark colour.

(1000X, Gram Stain, DMD-108)

I took several photos of this structure yet all seem to be somewhat out of focus. This appears to be the intact 'fruiting head' of an Aspergillus species as seen in the direct preparation of a broncheal wash. (1000X, Gram Stain, Nikon)

Here we have a fungus (dermatophyte) seen in an unstained nail preparation. The nail specimen is covered with 10% potassium hydroxide (KOH) which both softens & clarifies the nail for enhanced viewing as well as kills the fungus for safety.

(400X, 10% KOH, DMD-108)

Unspecified tissue containing fungal hyphae (arrows)

(400X, 10% KOH, DMD-108)

Aspergillus fruiting structure and dispersed conidia. The inset is from another photo of the same structure at an alternate focus. I believe this specimen was from a brocheal specimen.

(Specifics not noted, Nikon)

Fungus in a tissue sample. This histological section shows the hyphae sliced along their length (L=Longitudinal), as well as across their diameter (T=Transverse)

(1000X, Periodic Acid Schiff, Nikon)

Fungal elements seen in this histological specimen appear black against the green counterstain.

(1000X, Gomori Silver Stain, DMD-108)

Another tissue sample with fungal elements as above.

(1000X, GMS, DMD-108)

Cryptococcus neoformans in a blood culture. This direct stain shows the large capsule which surrounds the yeast cell of C.neoformans.

(1000X, Gram Stain, Nikon)

Cryptococcus neoformans again in the blood culture as above. Again, material surrounding the yeast cell clearly shows the capsule that surrounds the organism.

(1000X, Gram Stain, Nikon)

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