Saturday, 25 February 2012

Trichophyton rubrum

Trichophyton rubrum (Fungus, Dermatophyte) Teleomporph: None

Ecology, Epidemiology & Pathogenicity;

Trichophyton rubrum is a cosmopolitan, anthropophilic fungus meaning its primary reservoir is man. It is the most widely disseminated dermatophyte of man and most common cause of athlete’s foot (Tinea pedis), jock itch (Tinea cruris), ringworm (Tinea corpis), onychomycosis (Tinea unguium), and less commonly hair and scalp infections. Invasive infections in immunocompromised patients have been reported.

Trichophyton rubrum infections are of concern because nail infections caused by this dermatophyte are extremely difficult to cure.

Presented below is an isolate responsible for a case of onychomycosis, also referred to as Tinea unguium or simply stated a nail infection.

Rate of Growth;

Trichphyton rubrum is a moderately slow grower, reaching maturity within 14 days at 25o to 30oC.

Colonial Morphology;

Trichophyton rubrum species exhibits widely variable colonial morphology and the appearance is further influenced by the media on which it is isolated. Most typical strains are downy to cottony in texture with fine white aerial mycelium at the surface. The overall surface is white, sometimes becoming rose on ageing. The reverse is typically wine-red; however brown & yellow to olive-green hues may be present. SAB & Mycosel media tend to bring out the reddish-brown to yellow colours. Trichphyton rubrum often appears in literature as having two major forms of surface texture; a downy type and a granular type. Overlap between the two may be evident and even taxonomic relatedness is questionable. One source1 states that “deep red isolates with many macroconidia, once referred to as the ‘granular’ colony surface type, are now mostly recognized as representatives of the urease-positive T.raubitshekii.”

Trichophyton rubrum surface and reverse on SAB media after 10 days incubation at 30oC

Microscopic Morphology;

Trichophyton rubrum produces hyaline septate hyphae. The downy type, described in this post, is characterized by the production of moderate numbers of clavate (club shaped) or pyriform (tear-drop shaped) microconidia (3-5.5 X 3-3.5 µm) with rare if any macroconidia. (The granular form is characterized by the production of moderate to abundant numbers of microconida as well as moderate to abundant numbers of long, narrow, thin-walled cigar or pencil-shaped macroconidia (40-55 X 6-7.5 µm) with parallel sides.) Macroconidia (4 – 10 cells in length) may form directly on the ends of thick hyphae singly or in groups. It may also produce small chains of barrel-shaped arthroconida from both the hyphae and macroconidia and are similar in size to the macroconidia.

Photos below were taken with a Digital Nikon Coolpix 8400 Camera or the Leica DMD-108 microscope camera as indicated. The DMD-108 has a digital magnification factor of X10 which can be added to any optical magnification.

Structure size may vary between photographs of identical magnification due to selective cropping of the photograph.

* * *

(Click on any photo to enlarge for better viewing)

Toenail from which this Trichophyton rubrum was isolated. Portions of the nail specimen were placed on SAB and Mycocel media for isolation. Portion was also flooded with 10% KOH which both softens the nail and clarifies for better viewing of any fungal structure which may be responsible for the infection.

Photomicrograph of nail segment in KOH. Trichphyton rubrum fungal hyphae seen invading between nail cells. (dotted structure weaving through from upper left through to lower right) (DMD-108 Microscope X400)

Photomicrograph of T.rubrum fungal element (arrow) in toenail tissue (Magnification not noted)

Trichophyton rubrum microconidia on hyphae. This arrangement of microconida has been described as "birds on a wire" where the pyriform (teardrop shaped) microconidia are attached to the hyphae at the narrow end (tail).

Another view of the microconidia attached to the hyphae (DMD-108 LPCB X400)

Inset: isolated view of the microconidia on hyphae ( LPCB X250 Nikon)

Closer view of pyriform shaped microconidia on hyphae (DMD-108 LPCB X400)


This structure looks like I would expect the pencil-like macroconidia would appear, that is except for the size. Though I neglected to take measurements on this structure, it was clearly less than the 45-55 µm usually noted as the average length. Clearly visible are 4 cells within the structure. Perhaps this is still an immature macroconidium.

(LPCB X400 Nikon Camera - I suspect circular lines are from spherical aberrations on the Leica microscope lens at this particular magnification.)

Arrows; indicating presence of Arthroconidia, either free or forming at ends of hyphae,

(LPCB X400 Nikon)


T.rubrum can be differentiated from T.mentagrophytes by its typical microconidaia which are clavate (club shaped) to pyriform (tear-drop shaped), solitary, sessile alongside undifferentiated hyphae. T.mentagrophytes produces microconidia in clusters that are spherical.

Other Physiological Tests for T.rubrum;

  • Urease Negative
  • Hair perforation test negative.(1)
  • BCP-milk solids-glucose: no change in pH, restricted growth.
  • Potato glucose agar or cornmeal glucose agar enhance the production of red pigment on the reverse if the strain is unpigmented on Sabouraud Glucose (Dextrose) agar.
  • Requires no additional growth factors for growth. Unlike T.megninii, T.rubrum does not require histadine for growth.

Molecular Testing; has been used to reassess the classification of closely related Trichphyton species but remains beyond the scope of the average clinical laboratory and this post.

(1) Hair perforation test; sterile hair can be inoculated with Trichphyton species and examined after a period of growth to see if the fungus has physically invaded/perforated the hair.

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Friday, 24 February 2012

Absidia corymbifera (Lichtheimia corymbifera complex)

Mycocladus corymbifer (formerly Absidia corymbifera)
Lichtheimia corybifera complex (formerly Absidia corymbifera) Fungus
(Names seem to change monthly!!! - your choice!) 
Absidia corymbifera;
I’m going to refer to this organism throughout this post by its former name, “Absidia” corymbifera as most are still unfamiliar with the change in nomenclature.
Although uncommon, Absidia species have been implicated as an agent of human (sub)cutaneous mycosis as well as opportunistic pulmonary infections. As with other fungi previously considered to be relatively benign, they are now being isolated with greater frequency particularly in immunocompromised patients (AIDs, transplant patients, neutropenic patients, etc.) Absidia corymbifera is considered to be the only species of Absidia that is recognized as a human pathogen. Absidia corymbifera occasionally has been known to cause mycotic abortions in cows.
Absidia species are ubiquitous in nature and can be isolated from soils and decaying vegetation. As such they may be found as contaminants in cultures. Care must be taken in determining their significance when isolated.
Macroscopic Appearance:
Colonies grow rapidly, maturing in 3 – 4 days. They are white to a greyish-brown in colour with a woolly texture also described as resembling ‘cotton candy’. Reverse is uncoloured and non-descript. Absidia species are inhibited by cycloheximide and therefore will not grow on media such as mycosel. Maximum growth temperature for Absidia corymbifera is between 45 – 52oC. Absidia corymbifera is thermophillic in that it grows more rapidly at 37oC than at 25oC accounting for its human pathogenicity (body temperature). Most other Absidia species fail to grow at 37oC.
Absidia corymbifera on SAB media after 4 days incubation at 30oC
(Reverse not shown - rather nondescript colourless)
Microscopic Morphology:
Absidia’s hyphae are aseptate (rare septa) and relatively wide at 6 – 15 µm in diameter. Absidia species resembles Rhizopus species however the distinguishing feature is that the sporangiophores of Absidia arise from locations on the stolon that lie between the rhizoids and not from them. In other words, there is no rhizoid directly below the sporaniophore as would be observed in Rhizopus species.
Sporangiophores arise alone or in groups from the aerial hyphae and are rather long (up to 450 µm). Sporangiophores branch and widen apically where they form a conical apophysis immediately below the columella. Sporangia are about 20 – 90 µm in diameter are spherical to pyriform in shape. When mature the sporangial wall dissolves releasing round to oval (3 – 5 µm) sporangiospores leaving a small collarette where the apophysis and sporangium had met. Zygospores have been described.
Note: Apophysomyces elegans resembles Absidia corymbifera but produces white colonies and fails to sporulate on routine clinical laboratory media.
Photos below taken with the Leica DMD-108 Microscope.
Young slide culture Absidia corymbifera X100 LPCB
(Click on any photo to enlarge for better viewing)
Absidia corymbifera - Structures seen; (LPCB X100)
  1. Rhizoid
  2. Sporangiophore arises from stolon between rhizoids and not directly from a rhizoid though this appears to be more of a branch in this photo. Rhizoids were a bit of a challenge to find on this particular organism.
  3. Sporangiophore
  4. Sporangium
Ditto (LPCB X100)
Rhizoids were rather difficult to detect. Adhesive tape preparations failed to capture any. I suspect they were so well anchored to the media that the sporangiophores broke off from the stolon rather than pulling the stolon and rhizoids out with it. This photo was taken from a slide culture preparation where some of the agar media surrounding the rhizoid was removed along with the stolon and sporangiophore.
Numerous sporangia with loose sporangiospores are pictured. Notice that the sporangiophore widens apically where it becomes the columella and supports the sporangium. (LPCB X400)
Absidia corymbifera sporangium filled with sporangiospores. (LPCB adhesive tape preparation X400 + additional 1% digital magnification)
More views of the same. Apophysis structure can be seen in dissolving sporangium on left. (LPCB X400)
Sporangium dissolvingé breaking up releasing Sporangiospores
(LPCB X400)
Apophysis with remains of Collarette and a few remaining Sporangiospores.
More views of same - Stolon with branching sporangiophores bearing Sporangia in various stages of releasing Sporangiospores. Apophysis can be visualized on Sporangiophore at upper left. (LPCB X 400)
Dissolving sporangium releasing numerous sporangiospores with remaining apophysis on apical end of sporangiophore. (LPCB X400)

Wednesday, 22 February 2012

Strongyloides stercoralis Revisited (Sputum)

Parasite (Nematode)

While I already had a 2010 post on Strongyloides stercoralis , I decided to add a separate posting for this case as it differs in that the worm was not obtained from a fecal sample but rather from a sputum sample. It is precisely these twists that make microbiology both fun and challenging.

An astute colleague was examining a routine sputum culture for bacterial pathogens on this patient when he picked up on a clue that many might have missed. Small looping 'threads' (for lack of a better description) of bacteria which extended from the primary growth (see photo below). He immediately realized that this might be evidence of the presence of a worm. A viable (live) worm if present in the sputum sample would be inoculated onto the agar media along with respiratory bacteria. They can crawl just as an earthworm can crawl on a wet sidewalk. As they migrate around the plate they draw along bacteria that have adhered to their sides and deposit these along the way providing evidence of their travels. With continued incubation the individual deposited bacteria grow up into colonies outlining the worms journey.

Above (Arrows); A chocolate agar plate inoculated with a sputum sample showing curious looping trails of bacteria which were deposited by a worm migrating about the plate during incubation. Having noticed this curious evidence, the following morning the plate was examined under a plate microscope however the worm responsible for making this trails was not located.

Strongyloides stercoralis can migrate out of the gastrointestinal tract and pass through the lungs. The worm can be coughed out and/or re-swallowed to reinfect the patient. The attending physician had noted that this patient had produced "coffee ground" emesis (hematemesis). This is partially digested blood tinged sputum coughed up, or perhaps swallowed, then coughed up. It has the apperance of wet coffee grounds, hence the description. The sputum sample received by our lab no longer showed this coffee ground appearance. Our patient was an elderly gentleman with several other underlying medical conditions which will not be noted here.

Two Strongyloides stercoralis worms seen in this gram stain of sputum - one curled and one straight directly above. White blood cells dot the background.
Note micron bar in upper right corner. (DMD-108 Microscope X100)

Same worms as above (Gram Stain X1000)

Another Strongyloides stercoralis worm (note striated texture of body)
(Gram Stain of Sputum - DMD-108 Microscope: X1000)

Anterior end of worm with buccal cavity visible.
Compare size to gram negative bacilli bacteria present.
(Gram Stain of Sputum -DMD-108 Microscope X1000)

Strongyloides stercoralis in Iron Hematolxyin Stained Sputum Isolate.
(DMD-108 Microscope X1000)

Two Strongyloides stercoralis worms seen in this Iron Hematoxylin stained preparation of the sputum sample received. Numerous white blood cells present.
(DMD-108 Microscope X100)

Having requested additional specimen samples (and stool sample for interest sake), Strongyloides stercoralis eggs containing developing larvae were also found.
Above; Both a worm and egg are present with the egg trailing some incidental material attached to one end. (Concentrate of sputum sample DMD-108 Microscope X100)

Same egg as above with extraneous material trailing off to lower right
(DMD-108 Microscope X400)

Another Strongyloides stercoralis egg containing developing larvae
(Concentrate - DMD-108 Microscope X400)

Strongyloides stercoralis worm in sputum.
(Concentrate - DMD-108 X400)

A stool sample was received by the lab however diagnosis had already been made starting by those rather inconspicuous tracks left by the worm on bacterial agar media.

For further information on clinical symptoms, diagnosis etc, please refer to my previous post located here; Strongyloides stercoralis
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Sunday, 5 February 2012

Microsporum canis

Microsporum canis; (Fungus)


Microsporum canis is a cosmopolitan zoophilic dermatophyte usually acquired from infected dogs (hence canis) or cats. Most prevalent in children, it has been implicated in infections of the scalp and skin and occasionally nails. A scalp infection could be visualized by examining plucked hairs under a Wood’s (UV/black light) lamp for fluorescence.

Colony Morphology;

Surface growth has been described as downy to woolly, to fluffy, hairy and silky. Typically it exhibits a light yellowish pigment at the periphery and growth shows closely spaced radial grooves. The reverse is pale tan to yellowish (yellowish-orange –media dependent) which tends to turn brownish as it ages.

Microsporum canis on SAB at 5 days at 30oC

Microscopic Morphology;

Microsporum canis has septate hyphae that produce numerous macroconidia. The macroconidia are rather long (10-25 X 35-110 µm), spindle or fusoid in shape and are thick walled with a echinulte (rough) texture. The ends typically taper to a knob-like end that may be somewhat recurved at the apex. The macroconidia usually have six or more compartments when mature and few smooth walled club shaped macroconidia may be observed along the hyphae. Smooth walled, club shaped microconidia are infrequently seen forming along the length of the hyphae.

(all photos taken with DMD-108 digital microscope except where noted)

Microsporum canis growing at edge of cover slip from a slide culture preparation (LPCB X100)

(Click on any photo to enlarge for better viewing)


Microsporum canis (LPCB X100) Nikon

Microsporum canis (LPCB X250)

Microsporum canis (LPCB Magnification not noted)

Microsporum canis (LPCB X400) -Nikon

Microsporum canis - Macro & microconidia (LPCB X400)

(note 100 µm bar at upper right of photo)

Microsporum canis compartmentalized macroconidia showing rough surface with knob-like end (LPCB X1000 Adhesive Tape Preparation) -Nikon

Ditto (note curved ends)

Microsporum canis - 10% KOH* preparation (X400)

Microsporum canis - 10% KOH* preparation (X400)

(Inset; 2 macroconidia)


Microsporum canis needs no special growth factors or cultural requirements. I found that on the relatively nutritious Sabouraud Dextose agar, my isolate became sterile on repeated subcultures. Macroconida were produce when grown on Corn meal agar. The macroconidia are produced as a survival mechanism and are induced when conditions are not as rich or favourable for growth.

Microsporum canis can be inoculated onto sterile (autoclaved) polished rice grains where they produce a yellow pigment.

A hair perforation test (positive) can be performed in vitro. As previously stated, hairs infected with Microsporum canis will fluoresce under a Wood’s UV lamp.

* * *

*10% KOH - Potassium Hydroxide, kills and clears the preparation.

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Saturday, 4 February 2012

Aspergillus flavus

Aspergillus flavus (fungus)

Ecology & Pathogenicity;

Found worldwide. Ubiquitous in nature and is the second most common cause of invasive aspergillosis next to Aspergillus fumigatus. Has been implicated in pulmonary, systemic, sinus, ear and other infections. Most widely reported food-borne fungus and can be found colonizing decaying vegetation, crops and seeds. Has also been implicated as both an insect and animal pathogen. (For a slightly more expanded description of pathogenicity, see the intro to the Aspergillus fumigatus post.)

Macroscopic Colony Morphology;

Very rapid rate of growth, maturing in about three days. Surface is greenish-yellow to olive and may have a white border. Texture is often floccose, especially near the center and overall can be velvety to woolly. Unremarkable cream to tan to yellowish reverse on Sabouraud Dextrose media.

Aspergillus flavus on SAB media at 72 hour at 30oC

(note greenish-yellow colour with white edge)

(click on any photo to enlarge for better viewing)

Microscopic Morphology;

Septate hyphae with rather long conidiophores (~400-800 X 8-17 µm) which have a rather rough texture or even spiny, especially just below the vesicle. Vesicles are spherical to elongate and about 20 – 45 µm wide. Aspergillus flavus can be variable in seriation with most strains being about 20% biseriate however some strains can be almost entirely uniseriate. Metulae (8-10 X 5-7 µm) cover three quarters to the entire surface of the vesicle from which the phialides (7-12 X 3-4 µm) form. The metulae support the phialides and together form the biseriate structure. Conidia are globose to ellipsoidal (3-6 µm) with smooth to finely roughened walls.

(Photos below are of a ~30hr old slide culture preparation taken with the DMD-108 digital microscope - except where noted)

Aspergillus flavus mycelium &, conidiophores (LPCB) (X250)

Tangled web of the Aspergillus flavus mycelium as above (X250)

Aspergillus flavus conidiophore bearing vesicle (X1000)

look for the biseriate structure (metulae & phailide) on this and the following photos

Aspergillus flavus vesicle bearing metulae & phialides from which forms the conidia

Aspergillus flavus (LPCB X1000)

Look for the Biseriate structure - Rough surface of conidiophore at its apex (where it meets the vesicle ) is evident in this photo. This roughness is a diagnostic feature of Aspergillus flavus.

Aspergillus flavus (LPCB X1000) Biseriate structure may be easier seen in this photo.

Aspergillus flavus (LPCB X1000)

(Too many photos, but I just like them!)

Aspergillus flavus (LPCB X1000 + 10X digital magnification from DMD-108 Scope)

Though difficult to see, the slighly rough wall can be visualized on several of the conidia, particularly in the lower left of the photo. The conidiophore also has this rough or gently spiked texture particularly at the apex (where it meets the vesicle)

Also note that phialides radiate from vesicle in all directions as opposed to A.fumigatus where they tend found on the upper 2/3rds of the vesicle and extend parallel to the conidiophore.

Aspergillus flavus (LPCB X1000)

(again, enlarge photo and look for the rough texture of the conidia's surface)

Aspergillus flavus - more typical appearance with phialides radiating from vesicle in all directions. This photo taken from adhesive tape preparation of 72 hr fully mature culture. This technique was quite disruptive, dispersing conidia throughout the preparation.

(LPCB X400 -Nikon)


Many Aspergillus flavus isolates are capable of producing aflatoxins, very potent carcinogens. (Aflatoxin B1, cyclopiazonic acid, 3-nitroproprionic acid.)

Intended as Aspergillus flavus computer screen 'Wallpaper' (1024X768 when posted)

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