Monday, 23 February 2015

Ochroconis species

Ochroconis species (Hyphomycete)  Fungus

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

Ochroconis species 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. 
Ochroconis species 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.  Ochroconis species 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:
Ochroconis produces 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) 

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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Saturday, 24 January 2015

Aureobasidium pullans

Aureobasidium pullans  (Hyphomycetes) –Black yeasts

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

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.

A.pullans appears 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 30oC (Nikon)

 Aureobasidium pullans on SAB -progression of growth at 30oC.  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 30oC.  (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 - indivdual conidia 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)
  1. Free blastoconidia
  2. Dematiaceous, boxcar-shaped, arthroconidia
  3. Dematiaceous, round, intercalary chlamydoconidia
  4. 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)

Aureobasidium pullans:
·         Grows best at about 25o C and may be inhibited at 35oC.
·         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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