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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