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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Sunday, 14 December 2014

Hymenolepis nana

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 nana is 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.nana may 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 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.dimunata eggs 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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