Friday 1 November 2013

Eurotium herbariorum (Aspergillus glaucus)

Eurotium herbariorum (Aspergillus glaucus) Mould

Note:  This fungus has both a sexual and asexual means of reproduction.  When both are present, the sexual stage (teleomorph) name takes precedence over the asexual (allomorph) name.  Applied here, Eurotium herbariorum take precedence over Aspergillus glaucus.  However,  I will generally refer to the fungus here as Aspergillus glaucus as most references still continue to do so.

Aspergillus glaucus is a cosmopolitan fungus (worldwide distribution) and while it prefers drier environments, it can be isolated from soils, house dust, plants & dried foods.  A.glaucus is also osmophilic, meaning it can grow, and perhaps prefers to grow in environments containing a higher sugar concentration.  Growth is restricted or limited at 35˚C which may account for its limited pathogenicity.

A.glaucus is not very invasive and is rarely encountered in the clinical laboratory.  It has been implicated as a cause of ocular (eye) infections, particularly after some traumatic injury.  Cerebral, orofacial, cardiovascular and pulmonary infections are rare but have been reported.  May also cause sinusitis (nasal) and otitis (ear) infections.  May be considered an opportunistic fungus particularly with immunocompromised patients.

Macroscopic Morphology:
Growth is slow to moderate, maturing in about 7 to 21 days.  Colony size expands rather slowly.
Colony colouration is media dependent but is described as a dull to deep green to a greyish turquoise, with yellow to orange areas where cleistothecia are being produced.  The reverse is pale yellow to yellow.  The isolate presented below failed to grow at 30˚C but grew well at ambient room temperature (~20˚C).
 Eurotium herbariorum (Aspergillus glaucus) -SAB + ~20% Sucrose, Room Temperature, 14 Days
(this particular isolate failed to grow at 30˚C)

Note on Culture:  Our medical laboratory purchases all media from a commercial supplier and in these financially challenging times we stock only what can be clinically justified.  We have no media room where we can concoct media from basic ingredients.  Sabouraud Dextrose media has a sugar content (dextrose) of 4% which would favour the production of asexual conidia while a high osmotic concentration (~20%) would enhance cleistothecia production.  In order to enhance growth and increase the production of cleistothecia, I obtained a couple of tubes of Sabouraud Dextrose (slants) and melted them down in a boiling waterbath.  I did a rough 'back of an envelope' calculation of how much sugar I would have to add to bring the concentration up to about 20%.  With no lab supply of dextrose to add, I went down to our hospital 'Tim Horton' coffee shop (a Canadian franchise) and obtained a packet of sucrose.  Adding this to the melted SAB agar, I brought the total sugar concentration up to about 20% (glucose + sucrose) to raise the osmotic concentration.  I sacrificed a sterile media petrie dish contents (as we also do not stock sterile empty petrie dishes) and poured in my own concoction of "Tim Horton's Cleistothecia Enhancing Media", or THCEM for short.  For this reason you will see the media identification on the reverse plate blurred out. With no autoclave on site, I hoped that the boiled contents remained sterile and the high osmotic concentration would discourage other organisms from growing.  The THCEM media worked extremely well!  The photographs which follow are from this agar media.

Microscopic Morphology:
Hyphae are septate and hyaline.
Teleomorph – Sexual state is seen with the production of cleistothecia (ascomata).  These structures are globose to subglobose, about 60 µm to 150 µm in diameter.  In their natural state they appear yellow to golden in colour and their presence may be seen macroscopically as distinctly yellowish areas within the maturing colony.  Within the cleistothecia/ascomata, 8-celled asci are produced which are released at maturity or when ruptured.  The 8-celled asci (10 µm - 12µm diameter) are dehiscent (dissolve) and release individual ascospores on maturity).  The Ascospores themselves mature in about two weeks’ time and are lenticular (lens shaped) with a noticeable longitudinal furrow.  (On the side, they resemble a hamburger, with the patty being the furrow.)  They range between 5 µm to 7 µm by 3 µm to 5 µm in size).  The ascospores have a rather smooth surface texture which may help differentiate them from the ascospores produced by the Neosartorya species.

Teleomorph (Eurotium herbariorum) - Sexual State

Eurotium herbariorum (A.glaucus) - a first look with an adhesive tape preparation from the colony.  E.herbariorum produces numerous cleistothecia and production can be enhanced on media with a higher sugar content.  Cleistothecia production on the plate can be seen macroscopically as an enhanced yellowish band within the colony.
(250X, KOH, DMD-108)
 KOH= Potassium hydroxide

 Eurotium herbariorum (A.glaucus) -cleistothecia at a higher magnification.  The KOH kills the fungus so that the slide can be safely removed from the biological safety cabinet without fear of contamination.  It also clarifies the preparation to some degree and dose not alter the natural colour of the mould.
  (400X, KOH, DMD-108)

 Eurotium herbariorum (A.glaucus) -as above.
 (400X, KOH, DMD-108)

 Eurotium herbariorum (A.glaucus) -The cleistothecia vary in size as they mature but are generally in the range of 60 µm to 150 µm in diameter.  This size also aids in distinguishing Eurotium from other cleistotheia producing moulds.
(400+10X, KOH, DMD-108)

Eurotium herbariorum (A.glaucus) -the Aspergillus conidiophore (in rather poor shape) is seen on the left while a cleistothecium (also breaking up)  is seen on the right.
(400X, KOH, DMD-108)

 Eurotium herbariorum (A.glaucus) - A cleistothecium breaking apart and releasing ascospores.  The KOH does not react with the contents of the cleistothecium as it did with Emericella nidulans (Aspergillus nidulans) where the contents produced a purple colour.
(400X, KOH, DMD-108)

 Eurotium herbariorum (A.glaucus)  -Several cleistothecia seen with the released asci staining blue with the LPCB.
 (400X, LPCB, Nikon)

Eurotium herbariorum (A.glaucus) -a cleistothecium showing a break at the bottom and the asci within staining blue.
(400+10X, LPCB, DMD-108)

Eurotium herbariorum (A.glaucus) -another view as above. A released, intact 8-celled ascus can be seen on the bottom edge of the cleistothecium (or ascomata)
(1000X, LPCB, DMD-108)

 Eurotium herbariorum (A.glaucus) -another view of a cleistothecium (Ascomata) with an 8-celled ascus inside. (1000X, LPCB, Nikon)

  Eurotium herbariorum (A.glaucus) -two cleistothecia with the large one filling most of the upper right of the photo, filled with asci & apparently free ascospores.
 (1000X, LPCB, Nikon)

  Eurotium herbariorum (A.glaucus) -yet another view showing several 8-celled asci packets that have been released from the cleistothecia (Ascomata).
 (1000X, LPCB, Nikon)

  Eurotium herbariorum (A.glaucus) - a cleistothecium filled with asci.
 (1000X, LPCB, Nikon)

  Eurotium herbariorum (A.glaucus) -a free 8-celled ascus seen in the center of the photo with a cleistothecium on the right.
 (1000X, LPCB, Nikon)

 Eurotium herbariorum (A.glaucus) -two intact cleistothecia (ascomata) seen with a typical `cracked mud` appearance of the surface.
 (1000X, LPCB, Nikon)

  Eurotium herbariorum (A.glaucus) -A breach in the wall of a cleistothecium from where the asci contained within will escape.
(1000X, LPCB, DMD-108)

  Eurotium herbariorum (A.glaucus) -a single, free 8-celled ascus seen in the center of the photo.  This was released, with many others, when the cleistothecium matured and dissolved or broke apart. (1000X, LPCB, DMD-108)

   Eurotium herbariorum (A.glaucus) -a slightly different appearance of  a cleistothecium, with the point of attachement to the hyphae seen below it. 
(400+10X, LPCB, DMD-108)

  Eurotium herbariorum (A.glaucus) -two cleistothecia amongst the hyphae.  The photo appears oversaturated with the blue of the LPCB masking some of the detail.  I was unable to correct for this with using a photo editing program.
 (1000X, LPCB, DMD-108)

  Eurotium herbariorum (A.glaucus) -as above but here you now can see the rough-walled conidia which were produced by the Aspergillus glaucus allomorph.
(1000X, LPCB, DMD-108)

Allomorph – smooth walled conidiophores extend between 300 µm – 700 µm in length and are between 7 µm – 12 µm in width.  Vesicles are globose (spherical) to subglobose (subspherical) to pyriform (tear-drop) in shape and roughly 18-30 µm in diameter.  A.glaucus is uniseriate with phialides 7 – 11 µm to 3 – 7 µm in size and generally covers most of the vesicle.  The conidia (4 µm to 8 µm diameter) are spherical to ellipsoidal in shape and are echinulate to spinose (finely roughened/fine spines).

Allomorph (Aspergillus glaucus) - Asexual State

Aspergillus glaucus (Eurotium herbariorum) -so here is the allomorph which is the asexual Aspergillus glaucus component.  Both were present on this isolate.
(1000X, LPCB, DMD-108)

Aspergillus glaucus -a conidiophore and vesicle with the phialides bearing spiny, spiked or rough walled conidia.  The DMD microscope somehow tends to over saturate in the photograph while the image on the high definition LED screen looks pristine. Again, I find that my attempts at correcting for this with photo editing programs falls short.
(1000X, LPCB, DMD-108)

Aspergillus glaucus -Here you can easily see that Aspergillus glaucus is uniseriate.  If it were biseriate there would be an additional structure called a metulae between the phialide and the vesicle supporting it.  (Kind of a double-decker structure).  Again the rough, spiny conidia can be seen at the ends of of the phialides.
(1000X, LPCB, DMD-108)

 Aspergillus glaucus -just `cause I like the photo. Vesicles are globose (spherical) to subglobose (subspherical) to pyriform (tear-drop) in shape and roughly 18-30 µm in diameter.  Chains of conidia can be seen trailing from the apex of the phialides where they were produced.
(1000+10X, LPCB, DMD-108)

Aspergillus glaucus -a rather poor looking conidiophore and vesicle in the upper left however there is a very good example of the shape of the ascospores.  Here you see the original 8-celled packet (ascus) somewhat broken apart but the equitorial trough (furrow) and two lateral crests are clearly visible (inset).  The smooth texture helps to distinguish these ascospores from those produced by Neosartorya species.  The kind of look like a hamburger patty within a bun as viewed from the side!
(1000X, LPCB, Nikon)

Aspergillus glaucus -here is a whole mess of A.glaucus conidia showing the spiny texture of the surface.
(1000+10X, LPCB, DMD-108)

 Aspergillus glaucus -just to show the difference in size between the individual ascospores and the conidia produced by this species.  Depending on the orientation in the field, the ascospores may not show the equitorial trough or crests. (ie viewed from the top and not the side)
(1000+10X, LPCB, DMD-108)

Aspergillus glaucus -one last photo of an Aspergillus glaucus conidium with its rough wall.
(1000X, LPCB, DMD-108)

With the petrie dish under a stereoscopic dissecting microscope, the fruiting structures were both visible and distinguishable.  The Aspergillus conidiophores stood erect like trees in a forest, with the conidia appearing a pale sage green in colour.  The cleistothecia were interspersed and about the same size in total diameter but they appeared yellowish-brown in colour.  A really beautiful sight viewed between 16X to 40X magnification!
The Aspergillus glaucus group contains approximately 17 species (subject to continued molecular study and reassignment, I’m sure).  Of these, some species are extremely xerophilic (need little or no water to survive) and as such, may be found in greater numbers in tropical to subtropical regions as opposed to temperate zones.  This property also favours their growth on dry or concentrated substances.  As mentioned earlier, they are osmophilic and can grow in the presence of high sugar concentrations where the carbohydrate draws away the water.  That fungus growing on top of the jam jar may be Aspergillus glaucus!

Eurotium repens & E.rubrum are species closely related to E.herbariorum.  E.repens differs from E.herbariorum by the formation of ascospores without a distinct furrow or trough.  E.rubrum has ascospores with a more distinct furrow, and the hyphae tend to turn brick red with age.  Some consider these three species to be conspecific, referring to all of them as E.herbariorum.

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Wednesday 30 October 2013

Exserohilum rostratum

Exserohilum rostratum (Mould)

Exserohilum species are dematiaceous fungi (ie black mould), widely distributed in nature.  They are cosmopolitan, commonly found on many plants and grasses and can also be isolated from soils and water.

Exserohilum species are unlikely human pathogens.  Exserohilum species have been implicated in phaeohyphomycosis[i]. Most commonly they are isolated from nasal sinuses (sinusitis) and the eye (keratitis) after a scratch or traumatic injury.

In 2012, Exserohilum rostratum was implicated as the primary pathogen isolated from injectable methylprednisolone. Numerous patients receiving steroid therapy primarily for degenerative lumbar-disk and joint disease in the U.S., developed meningitis after injection. While normally not invasive, the fungus will cause illness when directly injected into the body. While sources vary in statistics, injectable methylprednisolone contaminated with Exserohilum rostratum appears to have been responsible for sickening upwards of 700 people, 30 of which died.  Many others continue to have ongoing neurological problems associated the infection or subsequent therapy.

Macroscopic Morphology:
E.rostratum exhibits rapid growth and matures within 4 to 5 days.
Surface growth is grey in colour, quickly darkening with the production of melanin, eventually developing shades from olive to brown to black.
The texture is woolly or cottony in appearance.

Exserohilum rotratum SAB 30oC, 72 Hours

Exserohilum rostratum SAB 30oC, 1 Week

Microscopic Morphology:
Hyphae are septate and darken with the development of melanin.  Conidiophores are rather long (up to 200 - 230 µm in length, 5 – 8 µm wide) and are also septate.  Conidiophores exhibit sympodial geniculate growth, where conidia are produced at bends (geniculate) as the conidiophore extends.  This gives the conidiophore a knobby, zigzag appearance where the conidia attach.  The mature conidia (ave. 14 X 80 µm or greater) are straight to slightly curved and are fusiform or ellipsoidal in shape with rather smooth walls.  Conidia are compartmentalized with between 7 to 11 septa and has a distinctive protruding dark hilum (scar) at the base where once attached.
More specifically, the conidia are ‘poroconidia’, a distinction where the conidia are produced through the extrusion or extension of the inner walls of the conidiogenous cells through a pore or channel.

 Exserohilum rostratum -First look.  Free conidia.  Tape mount at 250X (LPCB, DMD-108)

Exserohilum rostratum -Conidia attached to septate hyphae (400X, LPCB, Nikon)

Exserohilum rostratum - Large, compartmentalized conida which are somewhat fusiform in appearance and may appear slightly bent or curved.  (400X, LPCB, DMD-108)

Exserohilum rostratum - as above.  Showing variation in size and shape of conidia.  Brown pigmentation due to the production and accumulation of melanin. (400X, LPCB, DMD-108)

Exserohilum rostratum - Conidia attached to conidiophore.
(400X, LPCB, DMD-108)

 Exserohilum rostratum - as above, conidia attached to conidophore seen extending through the camera's plane of focus.  (400X, LPCB, DMD-108)

Exserohilum rostratum - a closer look at the septate conidiophore and the attachment of the conidia to the conidiophore.  Note the bent or zig-zag location on the conidiophore at the point of conidial attachment.  This appearance us referred to as geniculate growth.  (400X, LPCB, Nikon)

Exserohilum rostratum - Pigmented, fusiform shaped conidia, usually containing between7 to 11 internal septa. Note the prominent projection or hilum (arrow) which remains at the point of the attachment to the conidiophore. (400+10X, LPCB, DMD-108)

Exserohilum roatratum - single conidium with hilum visble on right side.  Length reads 76.01
µm. (400+10X, LPCB, DMD-108)

Exserohilum rostratum - another view of the geniculate growth / attachment of the conidia to the conidiophore. (400X, LPCB, DMD-108)

Exserohilum rostratum - Single conidium attached to conidiophore.
(1000X, LPCB, DMD-108)

Exserohilum rostratum - okay, I like photos!  Another loose conidium which is slightly bent.  This one has seven, possibly eight compartments.  The conidium is smooth walled and again, the hilum is clearly visible at one end.  (1000X, LPCB, DMD-108)

Exserohilum rostratum - More photos, just for the beauty of this organism.
(1000X, LPCB, DMD-108)

Exserohilum rostratum

Exserohilum rostatum - geniculate (zig-zag) conidiophore after the conidia have dispersed.
(400X, LPCB, DMD-108)
Exserohilum species may be confused with  Bipolaris and Drechslera species however Exserohilum has the protuberant hilum.

[i] A particular presentation of a fungal infection in tissue caused by certain dematiaceous fungi.  This presentation may be an initial clue as to the particular fungus responsible for the infection.  'Google' the term for a better definition.

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