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Short descriptions and photographs of some photogenic microorganisms.

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  • 11/29/14--19:05: Ascaris lumbricoides


  • Ascaris lumbricoides(Intestinal Nematode)  “Roundworm”

    Geographic Distribution:
    Ascaris lumbricoides can be found throughout the world but is more commonly found in moist temperate and tropical regions.  Ascaris is increasingly prevalent where poor sanitation exists.  In cultures where human excrement (night soil) is used as fertilizer, infection may again be greater.

    Pathogenicity:
    Infection occurs with the ingestion of fully embryonated eggs (ova).  Infection with Ascaris lumbricoides is termed Ascariasis and it affects more of the world’s population than any other parasitic disease.   Ingestion of small numbers of eggs may be asymptomatic for the host however larger numbers may cause Ascaris pneumonitis[i]or Loeffler’s[ii]syndrome.  Those infected with Ascaris lumbricoides may develop asthmatic attacks, shortness of breath, wheezing or a persistent cough.  A heavy infection in the small intestine may result in a bowel obstruction, especially in children.  Symptoms may include fever and generalized malaise possibly with abdominal distension, associated tenderness and possible vomiting.

    Life Cycle:
    Ingested embryonated eggs travel to the duodenum (small intestine) where they hatch and begin an obligatory migration throughout the body before they return to the duodenum to mature into adulthood.  Hatched larvae start the migration by penetrating the duodenum wall to enter the blood or lymphatic system which eventually carries the larvae to the liver and the heart to eventually enter pulmonary circulation.  In the lungs, the larvae break free of the capillaries to enter the aveoli where they continue to grow.  After about 3 weeks, they begin to migrate through respiratory passages to reach the esophagus where they are swallowed and once again reach the small intestine.

    At about two to three months post ingestion of the eggs, the now mature worms begin to lay their own eggs.  It has been estimated that mature female worms may produce an average of 200,000 eggs daily.  Ascaris lumbricoides females are oviparous[iii].  When passed, the eggs require about 2 to 3 weeks outside of the host to develop into the infective embryonated stage.  While the eggs are susceptible to excessive heat and drying, they can remain viable in moist soils for long periods of time.

    Ascaris Worm:
    The adult Ascaris lumbricoides worms area creamy-white in colour, occasionally with a pinkish cast.  Female worms range from 20 to 35 cm in length.  Males are generally shorter and usually do not exceed 30 cm.  Female worms are also generally thicker (3-6 mm) than the more slender males (2-4 mm).  Males can be distinguished from females by their incurved tail.  Females have a straight tail.  Adult worms are believed to live up to a year.  Ascaris worms do not attach themselves to the intestinal wall but rather maintain their position through constant movement.

     Ascaris lumbricoides: Adult male worm show with its distinctive curved tail.  This particular worm was about 24 cm or 10 in in length, (Nikon-Macroscopic)

    Ascaris Eggs (Ova):
    Diagnosis of ascariasis is by the demonstration of the characteristic eggs in the feces.
    Fertile eggsare broadly oval in shape and typically yellow-brown in colour, stained by the bile in freshly passed stools.  They measure 45 to 75 µm in length by 35 to 50 µm in breadth.  The outer albuminoid coat is coarsely mammillated covers a smooth shell which is difficult to distinguish in laboratory preparations.  Some confusion in diagnosis may occur if the Ascarisegg has lost its mammillated coat (decorticated) as it may somewhat resemble Hookworm or Trichostrongylus species ova.   Ascaris eggs contain only one cell when passed in the feces.
    Infertile eggsare elongate, about 85 – 95 µm by 43 – 47 µm in size.  Their mammillated layer may vary from being coarsely irregular to a relatively smooth layer, almost devoid of mammillations. The internal contents of infertile eggs appear as a mass of disorganized, refractile granules.

    Note:  All the following photos are take from the re-suspended sediment after fecal concentration.

     Ascaris lumbricoides egg seen in the center of the photograph.

      Ascaris lumbricoides egg  has a distinctive appearance.  This egg shows the rough looking surface due to the mammillated albuminoid coat.
    (DMD-108, 400X)

     Ascaris lumbricoides: another photo of the egg with its rough mammillated coat.
    (Nikon, 400X)

     Ascaris lumbricoides: a photo of the egg with an alternate focus inserted for comparison.  As the egg is three-dimensional, adjusting the focus can aid in visualizing the egg's structure.
    (Nikon, 400X)

     Ascaris lumbricoides: as above.
    (DMD-108, 400X)

     Ascaris lumbricoides:Two A.lumbricoides eggs seen in the same field.
    (Nikon, 400X)

     Ascaris lumbricoides: Typical appearance of the Ascaris egg.  The presence of these eggs in a fecal specimen is diagnostic for an Ascaris infection.
    (DMD-108, 400+10X)

     Ascaris lumbricoides: The thick albuminoid mammillated layer is quite evident on this fertile Ascaris egg.  (DMD-108, 400+10X)

     Ascaris lumbricoides: As above with a slightly altered focus.
    (DMD-108, 400+10X)


      Ascaris lumbricoides:  Another photo of the Ascaris egg in a fecal concentrate.
    (DMD-108, 400+10X)

     Ascaris lumbricoides: As above but with an alternate focus of this fertile Ascaris egg.  I post these alternate focus photos in an attempt to demonstrate the three-dimensional nature of the egg and their complexity.  (DMD-108, 400+10X)

     Ascaris lumbricoides:  Yet another example, 'cause more is better!.
    (DMD-108, 400+10X)

     Ascaris lumbricoides: At a higher magnification.  The large single cell interior is clearly visible.
    (Nikon, 1000X)

     Ascaris lumbricoides ova:  Okay, more is better, right?  Another photo showing the thick outer albuminoid mammillated layer.  The inner wall often cannot be visualized unless the egg loses this outer layer (decorticated).  The single celled contents are clearly visible.
    (Nikon, 1000X)

     Ascaris lumbricoides  Too many photos?  I have to do something with them!  Another photo of the Ascaris egg however it appears as if the interior cell has divided into four or more individual cells.
    (Nikon, 1000X)

     Ascaris lumbricoides: An Ascaris egg with the inset enlargement of the Ascaris egg.  There appears to have been a number of divisions of the original cell contained within the egg.
    (Nikon, 250X)

    Too many photos?  Just a few more to go..

    While the Ascaris lumbricoides ova can be found in iron-hematoxylin stained preparations, the eggs usually stain so intensely that they appear as silhouettes and very little structural information can be discerned.  They may be missed by an inexperienced eye.  A thinner part of the stained slide may offer the best chance of recognizing and finding these eggs.  The sedimentation concentration wet preparation technique is preferable in visualizing these eggs when present.

    Note: The following photos are taken from Iron-Hematoxylin stained fecal preparations.



      Ascaris lumbricoides:Two Ascaris eggs are seen in this photo which appear as two large dark masses on either side of the picture.  The dark blob in the lower center is just an artifact present in the stool and clearly does not have the outline of the Ascaris egg.
    (Nikon, 250X)

     Ascaris lumbricoides: As with the concentrates, if a good microscopic field is found in the stained preparation, the Ascaris egg exhibits different textures as one focuses through it.
    (Nikon, 1000X)

     Ascaris lumbricoides: Same photo as above but with altered focusing.  The mammillated layer appears as a scalloped edge surrounding the single celled contents of the egg.
    (Nikon, 1000X)

     Ascaris lumbricoides: In most iron-hematoxylin stained preparations the Ascaris ova will be heavily stained and often appear in silhouette with very little structure revealed.
    (Nikon, 1000X)

     Ascaris lumbricoides: As above.  If a thin field can be found, the stained ova may show some detail.
    (Nikon, 1000X)

     Ascaris lumbricoides: Okay, last one.  This egg doesn`t look particularly healthy.  The `scalloped`edge of the mammillated coat is somewhat visible along the upper edge.
    (Nikon, 1000X)

    Diagnostic Problems:
    Both fertile and infertile Ascaris eggs concentrate well however well using sedimentation techniques, however, if the laboratory only uses a flotation method for concentrating, infertile eggs may be missed by this technique.



    [i]Pneumonitis refers to an inflammation of the lungs which can be cause by a variety of agents, from allergic, chemical or an infection.  Ascaris pneumonitis is an inflammation caused by the presence of Ascaris lumbricoides migration through the lungs.
    [ii]Löffler's syndrome or Loeffler's syndrome is a disease in which eosinophils accumulate in the lung in response to a parasitic infection.
    [iii]Oviparous –egg development and maturation occurs outside of the female worm.


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  • 12/13/14--21:01: 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 nanais 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.nanamay 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:
    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.dimunataeggs 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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  • 01/24/15--09:02: Aureobasidium pullans


  • Aureobasidium pullans  (Hyphomycetes) –Black yeasts

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

    Ecology:
    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.

    Pathogenicity:
    A.pullansappears 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 - indivdualconidia 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)

     Physiology:
    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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  • 02/23/15--07:26: Ochroconis species


  • Ochroconis species(Hyphomycete)  Fungus

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

    Pathology:
    Ochroconisspecies 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. 
    Ochroconisspecies 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.  Ochroconisspecies 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:
    Ochroconisproduces 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) 

    Physiology:
    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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  • 07/03/14--19:37: Scopulariopsis brumptii


  • Scopulariopsis brumptii(Mould/Fungus)

    Compare this species to Scopularis brevicaulis.

    Ecology:  A cosmopolitan fungus (found just about everywhere), particularly in soil and in household dust.



    Pathogenicity:Scopulariopsis brumptii is rarely implicated in disease; however, as with many other ‘non-pathogenic’ fungi, they may be considered opportunists and may be increasingly found as the cause of pulmonary (lung) infections in severely immunocompromised patients.  Encountered in the laboratory as a possible contaminant, they must be distinguished from more pathogenic fungi.



    Macroscopic Morphology:  S.brumptii is a moderately rapid grower, usually maturing within about 5 days.  Surface growth may initially be whitish, then darkening from a light grey to a sepia-grey (reddish overtones) or fuscous (darker brown-grey).  One source described the colour as that of cigarette ash.  The grey overtones distinguish it from the cinnamon, tan or buff brown colour of Scopulariopsis brevicaulis.  The texture is described as velvety to powdery.

    Scopulariopsis brumptii - Sabouraud-Dextrose Agar
    (SAB, 12 Days, 30oC, Nikon)



    Microscopic Morphology:  S.brumptii produces septate hyphae that are hyaline when young but darkens as it matures. Conidiogenous (conidia producing) cells develop from the hyphae and can appear singly or in brush-like arrangements.   These conidiogenous cells are more specifically referred to as annellides as they produce conidia (annelloconidia) in succession, each leaving a ring-like collar on the annellide when the annelloconidia is released.   The annellides (5 µm – 10 µm X 2.5 µm to 3.5 µm) are somewhat flask-shaped, with a swollen base which tapers near the apex, however they are generally more cylindrical that those of S.brevicaulis.  The annelloconidia (4.0 µm – 5.25 µm X 3.5 µm – 4.5 µm) in size and although they can be smooth, the texture is usually described as finely roughened.  They are usually pyriform (pear-shaped), dark brown in colour and have a flattened or truncated base where once attached to the annellide.  The annellations (scar/ring) produced by S.brumptii may not be very conspicuous.  Annelloconidia are produced in loose chains from the apices of the annellides.


    Scopulariopsis brumptii - darkly pigmented annelloconidia.
    (KOH, 400X, Nikon)

    Scopulariopsis brumptii -hyaline hyphae become pigmented as are the ellipsoidal annelloconidia.
    (KOH, 1000X, Nikon)

    Scopulariopsis brumptii - conidiogenous cells develop from the hyphae either singly or in brush-like arrangements.  These annellides produce the oval or ellipsoidal, darkly pigmented annelloconidia.
    (KOH, 1000+10X, DMD-108)

     Scopulariopsis brumptii - anannellide is seen in the center of the photo extending from the hyphae.  The "three-fingered" brush-like arrangement at the apex has anelloconidia still attached and some which have been released.  (KOH, 1000+10X, DMD-108)

    Scopulariopsis brumptii-  appear to be two single annellides, with annelloconidia being produced at the apex.  (KOH, 1000X, Nikon)

    Scopulariopsis brumptii -the hyphae is septate and has produced a single annellide, bearing a single annelloconidium, at the center of the photo. (KOH, 1000X, Nikon)

    Scopulariopsis brumptii -two brush-like arrangements of the annellides of S.brumptii are seen here in the center-left of the photo.  Numerous annelloconidia, both attached and free are seen.
    (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii - two bunches of annellides (the right one more brush-like than the left) are seen in the center of the photo.  Pigmented annelloconidia are seen at the apex.
    (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii -More brush-like annellides showing their arrangement and attatchment of annelloconidia.  (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii - another branching annellide, thinned out a bit so the arrangement of the annellides are more easily seen.  The ring or collar is clearly visible on the annelloconidium seen at the lower middle of the photo.  It appears as a scar or flattened base where the annelloconidium was once attached to the annellide.   (LPCB, 1000X, Nikon)

    Scopulariopsis brumptii -okay, too many photos, but what else am I going to do with them?
    Hyphae, annellide & annelloconidia extending from the tips.
    (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii -dittoLots of annelloconidia produced by this brush-like structure.
    (LPCB, 1000+10X, DMD-108)

    Scopulariopsis brumptii -two single annellides seen extending from the hyphae traversing the center of the photo.  Each has an annelloconidium still attached to the apex.  Look closely at the darkly pigmented mature annelloconidia and you will see the collarette (annellide ring) on many.
    (LPCB, 1000+10X, DMD-108)

    Scopulariopsis brumptii -a poorer example of the photo explanation above.  Two single annellides producing annelloconidia.  Many more single annellides are present in S.brumptii cultures than in S.brevicaulis cultures where the arrangements are primarily brush-like.
    (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii -again, as above, single, rather than brush-like arrangements might be seen more frequently in S.brumptii, than in S.brevicaulis.
    (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii -ditto - more single, rather than brush-like annellides may be seen in S.brumptii cultures.  (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii -and a single annellide with an attached annelloconidium seen in the center of the photo.  A single free annelloconidium is seen in the upper left-third of the photo - the annellide ring or collarette visible at the bottom.  (LPCB, 1000+10X, DMD-108)

    Scopulariopsis brumptii - a crowded photo showing at least two brush-like arrangements of annellides within a 'sea' of pigmented annelloconidia.
    (LPCB, 1000X, DMD-108)

    Scopulariopsis brumptii -oh, what the heck! -here's one more.  A brush-like arrangement of annellides and darkly pigmented, oval to ellipsoidal annelloconidia.
    (LPCB, 1000+10X, DMD-108)


    Note: Scopulariopsisspecies may show variable sensitivity to cycloheximide and therefore may or may not grow on Mycosel/Dermasil® agar.

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  • 03/27/15--08:20: Acremonium species


  • Acremonium species -Hypocreaceae Family (obsolete name Cephalosporium spp.)

    Ecology:
    Acremoniumspecies are yet another cosmopolitan fungus (ie. found just about everywhere) which can be isolated from soils as well as decaying plant material.  There are about one hundred recognized species of Acremonium.

    Pathogenicity:
    Acremoniumspecies is associated with ‘white grain mycetoma’[i], an infection most commonly of the foot.  Acremonium has also been implicated in meningitis, endocarditis, endophthalmitis and corneal ulcers.  Onychomycosis or ‘tinea unguium’ (fungal infections of the nail) may also be caused by localized infection with Acremonium.  Disseminated infections are rare and may result from traumatic injury and may be more likely in immunocompromised hosts.

    Macroscopic Morphology:
    Acremonium exhibits moderately rapid growth, rather flat colonies that may be slightly raised in the center.  The colony has been described as glabrous to membrane-like initially, becoming powdery, cottony or even felt-like as it matures.  The colony may be white to cream in appearance or even yellowish to coral or pinkish in colour.  The reverse is pale, yellowish to pinkish in colour.

     Acremonium species -Sabouraud Dextrose Agar (SAB) incubated at 30˚C for 18 days. (Nikon)

      Acremonium species -Sabouraud Dextrose Agar (SAB) incubated at 30˚C for ~21 days. (Nikon)

    Note -On the Challenges of Plate Photography:
    This is the same species as above with a few more days of incubation.  The reason I posted both these photos is to show the difficulty I have in expressing the true colour (and often texture) of the fungus even when grown on the same media and under identical conditions.  The first photo was taken with the plate placed against a white background and the second against a black background.

    The other challenge is lighting.  I take these photos for my own entertainment and education - for "fun" as the title of the blog states.  The acute care lab I work out of is interested only in a quick and accurate identification of fungi in clinical specimens.  Documentation by photography is not a concern and we have no professional set up for taking photographs.  I have rigged up my own apparatus for use as a camera stand for use within a biological safety cabinet (BSC) (see the post entitled 'Toys').  Photography of macroscopic plates is confined to the BSC as the lab could quickly become contaminated with spores if fungal plates were to be examined outside of the BSC.  Many fungi produce vast quantity of spores which can become airborne with only the slightest breeze.

    I have little control over the lighting.  I often take a large number of photos using both white and black backgrounds.  I simply use a sheet of white paper which I have run through a photocopier without a "target".  In other words, I leave the cover of the photocopier open and "copy the air" which results in a sheet of paper covered with back toner on one side but white on the back.  I can flip this paper over to the black or white side as I wish and as the background paper may be contaminated with spores, I can discard it safely when finished.

    I use the fluorescent tube lighting of the BSC itself, I have an incandescent source of lighting I can bring into the cabinet, and I can use the flash unit on the camera itself in order to manipulate lighting.  Light coloured fungi often show more detail when on a dark background and dematiacious fungi (darkly pigmented) stand out better on white backgrounds - but not always!  I try to avoid cast shadows.  I also find that the flash often produces such glare and reflection off of the petrie dish and agar surface that it obscures the true nature of the organism.  By taking a large number of photos on various backgrounds and with various combinations of the lighting I have available, I can usually find a photograph that reflects the true characteristics of the fungus I am attempting to document.

    Microscopic Morphology:
    Acremoniumproduces septate hyphae from which erect, unbranched and tapering phialides extend.  Most phialides (but not necessarily all) have a basal septum which delimits them from the hyphae proper.  Conidia are oblong (2–3 X 4–8 µm) are usually one-celled, however bicellular conidia may occur.  The conidia are produced and accumulate as balls (rarely as chains) at the apices of the phialides but they are fragile and easily disrupted.

    Phialide: A specialized conidiogenous cell (conidiophore) that produces conidia in basipetal succession without increasing in length. (Mycology Online)


     Acremoniumspecies - above is a little piece of agar which adhered to the slide culture microscope cover slip when removed from the agar block (See Post on Slide Cultures).  From this little flake of agar you can see phialide extending outwards with little "balls" of conidia attached at their apices. (LPCB, DMD-108, 250X)

    Acremoniumspecies - a collection of hypha run through the center of the photo from which you can see the phialide bearing conidia at their apices.  This is like "micro-botany" with the fungi as tiny plants, the 'seeds' (conicia) at the tips of the stems.  (LPCB, DMD-108, 400X)

    Acremoniumspecies - As above but at a slightly higher magnification.
    (LPCB, DMD-108, 400+10X)

    Acremoniumspecies - the individual conidia can now be seen, gathered at the tapering tips of the phialides.  (LPCB, DMD-108, 1000X)

    Acremoniumspecies - not the greatest shot but try to picture the three 'balls' of conidia siting at the top of the phialides which are extending upwards, towards you, the viewer.  The phialides are blurred (out of focus) as they are out of the focal plane of the camera, lying beneath the balls of conidia, where they attach to the hyphae.  (LPCB, DMD-108, 1000X)

    Acremoniumspecies - The same as above but viewed more from the side.  The originating hyphae and tapering phialides are slightly out of the focal plane of the camera.  The conidia that were produced at the apex of the phialide remains undisturbed as a ball around the top.
    (LPCB, DMD-108, 1000_10X)

    Acremoniumspecies - The previous photos show the phialides and conidia almost in three-dimensions.  In the photos that follow, they are lying fairly flat, making it easier to see the features.
    (LPCB, Nikon, 400X)

    Acremoniumspecies - in the center of the photograph, there are two phialides, both with a collection of conidia at their apices, where they remain after being produced.
    (LPCB, DMD-108, 1000X)

    Acremoniumspecies - another view of the tapering phialides extending from their parent hyphae where elongated, ellipsoidal conidia lay gathered.
    (LPCB, DMD-108, 1000X)

    Acremoniumspecies - pretty much the same as in the above photo.  The phialide at the very top of the photo appears to have produced only one conidium.  Perhaps it is younger than the others.
    (LPCB, DMD-108, 1000X)

    Acremoniumspecies - another photo of tapering phialides and the ellipsoidal conidia gathered around the apices.  (LPCB, DMD-108, 1000X)

    Acremoniumspecies - insert -one tapering phialide with individual conidia gathered at the apex.
    (LPCB, DMD-108, 1000X)

    Acremoniumspecies - Two phialides, side by side producing large quantities of conidia.
    (LPCB, DMD-108, 1000+10X)

    Acremoniumspecies - tapering phialide with single ellipsoidal conidia at the apex.  A slight collarette can be seen remaining around the apex.  Conida already produced almost seem to have fallen down and accumulated around the base of the phialide and hypha.
    (LPCB, DMD-108, 1000+10X)

    Acremoniumspecies - as previously -another view of the tapering phialides and the balls of conidia adhering to the apex of the phialides where they were produced.
    (LPCB, DMD-108, 1000+10X)

    Acremoniumspecies - As previously.
    (LPCB, DMD-108, 1000X)

    Acremoniumspecies - a few barren phialides can be seen and the large mass of dispersed, primarily single celled conidia seen throughout.
    (LPCB, DMD-108, 1000X)

    Acremoniumspecies may be confused with Verticilliumand some isolates of Fusarium where macroconidia are not present.  Rate of growth and colony morphology differs from that of Acremonium species and may provide initial clues for differentiation.


    [i]Mycetoma, or maduromycosis, is a slow-growingbacterial or fungalinfectionfocusedin onearea of thebody,usuallythefoot.  Approximatelyonemonth or moreaftertheinjury,a painless noduleformsundertheskinsurface. The nodules develop into a tumor which produces sinuses to drain fluid. Thefluidcontainstinygrains, which may be a clue as to the type of organism is causing the infection.


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  • 04/16/15--08:12: Exophiala jeanselmei



  • Exophiala jeanselmei-Mitosporic fungi; Hyphomycetes

    Note:  With the advent of molecular testing, some sources are stating that Exophiala jeanselmei should more correctly be referred to as the Exophiala jeanselmei complex.  See explanation at the bottom of this post.

    Also: Compare Exophiala jeanselmei with Exophiala dermatitidis elsewhere in this blog by clicking here.

    Ecology:
    Exophiala species can be found worldwide and may be isolated from soil, decaying wood and fresh water sources. 

    Pathogenicity:
    Exophiala jeanselmei may be an agent of mycetoma[i]and phaeohyphomycosis[ii]has also been implicated as an agent of back grain mycetoma and chromoblastomycosis[iii].  While occurring worldwide, some evidence suggests that infections, particularly neurotropic infections, may be more prevalent in East Asia.

    Macroscopic Morphology:
    Colonies are initially moist and yeast-like in texture; however they soon form a velvety surface due to the production of aerial mycelia.  Some isolated can remain yeast-like without further development.
    The colony can be brownish-black to greenish-black with the reverse being black in colour.
    Exophiala jeanselmei grows slowly, maturing within about 14 days at 25ᵒC to 30ᵒC.

    Exophiala jeanselmei on SAB after incubation for 3 weeks at 30ᵒC.
    While this isolate matured rather quickly, compared to the "up to 14 days" stated by some sources, the colony expanded outward slowly. (Nikon)

     Exophiala jeanselmei on SAB after incubation for ~3 weeks at 30ᵒC.
    Heaped up, folded appearance.  (Nikon)
     
    Microscopic Morphology:
    Young cultures with budding yeast-like cells may eventually develop septate hyphae.  Hypha, initially hyaline (clear) become pale brown to olivaceous as the colony ages.  The conidiogenous annellophores (structure producing annelloconidia) are slender, sometimes branched, and narrow towards the apex (tip).  The smooth annelloconidia (2.5 – 6.0 µm X 1.2 – 2.5 µm) are oval or ellipsoidal in shape and if undisturbed, accumulate in clusters around the tip and upper sides of the annellide.  Potato Dextrose Agar (PDA) or Corn Meal Agar (CMA) may enhance conidia production, however all the photos on this post were taken from isolates grown on Sabouraud Dextrose Agar (SAB) with copious conidia produced.

    Exophiala jeanselmei - mycelial growth adhering to small piece of agar from a slide culture after 4 days of growth.  (250X, LPCB, DMD-108)

    Exophiala jeanselmei - small clusters or balls of conidia become evident, dispersed along the mycelia at this magnification. (250X, LPCB, Nikon)

    Exophiala jeanselmei -Individual conida can now be made out at this magnification.
    (400X, LPCB, DMD-108)

    Exophiala jeanselmei -septate hyphae appear as if vacuolated.  Conidiogenous structures (annellophores) do not appear to be highly differentiated from the hyphae themselves and can be intercalary (along the hyphae) or terminal (at the end of the hyphae).
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -as above, with typical accumulation of the oval to ellipsoidal annelloconidia around the top, and along the sides of the annellophore.
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -again, intercalary, and terminal production of annelloconidia.
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -and another.
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei -Both clusters of conidia and individual conidia can be seen at the tips of the annellophores.  (1000, LPCB, DMD-108)

    Exophiala jeanselmei -Branched annellophore with (annello)conidia.
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei - and another...
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei - ditto, as above.
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei -branched conidiophore at various stages of conidia production.
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei -okay, too many photos of the same structures!!
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei -the annellophore appears undifferentiated from the hyphae from which it arises.  Nice photo showing the annelloconidia accumulated around the apex of the annellophore from which they were produced.  Conidia are single-celled with the darkly staining band across the center of the conidia not being a septum. Also visible on many of the conidia is the remnants of a fringe, the scar that remains from where they were attached to the conidiophore.  This represents a specific method of reproduction and fringe or scar (annellide) is why we specify the conidiophore as an annellophore, and the conidia as an annelloconidia.  (1000+10X, LPCB, DMD-108)
     
    Exophiala jeanselmei -as the colony matures, the hypha become pigmented which gives the colony its distinctive colour. (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -as above, just another view,
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -an interesting shot showing the annellophore rising upwards from it's parent hyphae with the annelloconidia clustered about the apex.  Almost three-dimensional.
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -copious amounts of conidia are produced.
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -as previously stated, the darkly staining band across the center of the annelloconidium is not a septum.  The single-celled annelloconidia have the fringe or scar which remains at one end of the cell which is why the conidia are specifically referred to as annelloconidia.
    (1000+10X, LPCB, DMD-108)

    Exophiala jeanselmei -conidium germinating.
    (1000X, LPCB, DMD-108)

    Exophiala jeanselmei -annelloconidia (1000+10X, LPCB, DMD-108)



    [i] Mycetoma is a chronic, progressively destructive morbid inflammatory disease usually of the foot but any part of the body can be affected. Infection is most probably acquired by traumatic inoculation of certain fungi or ‎bacteria into the subcutaneous tissue. (WHO)

    [ii] Phaeohyphomycosis is a heterogeneous group of mycotic infection caused by dematiaceous fungi whose morphologic characteristics in tissue include hyphae, yeast-like cells, or a combination of these. (Wikipedia)

    [iii] Chromoblastomycosis is a chronic fungal infection of the skin and the subcutaneous tissue caused by traumatic inoculation of a specific group of dematiaceous fungi. (Medscape)

    Note:  As with most fungi, speciation of the genus Exophiala is undergoing change brought about by molecular analysis.  Some sources are now referring to the ‘Exophiala jeanselmei complex’.
    Isolates from the United States that in the past had been identified as E. jeanselmei based on morphologic and physiologic characteristic have been shown by molecular methods to be mostly two newly named species closely related to E.jeanslemei, i.e. Exophiala oligosperma and Exophiala xenobiotica.  A lower percentage were found to belong to several other species of Exophiala.  The precise species in the complex can only be determined by molecular testing.


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    Sporothrix schenckii Complex –Hyphomycetes  (Dimorphic Fungus)

    Note1:I first added a post entitled Sporothrix schenckii back in November of 2008 while bedbound, recovering from a serious injury.  I had just discovering ‘blogging’ and toyed with the idea of posting a few film photos that I had tucked away in a drawer – just for the fun of it.  The poor quality photo demanded it be upgraded once I decided to keep up my blog ‘Fun with Microbiology’.  Finally, here is the upgraded blog post, perhaps more accurately entitled Sporothrix schenckii complex Revisited.

    Note2: In recent years, gene sequencing studies have revealed that the species previously known as Sporothrix schenckii is actually composed of several species.  The currently accepted species names of the species which comprise the complex are as follows:  S.albicans (formerly Sporothrix pallida), S.brasiliensis, S.globosa, S.luriei, S.mexicana, and S.schenckii , ‘sensu strictu’.  As many clinical laboratories may not have routine access to molecular technology for specific speciation, this blog simply describes Sporothrix schenckii complex with macroscopic, microscopic and physiological features found in most current textbooks.

    Ecology:
    Sporothrix is a cosmopolitan (found just about everywhere) fungus which is commonly isolated from soil and decomposing plant matter.  Peat moss is a particularly well known source of Sporothrix.  It may also be found on living plants such as rose bushes leading to what has been termed ‘rose handler’s disease’ where the fungus gains entry to the host through thorny pricks.


    Pathogenicity:
    Sporothrix schenckii is the agent responsible for sporotrichosis, a chronic infection that most frequently begins as a skin puncture with introduction of the fungus into the subcutaneous tissues.  Eventually it will involve the lymph nodes and lymphatic channels that drain the infected area.  Implantation of the fungus is usually by puncture by items contaminated with plant material harbouring the fungus such as wood splinters, sphagnum moss, hay or thorns as previously mentioned.  Pulmonary (respiratory) infection may also develop in predisposed individuals after inhaling fungal spores.  Rare cases of disseminated Sporothrix infection with a fatal outcome have been reported.
    Laboratory acquired infections have also been reported.

    Sporothrix schenckii is a thermally dimorphic fungus, meaning it can take on one of two forms depending on the temperature it finds itself in.

    ·         At 25ᵒC exhibits the mould (filamentous) form, with a glabrous, moist texture.  Initially white or cream coloured, the colony may acquire a black colour with aging
    ·         At 37ᵒC exhibits a yeast-like form, with a creamy texture, cream to beige in colour.

    Macroscopic Morphology:
    The mould or filamentous phase in greater detail:
    Growth is described by most sources s moderately rapid to rapid, with the colony becoming mature within 7 days.
    The filamentous phase grown on SAB or PDA is generally cream coloured with some sources describing orange to orange-grey colouration.  As the fungus matures, a salt & peppery brown or black colour develops with the colony retaining a narrow whitish border.  Isolates may vary in their colour, some being dark/black from initial growth.  Stock cultures kept for long periods may lose their dark colour completely.
    The reverse of darkly pigmented colonies is usually dark in the center with a progressively lighter periphery.
    Sporothrixinitially has a moist appearance but becomes wrinkled and leather to velvety in texture as it ages.

    Sporothrix schenckii - SAB, 30ᵒC, 3 weeks incubation (Nikon)

    The Yeast Phase:
    The yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures).  At 37ᵒC, Sporothrix exhibits a yeast-like form, with a creamy texture, cream to beige in colour which also may darken with aging.
    (Yeast Phase Photo further below)

     

    Microscopic Morphology:
    Fillamentous Mould Phase:
    Sporothrixproduces narrow (1 – 2 µm dia.) hyaline, septate and branching hyphae.
    Sporothrixproduces two types of conidia:
    ·         Slender, tapering conidiophores arise at right angles from undifferentiated hyphae.   Hyaline conidia are produced at a small swelling at the conidiophores apex by sympodial growth resulting in a “rosette-like” appearance.  These conidia (2 – 3 X 3 – 6 µm) are tear-drop shaped to round in appearance and unless disturbed, remain attached to the conidiophore in young cultures via thread-like denticles. (Rosette)
    ·         Single thick-walled brown to black (dematiaceous) sessile conidia (2 – 4 µm dia.) can also be present, arising directly from the hyphae.

     Sporothrix schenckii -Initial look at the growth attached to a cover slip from a slide culture.
    (100X, LPCB, DMD-108)

    Sporothrix schenckii - A closer look where detail begins to emerge.
    (400X, LPCB, DMD-108)

    Sporothrix schenckii - ditto
    (400X, LPCB, DMD-108)

    Sporothrix schenckii - At yet a higher magnification, fine structures emerge. Conidia are easily seen at the tips of conidiophores.  At the center of this photo one such structure is seen in a 'rosette' arrangement, typical for this fungus.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii - hyphae bearing conidiophores with conidia being produced sympodially at the apex.  (1000X, LPCB, DMD-108)

    Sporothrix schenckii - appearing somewhat in 3-D, the hypha is seen running across the photo with conidiophores extending at right angles from the hypha.  Conidia formation is is seen at different stages with the conidiophore at the right having accumulated numerous conidia.
    (1000X, LPCB. DMD-108)

    Sporothrix schenckii - again in "3-D" perspective, you can see a number of hyphae below, and out of the plane of focus of the camera, bearing conidiophores which appear to be rising upwards, towards the camera/viewer.  Conidia are present at the apex.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii -septate hyphae are show and two well defined conidiophores are shown with a 'rosette' of conidia at the apex.
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii -another look at the conidiophores with conidia accumulated at the apex (tips).  Fine thread-like denticles can be seen attaching the ellipsoid or tear drop shaped conidia to the conidiophore.
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii -yet another view, as above.
    (1000+10X. LPCB, DMD-108)

    Sporothrix schenckii -what is meant as a "rosette" configuration of conidia.  This is typical of Sporothrix schenckii and assists in its identification.
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii - I particularly like this photo.  Like a shower of delicate flowers.  What is seen are the conidia rosettes at the tips of unseen conidiophores and hyphae, below the plane of focus.  (1000X, LPCB, DMD-108)

    Sporothrix schenckii -a single hypha running through the photo, bearing conidia in typical rosette pattern.  (1000X, LPCB, DMD-108)

    Sporothrix schenckii -single, sessile and begining to show dark pigment (dematiaceous), are seen along the hypae.  (1000X, LPCB, DMD-108)

    Sporothrix schenckii -ditto.  Black pigment much more pronounced in this photo.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii - again, as above.
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii - just another photo...
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii -Sessile (attached directly to hypha) with many developing a dark pigmentation (dematiaceous) which gives the macroscopic colony its distinctive colour.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii - dematiaceous cells are more abundantly produced as the colony ages.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii -sessile dematiaceous conidia are seen lining a hypha running through the photo, as well as some conidiophores bearing the tear-drop shaped conidia.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii -another view of the sessile, dematiaceous conidia lining the hypha with a few conidiophores extending from the hypha showing the delicate rosette arrangement at the tips.  (1000X, LPCB, DMD-108)

    Sporothrix schenckii -and just another photo showing what was described in the last few photos.
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii -a nice rosette formation in the center of the photo.
    (1000+10X, LPCB, DMD-108)

    The Yeast Phase:

    As previously mentioned, the yeast phase is best induced by growing the fungus on Brain-Heart Infusion (BHI) agar at 37ᵒC, and observing after several generations (subcultures).  Yeast cells are round to ovoid of varying size (1 – 3 X 3 – 10 µm), producing single or multiple buds often resembling rabbit ears or ‘Mickey Mouse’ ears extending from the primary yeast cell.

     Sporothrix schenckii -the very same organism added to the nutritionally rich Brain-Heart Infusion (BHI) agar and incubated at 37ᵒC for ten days.  A typical pasty, yeast form develops which is why this organism is 'dimorphic'. (Nikon)

    Sporothrix schenckii -yeast cells seen in a suspension in Lactophenol Cotton Blue.
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii - Sporothrix in the yeast phase may show single yeas like cells with some of the population budding with typical "rabbit ears" or "Mickey Mouse" ears.  They appear as two elongated cells projecting from the same surface of the parent cell.  One can be seen here, one-third in from the center left edge of the photo.  (1000X, LPCB, DMD-108)

    Sporothrix schenckii -"Mickey Mouse' ear configuration in the cell at the upper center.
    (1000X, LPCB, Nikon)

    Sporothrix schenckii -again, yeast cells with two attached daughter cells on the same side giving the apearance of "rabbit ears" or "Mickey Mouse" ears.
    (1000+10X, LPCB, DMD-108)

    Sporothrix schenckii -ditto (as above) -inset -floppy 'rabbit ears"
    (1000X, LPCB, DMD-108)

    Sporothrix schenckii - that's it, I'm done!
    (1000+10X, LPCB, DMD-108)


    Physiology:
    Sporothrix is resistant to cycloheximide; however growth is inhibited at temperatures of 39 - 40ᵒC.

    *   *   *

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    Apophysomyces elegans complex(Zygomycetes) Order Mucorales

    Note:As of this blog post, most textbooks still list Apophysomyces elegans as the sole species in this genus.  Recent studies [i]have demonstrated a high variability among the 5.8S rRNA gene sequences of clinical strains of A.elegans.  This study suggests that A.elegans is actually a complex composed of several (three) newly proposed species.  They have proposed the species names A.ossiformis, characterized by bone-shaped sporangiospores, A.trapeziformis, with trapezoid-shaped sporangiospores, and A.variabilis with variable-shaped sporangiospores.  A.elegans remains as the fourth species of the Apophysomyces complex.  Physiologically, A.elegans is able to assimilate the glycoside esculin, whereas the three newly proposed species failed to assimilate esculin.  Recent studies suggest that A.elegans may actually be a non-pathogenic environmental species, while the remaining three species are etiologically linked to clinical disease.  Further studies are warranted.

    Ecology:
    Although the ecology of Apophysomyces is rather poorly described, this fungus appears to have widespread distribution and has been isolated from soils and decaying vegetation in India, Australia, Southeast Asia and the United States.

    Pathology:
    Apophysomyces is an occasional agent of Zygomycosis.  Infection by Apophysomyces species differs from other Zygomycetes infections in two ways.  Firstly, it occurs more frequently in immunocompetent than immunocompromised individuals, whereas other Zygomycetes primarily infect hosts with weakened immune systems.  Secondly, Apophysomyces infections are acquired directly by traumatic implantation rather than by inhalation of spores which then progress to rhino-cerebral or other disseminated infections.

    Apophysomycesinfection may result in necrotizing cellulitis or fasciitis (flesh eating) requiring aggressive surgical debridement and antifungal therapy.  Despite immediate and aggressive intervention, the prognosis is often poor as once established the fungus can quickly spread to adjacent tissues and distant sites via the blood stream. 

    In 2011, a tornado carved a path of destruction through the American town of Joplin Missouri killing 160 citizens.  Many others were received traumatic injuries and of those, there were there were thirteen cases of necrotizing cutaneous zygomycosis due to Apophysomyces trapeziformis, five of which were fatal.

    While the descriptions of Apophysomyces in this blog is gathered from a variety of sources under the name of Apophysomyces elegans, the species presented photographically in this blog post is Apophysomyces variabilis.

    Macroscopic Morphology:
    As with other Zygomycetes, Apophysomyces exhibits very rapid growth, often filling the petrie dish with profusely woolly mycelia in two to three days.  Growth (SAB, 30ᵒC) initially appears white or off-white in colour but may acquire a slight brownish-grey colour as the colony ages.  The reverse is white to pale yellow.

     Apophomyces variabilis - on SAB, 48 Hours at 30ᵒC (Nikon)

     Apophomyces variabilis - on SAB, 48 Hours at 30ᵒC (Nikon)

    Microscopic Morphology:
    Note: The isolate presented here arrived in our laboratory as a proficiency testing challenge.  An initial direct tease mount revealed very broad, aseptate hyphae, suggesting Zygomycetes.  The isolate grew exceedingly well (SAB, 30ᵒC) yet failed to produce any fruiting structures.  The combined observations of broad aseptate hyphae and failure to produce fruiting structures on routine mycological media raised suspicions that this isolate belonged to either an Apophysomycesspecies, or was Sakseneavasiformis.

    Both Apophysomyces species and Saksenea vasiformisare known to be notoriously resistant to efforts attempting to induce sporulation.  Study of the structures associated with sporulation greatly assists the identification of the Zygomycetes and as such, techniques have been developed in an attempt to induce the spore production[ii].   Unfortunately, our laboratory supports an acute care hospital and we lack the facilities for any experimentation outside of the routine clinical protocol.  (ie. purchased prepared media: no autoclave, media components, etc.)  However this blog is entitled “Fun With Microbiology” and I had “fun” improvising the ‘sterile water and yeast extract sporulation media’ outlined in endnote ii with some success.

    Microscopic Morphology Continued:
    Apophysomyces species produce broad based (up to 10+ µm dia.) hyaline (clear, non-pigmented) hyphae which are almost entirely aseptate.  Sporangiophores are quite long (up to 540 µm), unbranched and usually produced singly from aerial hyphae.  Sporangiophores may be attached to the hyphae with a prominent structure resembling the ‘foot cell’ found in Aspergillus species.  The apex of the sporangiophore widens to form a structure called an apophysis (hence the genus name) which may be distinctly bell-shaped or vase-shaped (20 -58 µm dia.) distinguishing it from that of other Zygomycetes.  Yet other sources describe the apophysis as champagne glass shaped or perhaps more common to other Zygomycetes, funnel-shaped.  The columella (18 – 28 µm) is hemispherical (half of a sphere) in shape.  Sporangiospores (5.4 – 8.0 X 4.0 – 5.7 µm) have been described as smooth and subspherical to cylindrical in shape.  Recall from the initial ‘Note’ at the start of this blog, the newly proposed species are molecularly distinct but also appear to produce distinctly shaped sporangiospores.  Rhizoids are produced and are generally located beneath or to the side of the sporangiophores.

    An apology: as I lacked the facilities to properly induce sporulation in this fungus, these photos are as good as I'm going to get.

     Apophomyces variabilis - edge of tease mount showing single, well defined sporangiophore with attached sporangium.(250X, LPCB, Nikon)

     Apophomyces variabilis -direct tease moount.
    (400X, LPCB, Nikon)

    Apophomyces variabilis - apophysis without sporangium visible.  Several sporangiospores still visible in the bottom of the rather funnel-shaped apophysis.
    (400X, LPCB, Nikon)

    Apophomyces variabilis -again, from the direct tease mount showing a rather damaged apophysis remaining at the apex of the sporangiophore.
    (400X, LPCB, Nikon)

    Apophomyces variabilis - young sporangiophore at the tip of the sporangiophore.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -as above but appears to be some development of the sporangiospores within the sporangium.  (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -maturing sporangium with sporangiospores visible within.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -much the same.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis - produces long, un-branched sporangiophores.  One unique characteristic described in Apophysomyces elegans is a dark area (arrow) often found on the sporangiophore slightly below the apophysis.  (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -two sporangiophores.  The one on the bottom might be described as champagne flute-like in shape and there is at least one sporangiospore present within.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -probably an immature sporangiospore.
    (1000X, LPCB, DMD-108)

    Apophomyces variabilis -two columellas (?) with the one at the top of the photo showing sporangiospores still clinging to the surface.  The sporangium appears to have

    dehisced (dissolved, split open).  (1000+10X, LPCB, DMD-108)

    Apophomyces variabilis -looking down on a sporagium with a number of sporangiospores still attached to the surface.  Average size of the sporangiospores along the longer axis averaged 5.22 µm.
    (1000+10X, LPCB, DMD-108) 

    Apophomyces variabilis -an immature sporangium.
    (1000+10X, LPCB, DMD-108)

    Apophomyces variabilis -more of the same.  The large width of the zygomycete hyphae is evident here as a hypha runs through the photo.  (1000+10X, LPCB, DMD-108)

    Apophomyces variabilis -again, the broad hyphae of zygomyces in general.  The dimension reads 10.74 µm.  (400X, LPCB, DMD-108)

    Apophomyces variabilis -the apophysis looks distinctly bell-shaped in this microphotograph.  Sporangiospores appear to be present within the developing sporangium.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -to try to convince you of the bell-shaped apophysis.  Rather than a funnel or cone shape where each wall runs on one continuous angle, the walls here start on one angle, the abruptly flare out at a greater angle, somwhat resembling a bell.
    (400X, LPCB, DMD-108)

    Apophomyces variabilis -a rather shorter sporangiophore with a rhizoid seen at the top of the photo and apophysis and broken sporangium spewing sporangiospores at the bottom of the photo.
    (400X, LPCB, DMD-108)

    Apophomyces variabilis -dissolved or broken sporangium seen at center-right of photo, again releasing sporangiospores.  Large bubble at lower left of picture spoils this photo.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -champagne flute-like apophysis with few sporangiospores still clinging to the interior.  (1000+10X, LPCB, DMD-108)

    Apophomyces variabilis -sporangium is missing with the apophysis remaining at the apex of the sporangiophore.  Not at the base of the sporangiophore (right side), the foot-cell is clearly evident and has picked up an intense blue colour from the LPCB.
    (400+10X, LPCB, DMD-108)

    Apophomyces variabilis -foot-cell of Apophysomyces which resembles the same feature more commonly found in Aspergillus species.
    (1000X, LPCB, DMD-108)

    Apophomyces variabilis -a sporangium breaking apart to release the sporangiospores within.  They really do look variable in shape in this photo, true to the species name, 'variabilis'.
    (1000+10X, LPCB, DMD-108)

    Apophomyces variabilis -rhizoid, appears to have wrapped itself around a hyphal element.
    (400X, LPCB, DMD-108)

    Apophomyces variabilis -small rhizoid at center.
    (400X, LPCB, DMD-108)

    Apophomyces variabilis -okay, now that's a rhizoid! 
    (1000X, LPCB, DMD-108)

    Zygomycetes - some of the structures mentioned here and common to most zygomycetes



    Physiology:
    Apophysomyces, unlike other Zygomycetes, exhibits resistance to cycloheximide and therefore should grow on Mycosel® and Dermasel ® media.

    Apophysomycesgrows well at 30ᵒC, 37ᵒC & 42ᵒC


    Eduardo Alvearez, Alberto M. Stchigel, Josep Cano, Deanna A. Sutton, Annette W. Fothergill, Jagdish Chander, Valentina Salas, Michael G. Rinaldi and Josep Guarro
    Rev. Iberoam Microbiol., 2010 27(2) pg. 80 - 89 (for purchase)
    Arvind A. Padhye and Libero Ajello
    Journ. Clin. Microbio., Sept. 1988, pg. 1861 – 1863.   (free PDF)                                                                 

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  • 06/14/15--13:04: Sepedonium species


  • Sepedonium species

    Ecology:
    Sepedoniumspecies are cosmopolitan saprobes commonly found in temperate soils worldwide.

    Pathogenicity:
    Sepedonium species are considered to be of low virulence with only three possible cases cited in the literature.  No animal infections are known. When isolated, they are generally considered to be a contaminant.  As with any fungus, they may be opportunistic particularly if the host is immunocompromised.
    They are a known parasite of higher basidiomycetes, particularly Bolotales (mushrooms).

    Macroscopic Morphology:
    Sepedoniumexhibits rapid growth at 25-30ᵒC.  Colonies initially appear white or creamy in colour with a rather waxy texture.  As the colony matures it acquires a cottony to woolly texture and develops a golden yellow colour.  A white to cream coloured fringe may remain.  The reverse is frequently described as white or pale, however the isolate presented here has a bright yellow reverse.

     Sepedonium species on Saboraud-Dextrose agar (SAB) incubated at 30ᵒC for 5 days. (Nikon)

     Sepedonium species on Saboraud-Dextrose agar (SAB) incubated at 30ᵒC for 10 days. (Nikon)

    Microscopic Morphology:
    Hyaline hyphae are septate and may bear simple or branched conidiophores.  They show minimal differentiation from the hyphae from which they arise.  Immediately distinguishing features are the rather large (7 – 17 µm) conidia produced at the apex of the conidiophore.  Conidia are solitary, single-celled, globose to ovoid in shape and have a rather thick, often echinulate or verrucose (roughened) wall.  A phialidic conidial (phialoconidia) state that produces ellipsoidal or cylindrical, smooth walled conidia may sometimes be present, particularly when the colony is young.

    Notes on Photography:
    Sepedonium offered a few photographic challenges with the techniques and photographic equipment I was using.  
    Slide vs Adhesive Tape Technique:  Firstly, I like to take photographs using the slide culture technique as the glass cover slip, if possible, as it offers better transparency than does adhesive tape technique.  The adhesive tape, unless attached to the glass microscope slide smoothly may give an uneven, wavy picture.  The glue surface itself by be visible and deter from the quality of the photo.  The again, on some moulds, the sticky tape surface preserves the delicate arrangement and keeps in place structures which might be dispersed when manipulating the cover slip from a slide culture.  In the case of Sepedonium species, I found that this fungus adhered to the cover slip rather poorly.  The photos below are from both slide and adhesive tape preparations.  I have not stated which as it is the structure which is important.
    Choice of camera: I find that both the DMD-108 digital imaging microscope and the Nikon Coolpix camera have advantages and disadvantages when photographing microorganisms.  In the case of moulds, the DMD-108 sometimes tends to over-saturate the blue from the Lacto-phenol Cotton Blue (LPCB) dye and I have not been able to correct for this to my satisfaction at either the instrument itself or with any of several photo-manipulation computer programs.  Structures (conidia below) may appear very dark and blotchy with details obliterated.  The Nikon, paired with the Leica 2000 microscope I routinely use should produce excellent photos, however, even after proper maintenance by a professional service, I often find the quality lacking.  What I find most distracting is the spherical aberration which appears as concentric light and dark lines running through the photograph.  While these rings or ripples running through the image are not seen through the oculars, they are blatantly visible in the digital image, particularly at higher magnifications.  A frosted filter between the light source and condenser minimizes this distracting `bulls-eye`pattern, however it is still present.  It seems to me that the camera lens magnifies any defects in the microscope lens or that somehow they conflict even after Köhler illumination.

    Here, as throughout this blog, I use whichever technique (or combination of) most efficiently captures the organisms characteristics.

      Sepedonium species -edge of a slide culture at low magnification.  Hyphae and conidia are visible but too small to show any useful detail for identification.  (250X, LPCB, DMD-108)

    Sepedonium species -Large echinulate or verrucose (rough-walled) conida very apparent. 
    (400X, LPCB, DMD-108)

    Sepedonium species - another view, as above.
    (400X, LPCB, DMD-108)

    Sepedonium species - a phialidic conidial state which produces ellipsoidal or cylindrical, smooth walled conidia (phialoconidia) may sometimes be present, particularly when the colony is young.  These forms are visible at the center of the photo extending to the left with the phialoconidia at the tips.  (400X, LPCB, Nikon)

    Sepedonium species - loose phialoconidia are seen throughout the photo as well as two large rough-walled conidia seen near upper center.  (400X, LPCB, DMD-108)

    Sepedonium species - single oval or ellipsoidal phialoconidium seen at the end of a conidiophore, with a few loose phialoconidia at upper center.  (1000X, LPCB, DMD-108)

    Sepedonium species -and another view of the phialoconidia in the center of the photo and the relationship in size to the rough conidium seen at the center-left of the photo.
    (1000X, LPCB, DMD-108)

    Sepedonium species -another view, as above.
    (1000X, LPCB, DMD-108)

    Sepedonium species - large, young, still rather smooth,  conidium at the apex of the conidiophore.  Two free phialoconida seen at upper left.  (1000X, LPCB. DMD-108)

    Sepedonium species - two large roughly textured conidia seen with their conidiophores receding out of the focal range of the camera.  The conidia have a rough surface texture which is described by various texts as warty, tuberculate, echinulate or verrucose.  It is this feature which may initially confuse this non-virulent mould with the highly virulent Histoplasma capsulatum.
    (1000X, LPCB, DMD-108)

    Sepedonium species -large, still rather smooth, conidium seen at the end of its conidiophore.  The thick wall of the conidia can be readily seen in this photo.
      (1000X, LPCB, DMD-108)

    Sepedonium species - long conidiophore with a large, rough-walled conidium at the apex.
    (400+10X, LPCB, DMD-108)

    Sepedonium species - single rough-walled conidium seen in center of photo.
    (400+10X, LPCB, DMD-108)

    Sepedonium species - conidia attached to conidiophores of varying length.  Insert is just an alternate focus of conidiophore & conidium.  Distinctive, septate hyphae seen.
    (400+10X, LPCB, DMD-108)

    Sepedonium species - large (7 – 17 µm), rough-walled conidium seen.
    (1000X, LPCB, DMD-108)

    Sepedonium species - a number of large, rough-walled conidia taken from a mature colony.  This is from an adhesive tape mount.  (1000X, LPCB, DMD-108)

     Sepedonium species - numerous rough-walled conidia.
    (400+10X, LPCB, DMD-108)

    Sepedonium species - as above.  Conidia are in different planes of focus and therefore the rough texture often appears different on different conidia.
    (1000X, LPCB, DMD-108)

    Sepedonium species - large conidia still attached to their conidiophores.  Rough, tuberculate surface evident.
      (1000+10X, LPCB, DMD-108)

    Sepedonium species - perhaps the most dramatic of the photos showing the rough & thick-walled conidia.
      (1000+10X, LPCB, DMD-108)

    Sepedonium species - suitable for framing.
    (1000+10X, LPCB, DMD-108)

    Sepedonium species - rough & thick-walle conidium attached to what appears to be a short conidiophore.
      (1000+10X, LPCB, DMD-108)

    Sepedonium species
    (1000X, LPCB, DMD-108)

     Sepedonium species - conidium still attached to a segment of it`s conidiphore.
    (Cropped photo, LPCB, Nikon)
    Notes:
    One source states that Sepedonium may grow poorly if at all at 37ᵒC.  Caution: not a definitive test.

    Differentiation from other moulds:
    Sepedoniumsuperficially resembles the highly pathogenic mould Histoplasma capsulatum.
    ·         Growth rate – Sepedonium exhibits rapid growth at 25 - 30ᵒC and may be fully mature after one week while Histoplasma capsulatum grows slowly and may be barely visible after a week’s incubation
    ·         Sepedoniumspecies are inhibited by cycloheximide and therefore will not grow on Mycosel® or Dermasel® selective media.
    ·         Sepedoniumis not a dimorphic fungus (yeast & mould phase) and will not convert to a yeast phase at 37ᵒC as would Histoplasma.
    ·         Histoplasma capsulatum –specific GenProbe molecular testing.  Sepedonium negative.
    ·         Sepedonium species can be distinguished from Chrysosporiumspecies by its conidia’s tuberculate* (echinulate or verrucose) cell wall.
      
      *all terms are descriptions of the rough cell wall.

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  • 07/04/15--09:47: Pithomyces species



  • Pithomyces species  -Hyphomycete

    Ecology:
    The genus Pithomyces has approximately 50 recognized species to date.   Speciation is most accurately achieved by molecular means; however, careful observation of morphological features can identify this mould to the genus level.
    Pithomyces species are dematiaceous saprobes (darkly pigmented moulds which commonly grow on dead organic matter) and may be found on the leaves and stems of a variety of plants. They have also been isolated from decaying wood, tree bark (Acacia) and from soil.

    Pathogenicity:
    Pithomyces species have been implicated in human disease however their role has not been sufficiently substantiated.  Pithomyces has reportedly been isolated from finger and toe nails, a hand lesion (skin scrapings), peritoneal fluid, bronchial washings, and from a chronic nasal polyposis. The mould may also contribute to general allergic reactions.  In the United States, the most commonly isolated Pithomycesspecies appear to be P. chartarum,P. sacchari, and P. maydicus.
    Pithomyces species have been implicated in pithomycotoxicosis (facial eczema) of ruminants such as sheep, cattle and goats.  P.chartarum, in particular is considered the cause of facial eczema in sheep.
    Pithomyces species are commonly considered to be laboratory contaminants, however, they should not be ruled out without careful consideration, particularly in immunocompromised patients.

    Macroscopic Morphology:
    Pithomyces exhibits fairly rapid growth, maturing in about five days to a week.  Colonies on SAB at 30ᵒC are olivaceous, light to dark brown to brownish-black.  Colour is species and media dependant.  Dark brown to black areas, may be seen macroscopically on some species (P. atro-olivaceus), revealing sporodochia (pleural of sporodochium), which are areas of greater conidial production.  The overall colonial texture is downy to cottony, with a short feathery nap (effuse).  The reverse is brown to brownish-black in colour.
    The isolate presented in this blog (SAB 30ᵒC) post had a lighter cream coloured fringe or edge to the colony.

     Pithomyces species - Sabouraud-Dextrose Agar (SAB), 1 Week, 30ᵒC (Nikon)

    Microscopic Morphology:
    Pithomyces species produce septate, sub-hyaline (pale to light brown) hyphae.  Conidiophores are generally short (peg-like, ~10 µm length), and rather poorly differentiated from the vegetative hyphae from which they extend.  Conidia (10 – 17 µm X 18 – 30 µm) are produced singly at the apex of the conidiophore where they are attached by a short denticle.  After conidial dehiscence (release of conidia), a visible annular frill may remain at the conidial base where once attached to the conidiophore.  Conidia are muriform (have both longitudinal and transverse septations) and are broadly ellipsoidal to ovate (egg shaped) or pyriform (pear shaped) in shape.  P.chartarum usually exhibits 2 – 5 transverse septa with 0 – 3 longitudinal septa. The muriform or septation pattern may be species dependant; P. atro-olivaceus may only produce horizontal septa.  Conidia are dark brown in colour when mature and usually have an echinulate (spiny or prickly) or verruculose/verrucose (warty) texture. 

    Caution: Micron scale (µm) may change between 50 or 100 µm at higher magnifications.

     Pithomyces species - Initial view -growth from the edge of a slide culture.
    (250X, LPCB, DMD-108)

    Pithomyces species - darkly pigmented conidia with internal septations are seen.
    (400X, LPCB, DMD-108)

    Pithomyces species - Numerous, pigmented conidia seen.  Insert shows the muriform (both longitudinal and transverse septa) septations.
    (400X, LPCB, DMD-108)

    Pithomyces species - after conidial dehiscence (release of conidia), a visible annular frill may remain at the conidial base where once attached to the conidiophore.  (400X, LPCB, DMD-108)

    Pithomyces species - conidia are broadly ellipsoidal to ovate (egg shaped) or pyriform (pear shaped) in shape. (400X, LPCB, 400X)

    Pithomyces species - Conidia are borne on short stalks. Brownish pigment has exuded from the hyphae and can be seen as the brown haze alongside the hypha. (400X, LPCB, DMD-108)

    Pithomyces species - conidiophores are generally short (peg-like, ~10 µm length), and rather poorly differentiated from the vegetative hyphae from which they extend.  (400X, LPCB, DMD-108)

    Pithomyces species - as above.  (400+10X, LPCB, DMD-108)

    Pithomyces species - conidia (10 – 17 µm X 18 – 30 µm) are produced singly at the apex of the conidiophore where they are attached by a short denticle.  (400+10X, LPCB, DMD-108)

    Pithomyces species - septate hypha with pigment seen along the outer walls of several. Conidium on short stalk is seen at center right. (400+10X, LPCB, DMD-108)

    Pithomyces species - single, elongated conidium seen at the apex of a withering hyphal element or conidiophore. (400+10X, LPCB, DMD-108)

    Pithomyces species - some chains appeared to be formed by this particulate isolate. Only one source I consulted (Davone -see sidebar) stated that Pithomyces species do not chain. I isolate presented here conforms to the characteristics described for Pithomyces with the exception of chain formation by the conidia. This should not be confused with the chain-like formation of conidia as seen in Alternaria species. (400+10X, LPCB, DMD-108)

    Pithomyces species - ditto.
    (400+10X, LPCB, DMD-108)

    Pithomyces species - yet another photo at higher magnification...
    (1000X, LPCB, DMD-108)

    Pithomyces species - oval conidium at apex of a short stock which shows little differentiation from the vegetative hyphae. (1000X, LPCB, DMD-108)

    Pithomyces species - pigment escaping from the hyphae into the surrounding medium.
    Vegetative mycelium composed of thin-walled hyaline, septate, smooth or verrucose, septate hyphae, 4–7 µm diameter, which may give rise to chains of verrucose, one-celled, dark brown, intercalary chlamydospores 10 -20 µm X 8 - 18 µm[i].
    (1000X, LPCB, DMD-108)

    Pithomyces species - pigment escaping from the septate hyphae into the surrounding medium.  Annular frill can be seen attached to the anterior end of the conidium.
    (1000X, LPCB, DMD-108)

    Pithomyces species - more intercalary chlamydospores seen as described two photos above.
    (1000X, LPCB, DMD-108)

     
    Pithomyces species - Conidia are dark brown in colour when mature and usually have an echinulate (spiny or prickly) or verruculose/verrucose (warty) texture.  The conidium at center-right clearly shows a spiny or prickly surface.  Intense uptake of the Lactophenol Cotton Blue (LPCB) dye somewhat obscures the muriform septations within the conidium.

    Pithomyces species - appears to be attached at both ends which would make it an intercalary chlamydospore (?)
     (1000+10X, LPCB, DMD-108)

    Pithomyces species - short, peg-like, conidiophores arising from the vegetative hyphae at right-angles with single pigmented, muriform conidium at each apex.
    (1000X, LPCB, DMD-108)

    Pithomyces species -here you get it all as described in previous photos.  Pigmented septate hyphae with pigment escaping into the surrounding medium.  Prickly surfaced muriform conidia borne singly on short peg-like conidiophores.  The free conidium closest to the top shows the annular frill which remains from where it was attached to the conidiophore.
    (1000X, LPCB, DMD-108)

    Pithomyces species -two, rather smooth walled, conidia attached to the hypha by short conidiophores.
    (1000+10X, LPCB, DMD-108)

    Pithomyces species - some chaining (?) evident.
    (1000X, LPCB, DMD-108)

    Pithomyces species - single muriform conidium at the end of a short conidiophore.
    (1000+10X, LPCB, DMD-108)

    Pithomyces species - single muriform conidium at the end of a short, rather twisted,  conidiophore.(1000+10X, LPCB, DMD-108)

    Pithomyces species - conidia are borne singly at the ends of the conidiophores.
    (1000+10X, LPCB, DMD-108)

    Pithomyces species - I'm trying to figure this one out.  Are those two conidia arising from two, obscured, conidiophores, or is one an intercallary chlamydospore with a conidiophore & conidium arising from the same area of the hypha?.

    Pithomyces species - Sources state that Pithomyces conidiophores produce single conidia.  Does this photo show a single conidium at the end of a short, pale pigmented conidiophore or is conidiphore the LPCB stained structure arising from the hyphae below with the apex of the conidiophore branched, and one conidium missing?  You decide...
    (1000+10X, LPCB, DMD-108)

    Pithomyces species -muriform conidia at the end of short peg-like conidiophores.
    (1000+10X, LPCB, DMD-108)

    Pithomyces species -conidia texture described as echinulate (spiny or prickly) or verruculose/verrucose (warty) texture.
     (1000+10X, LPCB, DMD-108)

     
    Pithomyces species - conidia which aren't over-saturated with the LPCB stain and more clearly show the muriform septation within.
    (1000X, LPCB, DMD-108)

    Pithomyces species - thickened and roughened wall of the intercalary chlamydospores.
     (1000+10X, LPCB, DMD-108)

    Pithomyces species - chaining of conidia (?) at left.  Two, rather young conidia on short peg-like conidiophores at lower center of photo.
     (1000+10X, LPCB, DMD-108)


    Pithomyces species
      (1000+10X, LPCB, DMD-108)

    Pithomyces species have to be differentiated from closely related dematiaceous hyphomycetes such as Ulocladiumspecies,Stemphylium species, Alternaria species and Epicoccum species. (See Table Below)
     


    Too small to read?  Click on table to get image. Now right click on image and select 'view image'.  In Windows, cursor now has a + sign within it.  Click on image of table to now magnify the table.
    Alternatively, just click and download the damn thing...

    [i] The Genus Pithomyces in South Africa
    W.F.O. Marasas and Ingrid H. Schumann,
    Bothalia 10, 4: 509 – 516, 1972

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  • 07/04/15--12:38: Stemphylium species


  • Stemphylium species(Hyphomycetes)

    Ecology:
    Stemphylium species are widely distributed in nature and can be found in soils as well as plant parasites and as saprophytes on decaying plant material.  As a plant pathogen, Stemphylium species are implicated in the leaf spot of alfalfa (S.botryosum), black rot of carrots (S.radicinum), and grey leaf spot of the tomato plant (S.solani).


    Pathogenicity:
    Stemphyliumspecies found in the clinical laboratory are generally considered to be contaminants; however, they may contribute to allergic reactions in general and have been reported as agents of phaeohyphomycotic sinusitis. 

    Macroscopic Morphology:
    Colonies on SAB are velvety to cottony with a rather short ‘nap’ (does not extend very high above the agar surface).  They are olivaceous (grey-green-brown) to a brownish-black in surface colour.  The reverse is black.  Stemphylium species exhibit a moderate growth rate, becoming mature within five days.

    Stemphylium species - 1 Week on SAB at 30oC (Nikon)


    Microscopic Morphology:
    Hyphae are septate and develop a pale brown to deeper brownish colour as they age.  Conidiophores also show septations and the structure is simple or occasionally branched.  The cell at the apex of the conidiophore which bears the conidium may show a slight swelling in relation to the rest of the conidiophore.  The conidiophore which generally shows somewhat smooth and parallel walls when young may develop a much more knobby appearance as it ages and produces conidia.  Conidia (12 – 20 µm X 15 – 30 µm) are produced by growing from the tip of the terminal conidiogenous cell (poroconidia).  They can be smooth or rough (echinulate) in texture.  They have been described as oval, oblong, ellipsoidal, obclavate and subspherical.  More simply they may be described as ‘box-like’ with rounded corners.  The conidia may also show a marked constriction around a central septum or division within individual conidia. The conidia are muriform (both transverse and longitudinal septations or divisions), and acquire a dark brown pigment as they mature.  

    Caution: Micron scale (µm) may change between 50 or 100 µm at higher magnifications.

    Stemphylium species - not much to distinguish between other moulds in this photo.  I've added it just to show the tangled mass of the mycelium that fungi produce.  Conidia are visible as the dark spots but features are indistinguishable at this magnification. 
     (250X, LPCB, DMD-108)

    Stemphylium species - edge of a slide culture showing hyphae growing out from the edge of the SAB block from which conidiophores extend and produce pigmented conidia at the ends.
    (250X, LPCB, DMD-108)

    Stemphylium species -another shot showing numerous pigmented conidia sitting on the ends of the conidiophores which extend at right-angles from the supporting vegetative hyphae.
    (250X, LPCB, DMD-108)

    Stemphylium species -more detail emerges at this higher magnification.  Septations become visible in the pigmented conidia.
    (400X, LPCB, DMD-108)

    Stemphylium species -hyphae and conidiophores.
    (400+10X, LPCB, DMD-108)

    Stemphylium species -rather short conidiophores, extend primarily at right-angles from the parent vegetative hypha.  At the lower left corner there is a branched conidiophore with a rather young, still unpigmented, conidium developing on the left branch. Two more young conidia can be seen in the photo as well.
    (400X, LPCB, DMD-108)

    Stemphylium species -a few more photos to leave you with an impression of what Stemphylium looks like.  Conidia can be longer and branched which distinguishes it from Pithomyces species.  The shape of the conida also is different from Pithomyces - discussed in later photos.
    (400X, LPCB, DMD-108)

    Stemphylium species -brown pigmented conidia at the apices of individual conidiophores extending from an 'out of focus' hyphal element.
    (400X, LPCB, DMD-108)

    Stemphylium species -conidiophores extending from the supporting vegetative hyphae with conidia at various stages of maturity.  Young, blue-stained conidia and brown pigmented mature conidia are present.  (400X, LPCB, DMD-108)

    Stemphylium species -ditto. 
    (400X, LPCB, DMD-108)

    Stemphylium species -self indulgence here.  More photos to demonstrate the same,  Conidiophores with mature pigmented conidia at the tips.  Muriform (longitudinal & transverse) septation is apparent.  (400X, LPCB, DMD-108)

    Stemphylium species -the appearance in this photo is very similar to that of Pithomyces species in that there are conidia at the ends of short conidiophores arising at right-angles to the hyphae from which they extend.  Darkly pigmented conidia have internal compartments made by the muriform (both up and down and across) septations.  The pigmented hyphae to the left of the photo is clearly septate and appears to be releasing it's pigment into the medium (brown haze).
    (400X, LPCB, DMD-108)

    Stemphylium species -as above. 

    Stemphylium species -massive amounts of conidia can be produced as seen here.
    (400X, LPCB, DMD-108)

    Stemphylium species - a slightly closer look.  Muriform septations evident in the mature brown conidia.  The shape has been described as oval, oblong, ellipsoidal, obclavate and subspherical.  More simply they may be described as ‘box-like’ with rounded corners.
    (400+10X, LPCB, DMD-108)

    Stemphylium species - The conidiophore which generally shows somewhat smooth and parallel walls when young may develop a much more knobby appearance as it ages and produces conidia (lower left).  (400+10X, LPCB, DMD-108)

    Stemphylium species - mature brown box-like conidia at the ends of somewhat knobby conidiophores.  (400+10X, LPCB, DMD-108)

    Stemphylium species - conidia with muriform septations.
    (400+10X, LPCB, DMD-108)

    Stemphylium species - ditto, as above.
    (400+10X, LPCB, DMD-108)

    Stemphylium species -the conidia may also show a marked constriction around a central septum or division within individual conidia (arrows).
    (400+10X, LPCB, DMD-108)

    Stemphylium species - conidia (12 – 20 µm X 15 – 30 µm, when mature) are produced by growing from the tip (arrow) of the terminal conidiogenous cell (poroconidia).
    (1000X, LPCB, DMD-108)

    Stemphylium species - here on another conidiophore you can see the conidium (poroconidium) increasing in size as it develops.  Internal septations have yet to develop.
    (1000X, LPCB, DMD-108)

    Stemphylium species - branching conidiophore -mature brown conidia and a smaller and younger, pale blue conidium.  To follow along from the last two photos, the pale blue conidium is developing further and starting to develop a transverse septum (light line crossing the inside of the small blue conidium).  (400+10X, LPCB, DMD-108)

     Stemphylium species - Mature brown muriform septate conidia showing central construction along where the transverse septum crosses.  Conidiophores may develop a knobby appearance with maturity and the cell at the apex of the conidiophore which bears the conidium may show a slight swelling in relation to the rest of the conidiophore.
    (1000X, LPCB, DMD-108)

     Stemphylium species - as above.
    (1000X, LPCB, DMD-108)

    Stemphylium species -  As above.  Different stages of maturity.  Too many photos, I know!
    (1000X, LPCB, DMD-108)

    Stemphylium species - ditto.
    (1000X, LPCB, DMD-108)

    Stemphylium species - conidia can be smooth or rough (echinulate) in texture.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species - can be smooth or rough (echinulate) in texture. Conidiogenous cell at tip of conidiophore may be somewhat distended (wider, swollen) in relation to the rest of the conidiophore.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species - conidiophore shows septation.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species - again, conidiogenous cell closest to the tip may show a slight swelling in relation to the rest of the conidiophore.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species - conidia with various surface textures.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species - yet another photo!  You get the picture by now...
    (1000+10X, LPCB, DMD-108)

    Stemphylium species
    (1000+10X, LPCB, DMD-108)

     Stemphylium species

    Stemphylium species - poroconidium (doesn't show the annular frill which is seen on somewhat similar looking muriform conidia produced by Pithomyces species.
    (1000+10X, LPCB, DMD-108)

     Stemphylium species - looks like a degenerating, rather wilted conidiophore.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species- branching septate conidiophore.
    (1000+10X, LPCB, DMD-108)

     Stemphylium species - branching conidiophore.
    (1000+10X, LPCB, DMD-108)

    Stemphylium species
    (1000+10X, LPCB, DMD-108)

     Stemphylium species 
    (1000+10X, LPCB, DMD-108)

     Stemphylium species 
    (1000+10X, LPCB, DMD-108)

     Stemphylium species 
    (1000+10X, LPCB, DMD-108)

    Stemphylium species -Last one.  I don't know what I was thinking, uploading some 45 photos to illustrate Stemphylium species.  As with most of the posts in this blog, the photos posted represent only 10% of what I've taken.  Sorry for anyone who I lost with boredom -I find the little critters fascinating.
    (1000+10X, LPCB, DMD-108)

    Note: May be confused with other darkly pigmented moulds such as Alternaria species,Pithomyces species, and Ulocladium species (see table below).


     Too small to read?  Click on table to get image. Now right click on image and select 'view image'.  In Windows, cursor now has a + sign within it.  Click on image of table to now magnify the table.
    Alternatively, just click and download the damn thing...
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  • 08/01/15--15:28: Penicillium citrinum


  • Penicillium citrinum

    Ecology:  Penicillium citrinum is a commonly occurring filamentous fungus with worldwide distribution.  It has been isolated from a variety of sources including soils, decaying vegetation, foodstuffs (beans, coffee, cereals & spices) as well as a variety of indoor environments.

    Pathology:  While Penicilliumspecies are generally regarded as laboratory contaminants, or at best, opportunists, a number of species have been implicated as being involved in the disease process.  While Penicillium species may be isolated from clinical specimens, it is commonly believed that a true infection can only be established by histological demonstration of tissue invasion.  With that in mind, Penicillium citrinum has been reported in mycotic keratitis (eye), lung infections (pneumonia), a single case of a urinary tract infection (UTI) and one of pericarditis.  Their contribution to the disease process may be secondary an additional underlying illness.  As with all fungi, immunocompromised individuals may be at greater risk of infection including those rarely considered as pathogenic.

    Macroscopic Morphology:  Penicillium citrinum exhibits moderately slow growth on Sabouraud-Dextrose agar (SAB) at 30ᵒC.  Surface texture is velutinous (soft, velvety surface) to floccose (woolly tufts of soft “hairs”).  The colonial growth appears radially sulcate (narrow, deep furrows or radial grooves –like spokes on a wheel).  The mature colony has a central greyish-turquoise to greyish-orange colour with a white periphery (outer edge).  Exudates (extrolites) are frequently produced which appear as drops of liquid upon the surface of the colony.  These may appear clear, to pale yellow, to a reddish-brown in colour.  Some strains may also produce a soluble pigment which can diffuse into the surrounding medium.  The reverse is a pale yellow to a light yellow-brown. Colours and growth characteristics are, of course, media and strain dependent.

       Penicillium citrinum on Saboraud Dextrose Agar (SAB) after ~7 days incubation at 30ᵒC (Nikon)

    Penicillium citrinum-SAB, 14 days incubation at 30ᵒC (Nikon)
    Note the drops of exudate (extrolites) which have formed on the surface.
    Colour variation due to maturing of colony but also a difference in my lighting for photography.

     Exudates (or Extrolites): Some fungi can produce exudates as a by-product of their growth, many of which can be collected for commercial use.  Mycotoxins are by-products (secondary metabolites) which are potent poisons.  Penicillium citrinum produces Citrinin, a nephrotoxic mycotoxin which derives its name from the fungus.  It may also produce other extrolites such as tanzowaic acid A, quinolactacins, quinocitrinines, asteric acid and compactin.

    Microscopic Morphology:  Penicillium citrinumproduces septate, hyaline (clear, not pigmented) hyphae.  Smooth-walled conidiophores stipes are rather long (100 – 300 µm) and is biverticillate (see diagram at end of post).  Metulae are 12 – 15 µm in length which are found in whorls of 3 – 5 divergent structures.  Phialides are ampuliform (flask-shaped) and about 7 – 12 µm in length.  Conidia (2.2 – 3.0 µm dia.) are globose to sub-globose (round to off-round) and are smooth or have a finely roughened surface.  Conidia resist disruption and form rather long chains.  These characteristics: the metulae longer than the phialides and the conidia being both spherical and produced in well-defined chains, are distinguishing features of Penicilliumcitrinum.

     Penicillium citrinum-  distinguishing features of Penicillium'species' can already be made out at low magnification. (250X, LPCB, DMD-108)

     Penicillium citrinum-  distinguishing features of Penicillium'species' much more evident at 400X.
    Typical "fingers" made up of the metulae and phialide structures from which chains of conidia extend. (400X, LPCB, DMD-108)

    Penicillium citrinum- a mass of overlapping fruiting structures with copious amounts of conidia.
    (1000X, LPCB, DMD-108)

    Penicillium citrinum- a little less congested in this photo.  Conidiophores (stipes) seen from which extend the metulae and conidia producing phialides.  Conidia are globose (round) to sub-globose (somewhat off-round) in shape,  (1000X, LPCB, DMD-108)

    Penicillium citrinum- long metulae and the somewhat shorter phialides are clearly distinguishable in this photograph.  The conidia are generally smooth or can have a finely roughened surface.
    (1000+10X, LPCB, DMD-108)

     Penicillium citrinum- another view.
    (1000+10X, LPCB, DMD-108)

     Penicillium citrinum- exhibits biverticillate branching meaning that the conidiophore can branch and the metulae & phialides extend from these branches.  Triverticillate would have the conidia branching and then the branches also branching to finally produce the metulae & phialide fruiting structures.
    (1000X, LPCB, DMD-108)

     Penicillium citrinum- Phialides are ampuliform (flask-shaped) and about 7 – 12 µm in length.  Again, conidia (2.2 – 3.0 µm dia.) are globose to sub-globose (round to off-round) and are smooth or have a finely roughened surface. (1000+10X, LPCB, DMD-108)

     Penicillium citrinum- Here we see the proportions of the metulae (M) and the 'flask-shaped' phialides (P) with the metulae being substantially longer than the phialides,  The biverticillate structure is evident in this photo. (ie. each branch extending from the conidiophore (stipe), branches only once and then bears a fruiting structure consisting of the metulae and phialides.
    (1000+10X, LPCB, DMD-108)

    Penicillium citrinum- another example.
    (1000+10X, LPCB, DMD-108)

    Penicillium citrinum- a few more photos to finish up.
    (1000+10X, LPCB, DMD-108)

    Penicillium citrinum- suitable for framing!
    (1000X, LPCB, DMD-108)

    Penicillium citrinum
    (400+10X, LPCB, DMD-108)
    Penicillium citrinum
    (400X, LPCB, DMD-108)

    Penicillium citrinum- another colony showing the exudate (extrolites) which accumulate on the colony surface after extended incubation.  These metabolites may be potent poisonous mycotoxins or might have beneficial uses in industrial or pharmaceutical applications. (Nikon)



    Physiology:  The spores of Penicillium citrinum fail to germinate at 5ᵒC and may show restricted growth at 37ᵒC.
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  • 10/30/13--17:27: Exserohilum rostratum


  • Exserohilum rostratum (Mould)

    Ecology:
    Exserohilumspecies 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.

    Pathogenicity:
    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.rostratumexhibits 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)
     Caution:
    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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    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.

    Ecology: 
    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.

    Pathology:  
    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 Neosartoryaspecies.

    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 byNeosartorya 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)

    Notes:
    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!

    Differentiation:
    Eurotium repens& E.rubrum are species closely related to E.herbariorum.  E.repensdiffers from E.herbariorum by the formation of ascospores without a distinct furrow or trough.  E.rubrumhas 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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  • 01/04/14--09:31: Okay, Why so much Fungus???


  • This blog is called ‘Fun With Microbiology’, not’ Fun With Mycology’ – so what gives?  Why the disproportionate number of Fungal posts?

    Well, this blog came about primarily because, after my injury, I needed some way to fill my time after regular work hours while waiting for my ride home.  With my return to work, I discovered that the laboratory had acquired some new “Toys” for photographing interesting microbiological specimens.  Surprisingly, few were interested in utilizing them, whereas with my previous interest in photography, I was only too happy to take command of these tools.

    This blog is primarily about photography - about describing microbiology through pictures.  It is also about “interesting” cases – some of the less commonly encountered observations usually not described in text books.   (eg Mycobacteria in a gram stain, Strongyloides  tracks on agar plates, VDE –bacteria that need  antibiotics to survive.) Finally, it is about sharing my photographs with anyone who may find them of interest.

    So, why so much fungus?  Well, bacteria exhibit a limited number morphological variations, as I’ve attempted to illustrate (right).  Generally speaking, an E.coli cell looks pretty much like a Salmonella cell, looks like a Citrobacter cell.  While important, yet often subtle, differences in morphology do occur in bacteria, I have chosen not to pursue them on this site.  Bacteria will be documented when the topic best lends itself to photographic interpretation.  

     Generally speaking, there are a limited number of bacterial morphotypes to explore with photography.

    While I wish to pursue Parasitology a bit more extensively, I find myself limited to what cases present themselves in our acute care community hospital.

    Numerous fine textbooks on Clinical Mycology are in print (see sidebar); however I have frequently found that the description ‘in text’ relates poorly to the one or two small supporting photographs offered.  Fungi are three dimensional organisms whose structure varies as they mature.  I’ve attempted to document these fascinating organisms from all angles and in all pertinent stages of development.

    In summary, whether saprophytic contaminants or clinically significant isolates. fungi are the most photogenic microbiological organisms and present themselves in sufficient numbers to keep this blog going.

    Finally, I offer my apologies for the rather lame name of this Blog.  My wife suggested I explore ‘blogging’ as a way to pass the time while bed bound, recovering from a catastrophic injury.  I jokingly chose this name never thinking I’d develop it past the few print photos I had taken years earlier.  Too late to change it now…


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  • 01/09/14--09:17: Fasciola hepatica


  • Fasciola hepatica(Trematode – Parasite)

    Geographic Distribution & Pathogenicity

    Fasciola hepatica is commonly known as the sheep liver fluke and is a common parasite in herbivores.  With cosmopolitan distribution, human infections have been reported in many parts of the world.  Fasciola hepatica is most frequently found in countries where sheep raising is common, such as China, Taiwan, India, Indonesia and other parts of Asia. 

    Fasciola hepatica is responsible for the disease fascioliasis, also known as fasciolopsiasis or simply, sheep liver fluke infection.  The infection may have first been recognized as early as 1379 when the effects were noticed between certain water plants and the sheep that had eaten them.  Fascioliasis is considered a zoonotic disease (passed from animals to man).


    Symptoms:  The infection may produce symptoms of biliary obstruction and cholangitis.  Symptoms may include upper right quadrant pain, fever, chills and jaundice.  Symptoms may depend on the worm burden and light infections may be asymptomatic.

    Life Cycle & Morphology:

    Worms: The Fasciola fluke is quite large and may measure as large as 3 cm by 1.5 cm in size.  The anterior end of the worm (fluke) has a distinctive cone shaped projection. The interior organs of the worm appear extensively branched.  The adult worms live in the bile ducts of the liver and the gallbladder.

    Eggs: The eggs (ova) are large (80-150 µm by about 60 -90 µm) and broadly elliptical in appearance.  They are operuclated but the operculum is rather small in relation to the egg and rather inconspicuous.  The eggs are unembryonated when passed in the feces.  When passed into water, they undergo embryonation and subsequently miracidia are hatched (usually in 1 – 2 weeks).  Fasciola hepatica requires an intermediate host for development, which in this case is a freshwater snail (Lymnaea sp).  The miracidia within the snail mature and emerge as cercariae which then attach to aquatic vegetation (eg. watercress) where they undergo encystation.  Humans are infected by the ingestion of uncooked aquatic vegetation on which the metacercariae are encysted.  The metacercariae excyst (hatch) in the duodenum and migrate through the intestinal wall into the peritoneal cavity.  The larvae penetrate the liver and wander through the parenchyma for up to 9 weeks.  The larvae finally enter the bile ducts where they mature and in about three to four months and begin to produce eggs, which are ultimately passed out in the feces.  The adult worms may live for up to a year.

    Diagnosis:

    Diagnosis is made by the detection of the characteristic eggs in the patient’s faeces.  One problem in identification is that the species Fasciolopsis buski produces eggs which are almost indistinguishable from those produced by Fasciola hepatica.  Life cycles of these two trematodes are very similar.  In some areas of the orient where these two species overlap, the clinical evaluation of symptoms aids the diagnosis of these faciolid eggs.  The size of the operculum opening may also assist in diagnosis where the Fasciola hepatica’s operculum is larger than that of Fasciolopsis buski(measurements to follow).  Putting pressure on the coverslip of a concentrated faecal specimen with the eraser end of a pencil may be sufficient to cause the operculum to pop open and better reveal itself.  Molecular methods may provide a definitive identification.


     Fasciola hepatica egg in faecal concentrate.  Bile-stained shell and inconspicuous operculum.
      Egg measures 151µm  by 75 µm.  (DMD-108)

     Fasciola hepatica egg with Iron Heamatoxylin stain. (DMD-108)

    Fasciola hepatica egg showing the operculum (OP). Measurement reads 28.82 µm.  (DMD-108)


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    Vancomycin Dependant Enterococcus (VDE)

    Explanation of Enterococcus faecium’s curious response to the antibiotic Vancomycin

    So you try to determine the Minimum Inhibitory Concentration (MIC) of Vancomycin against an Enterococcus isolate by E-test (epsilometer test) methodology and after appropriate incubation you obtain this curious result:

    Enterococcus faecium's response to a Vancomycin E-test
    (Mueller-Hinton Agar - 24+hours, 37˚C)

    What the *$%#&!;.....??  The greatest growth is where the antibiotic concentration is the greatest and tapers off where the antibiotic concentration is the lowest.  It is kind of like shooting at a flock of ducks flying overhead, and only the ones that don’t get hit drop!!!

    So what is happening here?  Let’s back up a bit and get some history:

    Vancomycin is an important antibiotic as it is the last ‘common’ antibiotic active against most gram positive organisms.  Once an organism acquires resistance to vancomycin, the antimicrobial arsenal is greatly limited in what can be used to fight an infection.

    Vancomycin resistance is plasmid mediated, meaning that vancomycin sensitive enterococci and acquire resistance from other organisms already vancomycin resistant.  In turn, these Vancomycin Resistant Enterococci (VRE) can pass the plasmid on to other organisms.  This may result in an outbreak of resistant organisms which are challenging to treat and may be particularly devastating in severely debilitated patients.

    There are eight known vancomycin resistance genotypes in enterococci with those known as Van-A being most prevalent, followed by Van-B.  Van-C offers low level intrinsic resistance to E.gallinarum & E.casseliflavus.  The remaining genotypes have not proven to be significant in the clinical setting.

    Enterococci expressing the Van-A genotype are resistant to both vancomycin & teicoplanin.  Expression of the Van-B genotype conveys resistance to vancomycin but the enterococcus remains susceptible to teicoplanin.  Van-A resistance is generally higher (16 – 516 µg/ml) than that provided by Van-B (4 – 64 µg/ml).

    In order to prevent nosocomial (hospital acquired) infections, many facilities require a rectal swab be taken from newly admitted patients in order to screen for VRE.
    Various methods & media can be employed for this screening.  Our facility utilizes Oxoid® Brilliance Chromogenic VRE media.  On this media, E.faecalis appears as light blue colonies while E.faecium appears purple.  Other organisms are repressed or appear uncoloured.

     
    Enterococcus faecium on Oxoid ® Brilliance Chromogenic Media (24hrs at 37˚C)

    Suspicious colonies are investigated further by determining the actual MIC of vancomycin using the E-test as mentioned above.  Enterococci with MIC’s greater than 8 µg/ml are considered to be VRE.  (Identifications can be confirmed using common microbial identification platforms or traditional tests).
    Patients known to harbour VRE’s can be isolated and contact precautions implemented to reduce the likelihood of dissemination.



    The antibiotic sensitivity plate above shows three different organisms subjected to an e-test in order to determine their susceptibility to vancomycin.  Organism (1) is an enterococcus susceptible to vancomycin (VSE), (2) shows an enterococcus resistant to vancomycin (VRE), and (3) shows a curious response to vancomycin I had never before encountered.  This organism exhibits vancomycin dependence! (VDE).

    Note:the E-test is a strip impregnated with a continuously varying concentration of antibiotic along its length.   On the Vancomycin E-test strip the concentration varies from 0.016 µg/ml to 256 µg/ml.  The MIC value is where the growth/no-growth intersects the strip.  The zone of inhibition is narrowest as it approaches the point of intersection and widest at the top of the strip where the concentration is the greatest.

    Okay, what gives? First, let's explore Vancomycin Resistance a bit further:

    Vancomycin binds to the terminal D-Ala:D-Ala structure in the peptidoglycan layer of the enterococcal cell wall. This prevents the crosslinks from forming and the pentapeptide structures from extending during synthesis. Cell wall formation is terminated, or rather the cell wall is weakened without the cross-links and therefore the integrity is compromised and the bacterial cell is subject to variations in osmotic pressure.  Eventually the cell will burst if not strengthened with the cross-links.  Think of each peptidoglycan molecule as a brick and the pentapeptide bridge as the mortar holding the bricks together. (see diagram below)

    In both Van-A & Van-B genotypes, the gene cluster acts to a) detect the presence of vancomycin and start transcription of specific resistance genes, b) form and incorporate D-Ala:D-Lac into the growing peptidoglycan wall, and c) eliminate any D-Ala:D-Ala precursors, thereby eliminating the vancomycin sensitive pathway of peptidoglycan formation.

    In other words, vancomycin binds to D-Ala:D-Ala, however by the enterococcus substituting D-Ala:D-Lac into the structure, vancomycin will no longer "functionally" bind rendering the organism vancomycin resistant.


    Exactly why does the substitution of D-Ala:D-Lac make the enterococcus resistant to vancomycin?  My university biochemistry was rusty and I wondered if the Lactate molecule in D-Lac was larger or more complex than the Alanine molecule in D-Ala and the vancomycin was sterically inhibited or prevented from binding by shear size.  Did this substituted molecule block the vancomycin? In the sentence previous to the above diagram, I said that the vancomycin does not "functionally" bind to the D-Ala:D-Lac side chain of the peptidoglycan cell wall component.  Looking at the structure of D-Ala:D-Alanine compared to D-Ala:D-Lactate you can see in the diagram below that they are almost identicle.  The difference lies in the substitution of an oxygen molecule in the D-Ala:D-Lac for the amine group (NH) in D-Ala:D-Ala.


    Vancomycin is the large molecule at the top third of this graphic.  By substituting D-Ala:D-Lac for D-Ala:D-Ala, the amine (NH) is replaced by and oxygen (O) and the hydrogen bond shown in red reduces the total number of hydrogen bonds from 5 to 4.

    In more detail:

    Now, vancomycin binds to the D-Ala:D-Ala in the peptidoglycan side chain via 5 hydrogen bonds (kind of an electrostatic bonding which can only occur between Hydrogen and Nitrogen, Oxygen or Fluorine)  However, with the substitution of D-Ala:D-Lac into the side chain, and the removal of the amine group, there is one less hydrogen bond capable of forming.  You say "big deal"?  Well, actually it is a big deal as this loss of one hydrogen bond weakens the bond between the vancomycin molecule and the enterococcal peptidoglycan side chain by three fold, or 1000X.  While the vancomycin molecule still attaches to some degree, it is not in a configuration that can effectively block or prevent the Trans-glycosylase enzyme from forming the pentapeptide cross-link between the neigbouring peptidoglycan wall components


    Vancomycin Dependence (VDE):
    It has been proposed that vancomycin dependence may develop from the loss of a functional D-Ala:D-Ala ligase in the VRE strain, which is then unable to survive unless vancomycin induces the production of D-Ala: D-Lac ligase. This dependence involves mutations to the dll gene which encodes the enterococcal D-Ala:D-Ala ligase protein.

    In other words, Vancomycin induction of the Van A or Van B ligase would compensate for the absence of the native ligase by producing D-Ala:D-Lac allowing for cell wall precursor synthesis. Since these ligases are only induced in the presence of vancomycin, the organisms cannot grow in the absence of this antibiotic unless it reverts to the vancomycin resistant form.

    Revertant Mutant Enterococci:
    If a particular strain of enterococcus becomes dependent on vancomycin for its growth and survival, it would seem logical that removing vancomycin would cause the organism to die. Surprisingly, this is not always the case as the organism may undergo a ‘revertant’ mutation. The enterococcus may undergo another genetic change that restores the D-Ala:D-Ala ligase function. The organism may enter a cyclical mutational change allowing it to shift between resistant and dependant phenotypes.
    Withdrawal of vancomycin may not be adequate to eliminate vancomycin dependent strains.

    On the first photograph of this post, the colonies randomly scattered throughout the agar surface, away from the E-test strip may be revertant colonies.  These colonies were not apparent after 24 hours however these colonies appeared after sitting on the bench for approximately another 16 hours.

    Revertant strains have not been observed in clinical situations and the presence of VDE does not appear to affect the patient’s clinical outcome.

    These are the kind of microbiological oddities that give this blog its title “Fun With Microbiology”!

     References:


    1. Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story.
    C.T. Walsh et al: Chemistry & Biology: January 1996, 3:21-28

    2. Vancomycin resistance in enterococci: reprogramming of the d-Ala–d-Ala ligases in bacterial peptidoglycan biosynthesis.
    V.L. Healy et al: Chemistry & Biology: Volume 7, Issue 5, 1 May 2000, Pages R109–R119

    3. Vancomycin-Resistant Enterococci: Mechanisms and Clinical Observations.
    H.S.Gold; Clinical Infectious Diseases (CID)  33: 210-218, July 2001

    4. Crystal Structure of Vancomycin
    Martina Schäfer, Thomas R Schneider & George M Sheldrick.
    Structure: 15 December, 1996, 4: 1509-1515

    5. The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate.
    S. Handwerger et al: J Bacteriol. Sep 1992; 174(18): 5982–5984.

    6. Final Diagnosis – Vancomycin-Dependent Enterococcus VDE:  (no source address/author provided. ) http://path.upmc.edu/cases/case417/dx.html

    7. Vancomycin Dependent Enterococcus faeciumIsolated from Stool following Oral Vancomycin Therapy:  LIsa L. Dever, et al.) J Clin Micro. Vol. 33, No. 10  Pg. 2770 – 2773, 1995  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC228574/pdf/332770.pdf

    8. An Outbreak of Vancomycin-Dependent Enterococcus faecium in a Bone Marrow Transplant Unit.  B.D. Kirkpatrick et al: CID Vol 29: pg. 1268 – 1273, 1999  http://cid.oxfordjournals.org/content/29/5/1268.full.pdf+html

    9. Noscomial Infection with Vancomycin Dependent Enterococci:  Paul A. Tambyah et al, Emerg. Infect. Dis. Vol. 10, No.7 July 2004   http://wwwnc.cdc.gov/eid/article/10/7/pdfs/03-0993.pdf

    10. Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate.
    L.Chen et al: Proc Natl Acad Sci U S A. May 13, 2003; 100(10): 5658–5663.


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