Chrysonilia sitophila

Chrysonilia sitophila -Hyphomycetes (formerly Monilia sitophila)

Ecology:

Chrysonilia species are a cosmopolitan (widespread) saprobe (lives of dead or decaying plant matter).  Chrysonilia may be found within the home as ‘red bread mould’, named for the colour of growth found on contaminated bread.  Chrysonilia is the asexual state (anamorph) of Neurospora, its sexual or (teleomorph) state.  Neurospora’s ascospores are stimulated by applied heat and therefore Neurospora and the Chrysonilia anamorph may be the first organisms to repopulate areas devastated by grass or forest fires.   For the same reason, it is notorious for populating heat sterilized soils such as those found in greenhouses.  Chrysonilia’s teleomorph, Neurospora, is frequently used in the study of genetics and basic eukaryotic cell biology because of its desirable growth characteristics.

Chrysonilia sitophila was formerly called Monilia sitophila; however the genus Monilia now only encompasses plant pathogens.

Pathogenicity:

Chrysonilia is considered to be a contaminant and is not considered to be very pathogenic.  It has been implicated in peritonitis, eye infections as well as occupational asthma.

Macroscopic Morphology: 

·         Colonies exhibit extremely rapid growth, maturing within 72 hours.

·         Colonies can range in colour from white to a pale pink, a salmon colour to light orange.

·         Texture is very cottony which quickly fills the petrie dish, often referred to as a “lid-lifter” as it crawls up the sides of the dish and presses against the lid.

 Chrysonilia sitophila - 24 Hours growth on SAB agar plate incubated at 30˚C (Nikon)

 Chrysonilia sitophila - This composite shows how quickly this fungus grows.  Within one day the centrally inoculated plate has been filled with the mycelium.  By 4 hours it has already completely filled the standard petrie dish and the areal hyphae are pushing against the lid.  By day three, the slight pinkish to salmon to light-orange colour, characteristic of this fungus is evident.  After a week the growth may have collapsed back on itself and be even be trying to crawl out the sides of the petrie dish.  This organism is notorious for contaminating laboratories with the multitude of spores (conidia) produced. (Nikon)

Microscopic Morphology:  

 ·         Chrysonilia produces smooth-walled, hyaline, septate hyphae.

Sources differ on their description of the conidia produced.  The majority of sources I consulted only mention rectangular ‘arthroconida’, produced as the hyphae disarticulate or break-up.  Yet another source mentions blastoconidia in addition to the arthroconidia.  By definition these should emerge as a bud which is then cleaved off at maturity, often leaving a mark or scar at the point of attachment.  I have pointed these out where I believe they match the description.

·         Simple, poorly differentiated conidiophores can be single or branched.

·         Conidiophores produce branching chains of oval conidia (5-10 µm X 10-15 µm)

·         Mature hyphae break up forming thick-walled rectangular arthroconidia connected by disjunctors.

  Chrysonilia sitophila -a first look at low magnification.

(100X, LPCB, DMD-108)

 Chrysonilia sitophila -septate hyphae are seen with poorly differentiated conidiophores producing chains of rather round blastoconidia.  Rectangular arthroconidia are also evident, produced as the hyphae fragment or disarticulate.

(400X, LPCB, DMD-108)

 Chrysonilia sitophila - another view as above.

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -and another.  The rather round Blastoconidia (Bc) are being produced in addition to the rectangular Arthroconidia (Ac).  Are the so called 'Blastoconidia' referring to immature or developing arthrospores in one source?  The blastoconidum shown clearly does not appear to be the product of a fragmenting hyphal element.  The hyphae are septate (S), along the lines where they will disarticulate into separate arthroconidia,

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -Branching hyphae & conidiophores.

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -ditto

(400X, LPCB, DMD-108)

 Chrysonilia sitophila -whether these are considered to be blastoconidia or immature arthroconidia, chains are clearly shown to be developing from a branch extending from a main hyphal element.

(400X, LPCB, 400X)

 Chrysonilia sitophila -arthroconidia can be seen joined to each other via a disjunctor cell (Dj) which appears as a small bridge separating one cell from the next.  Various texts show the disjunctor as being more pronounced than seen here.

 (1000X, LPCB, DMD-108)

The photograph show above, as several of the previous, are taken from an adhesive tape preparation.  I have found at times that an adhesive tape preparation preserves features better than a slide culture and sometimes the opposite.  Use whatever technique works best for you.  Overall, I like slide cultures as it generally produces a much clearer photograph.  In tape preps such as this, you  can often detect the unevenness of tape surface and distribution of the adhesive.  Here the background is full of bubbles from trapped air and the adhesive.

 Chrysonilia sitophila -hyphae disarticulating into arthrospores (AC).

(1000X, LPCB, 1000X)

 Chrysonilia sitophila -once again, it appears to me that there is a distinct difference between the blastoconidia (BC) which often appear to develop from the side of the hyphae or rudimentary conidiophore and those of the already rather rectangular arthrospores at the apex of a fragmenting hyphal element. (Dj = Disjunctor)

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

 Chrysonilia sitophila - One last photo again showing what I call a round blastoconidium (BC) on a very basic conidiophore (Cp).  To my eye, these look distinctly different from the rectangular arthrospores forming by the disarticulation of the hyphae which they originate.

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

Physiological Tests:

·         +Growth at 37˚C.

·         +Growth on Cycloheximide Agar

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Candida albicans

Candida albicans  (Yeast)

(Note: For Candida dubliniensis, scroll to bottom of this post.)

I thought I’d start off my return from ‘sick leave’ by checking out the lab’s new digital camera. Just back, I have no “exotic” isolates to play with and explore so I’ve just retrieved a common, everyday Candida albicans. I’ve often, been disappointed in photographs found in most textbooks as they are small in size, black & white, poorly focused and often scrunched between a few lines of description. This yeast deserves a bit more respect as it is the most common cause of candidiasis, an infection which can range from acute, sub-acute or chronic. It may be present, yet unrecognized or have devastating, life threatening consequences. C.albicans may be present as commensal flora in the normal mouth, the skin and in the stool. Problems arise when, for what ever reason, the microbial balance is upset and Candida is allowed to overgrow other organisms which may assist in keep it in check. Antibiotic therapy may lessen normal bacterial flora allowing Candida to flourish. A change in the pH of the healthy vagina may result in a yeast infection. Immunosuppressive therapy may predispose a patient to infections by yeast.

Macroscopic Morphology:
Candida albicans grows rapidly in culture, reaching maturity in as little as three days. Colonies are cream coloured, raised, entire, smooth & butyrous. On enriched media such as Blood Agar, or Chocolate Agar, the colonies may develop small striations or outgrowths often referred to as “feet” which are indicative of the Candida albicans species.

Candida albicans on Sabouraud-Dextrose Agar at 48 hours at 30C
(click on photo and illustration at right to enlarge for better viewing)

Microscopic Morphology:
A smear made from colonies taken from Sabouraud-Dextrose Agar, or blood agar will appear as round to oval cells about 4 to 8 µm. Though the cell wall structure differs from that of gram positive bacteria, yeast cells retain the crystal-violet stain of the routine gram stain and therefore appear purple. The yeast cell divides by budding. On primary media (reduced nutritionally) the budding can create elongated cells which when lined up along the dividing plane, mimic the appearance of a hyphae however these inline individual cells are referred to as a pseudohyphae (false hyphae). Some true hyphae may also be formed.
Along side of the pseudohyphae, Candida albicans develops blastoconidia around the area of the ‘septa’ (division). These appear as smaller round ‘grape-like’ clusters.

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Candida albicans retaining crystal violet stain from routine gram stain taken from SAB Agar
(click on photos to enlarge for better viewing) X1000

Another structure is the thick walled, rather refractle chlamydospore which usually develops at the end of the pseudohyphae (terminal).

Refractile Chlamydospore production on Corn Meal Agar at 2 hrs at 30C
(click on photo to enlarge for better viewing)

Technique: Chlamydospores production is best induced using the Corn Meal Agar (CMA) or Oxgall Agar. Inoculate the plate by picking up a sample of the Candida albicans colony with a straight wire and scratching the surface of the agar with it. With the surface inoculated, cover the scratched area with a microscope cover slip. This has a two-fold purpose. The glass cover slip reduces the atmospheric tension under the surface and protects the microscope objective from damage when viewing the plate after incubation. A filter paper moistened with sterile water and incubation at room temperature in the dark, may further encourage the production of chlamydospores After incubation (24-48 hours) remove the petrie dish plate cover (and microscope stage if you wish) and view the growth by carefully lowering the objective over the cover-slipped agar (X100-250). Viewing the growth around the edge of the glass cover slip should yield the best results.

Refractile Chlamydospores and smaller 'grape-like' Blastoconidia (Blastospores) produced on Oxgal Agar and photographed through the microscope cover slip with the agar plate placed on the microscope stage.
(click on photo to enlarge for better viewing)

Yet another characteristic of Candida albicans is that it has the ability to produce 'Germ Tubes' when placed in a nutritionally rich horse serum. Inoculate about 1 to 2 ml of horse serum and incubate at 37C for about 2 to 3 hours (too long may result in the formation of pseudohyphae, mimicking germ tubes) . Examine under the light microscope for a protrusion growing out from the yeast cell. Germ tubes appear as outgrowths from the side of the yeast cell and although characteristic of Candida albicans, be aware that the closely related Candida dublinensis can also produce germ tubes. Other characteristics which won't be discussed here can easily be used to separate these two species. Germ tubes protrude from the originating cell with no "pinching" seen at the point where they extend. If there is evidence of pinching, this may not be a germ tube but rather the beginning pseudohyphal growth.

Candida albicans germ tube production in Horse Serum at 37C after 3 hours incubation.
Note; there is no constriction/pinching where the germ tube leaves the originating cell indicating that this is indeed a germ tube and not the beginning of a pseudohyphae. (wet prep X400)
(click on photo to enlarge for better viewing)

Below are a few photos of Candida albicans as seen in actual specimens;

Candida albicans in a vaginal swab seen in the form of a pseudohyphae and a few individual oval cells. (Gram Stain X1000)
(click on photo to enlarge for better viewing)

Candida albicans seen as a pseudohyphae at lower left and individual yeast cells at upper right.
(Gram Stain of Sputum specimen X1000)
(click on photo to enlarge for better viewing)

Candida albicans pseudohyphae and individual cells seen in an aspirate from a kidney.
Depending of various factors, individual yeast cells, pseudohyphae, or both may be present in any given specimen.
(click on photo to enlarge for better viewing)

New: April 01, 2012
An interesting photo of a fungus as it appeared in a direct blood culture gram stain. Identification proved it to be Candida albicans. Just had to share my beautifully bizarre photo - hard to imagine this spiny ball of spikes bouncing around inside someones blood vessels!

Candida albicans in direct smear of Positive Blood Culture.
(Usually seen as the round to oval cellular form and not as these clumps of pseudohyphae)

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New: April 18th, 2015

Candida albicans vs Candida dubliniensis

Okay, you think you have isolated Candida albicans.  But is it really?....and why should you care?  Well, Candida albicans & Candida dubliniensis are virtually indistinguishable using common physiological tests (see list which follows).  However, there may be one significant difference which should be considered.  Candida dubliniensis may exhibit increased resistance to the anti-fungal agent Fluconazole.  This is of particular concern when this species is isolated from sterile sites such as blood cultures where delayed or failure of treatment may be fatal.

• Both yeasts are virtually identical in their physiological characteristics

• Carbohydrate assimilation is virtually identical ( some variation +/- vs -/+ of little use in identification or differentiation)

• Glucose fermentation – both positive

• Chlamydospore production – both positive

• Germ tube production – both positive

• Urease – both negative

• Nitrate – both negative

• Cyclohexamide (0.04%) – both positive for growth.

• Growth at 30^oC & 37^oC -both positive

While some commercial identification systems claim they can distinguish between the two species, this is a costlier solution to a problem that can be solved using incubation temperature alone. 

C.dubliniensis
• Growth at 42^oC to 45^oC = -/w
• Fluconazole –may exhibit increased resistance.

C.albicans
• Growth at 42^oC to 45^oC = +/-
• Fluconazole – generally considered susceptible.

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