Monday 30 November 2015

Alternative Stains & Preparation of Moulds & Yeast

Common and Alternative Stains for Viewing Moulds &Yeast

While cleaning out some files, I came across a number of microphotographs which might be of interest to the reader.  Most of my posts show the organism in question after it has already been isolated and not how it may first clue the microscopist/technologist that an organism is present in a specimen.  A few photos of specimens containing fungi follow below.

I worked on some of these specimens personally while others were brought to my attention by colleagues in the microbiology or histology laboratories.  For those I did not work on personally, I have few specifics such as the final identification.

Yeast cell producing pseudohyphae in a sputum specimen.  Numerous white blood cells present.
(1000X, Gram Stain, Nikon)

Numerous yeast cells, many budding, along with other bacteria in this sputum sample.  A large epithelial cell is seen in the lower left.
(1000X, Gram Stain, Nikon)

Several yeast cells seen along the center of this sputum specimen photo. This sample also grew Streptococcus pneumoniae (pneumococcus) and Haemophilus influezae.  White blood cells (wbc) and epithelial cells also present as one might expect.
(1000X, Gram Stain, Nikon)

Several individual yeast cells and the long pseudohyphae present seen in a vaginal swab specimen.  Epithelial cells and commensal bacteria also present.
(1000X, Gram Stain, Nikon)

Yeast pseudohyphae in a kidney aspirate.
(1000X, Gram Stain, Nikon)

Another view (as above) of the yeast peudohyphae in the kidney aspirate, criss-crossing the photo.
(1000X, Gram Stain, Nikon)

Rather elongate yeast cell(s) in a blood culture.  Identification was not noted.
(500X, Gram Stain, Nikon)

Candida parapsilosis in a blood culture.
(1000X, Gram Stain, Nikon)

Another photo as above - budding Candida parapsilosis in a blood culture.
(1000X, Gram Stain, Nikon)

This bizzare looking structure was from a direct gram stain of a blood culture and grew Candida albicans.  It appears as if the yeast cells became clumped in this material (?) and were producing pseudohyphae seen as the 'branches' protruding outwards.
(250X, Gram Stain, DMD-108)

Another view as above.  Pseudohyphae protruding from material clumped together in a blood culture.
(400X, Gram Stain, DMD-108)

Fungal conidium germinating in an ear.  Two true hyphae can be seen extending from the conidium.  Single epithelial cell seen in upper center.
(400+10X, Gram Stain, DMD-108)

Another photo of the ear swab (as above), here showing the dichotomous branching of the fungal hyphae.  Septations within the hyphae are visible.
(400X, Gram Stain, DMD-108)

...And yet one more as above.
(400X, Gram Stain, DMD-108)

Fungus present in the gram stain from a nasal aspirate.  This isolate was identified subsequently identified as Aspergillus flavus.
(1000X, Gram Stain, DMD-108)

Another photo of the nasal aspirate showing the fungal hyphae.  Gram's crystal violet stain is strongly retained in this preparation resulting in the intense dark colour.
(1000X, Gram Stain, DMD-108)

I took several photos of this structure yet all seem to be somewhat out of focus.  This appears to be the intact 'fruiting head' of an Aspergillus species as seen in the direct preparation of a broncheal wash.  (1000X, Gram Stain, Nikon)

Here we have a fungus (dermatophyte) seen in an unstained nail preparation.  The nail specimen is covered with 10% potassium hydroxide (KOH) which both softens & clarifies the nail for enhanced viewing as well as kills the fungus for safety.
(400X, 10% KOH, DMD-108)

Unspecified tissue containing fungal hyphae (arrows)
(400X, 10% KOH, DMD-108)

Aspergillus fruiting structure and dispersed conidia.  The inset is from another photo of the same structure at an alternate focus.  I believe this specimen was from a brocheal specimen.
(Specifics not noted, Nikon)

Fungus in a tissue sample.  This histological section shows the hyphae sliced along their length (L=Longitudinal), as well as across their diameter (T=Transverse)
(1000X, Periodic Acid Schiff, Nikon)

Fungal elements seen in this histological specimen appear black against the green counterstain.
(1000X, Gomori Silver Stain, DMD-108)

Another tissue sample with fungal elements as above.
(1000X, GMS, DMD-108)

Cryptococcus neoformans in a blood culture.  This direct stain shows the large capsule which surrounds the yeast cell of C.neoformans.
(1000X, Gram Stain, Nikon)

Cryptococcus neoformans again in the blood culture as above.  Again, material surrounding the yeast cell clearly shows the capsule that surrounds the organism.
(1000X, Gram Stain, Nikon)

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Sunday 29 November 2015

Stachybotrys species

Stachybotrys species

Stachybotrys is a common cosmopolitan saprobe (lives on decaying vegetative material) which can be found in soil.  It is frequently found in the indoor environment, particularly in damp areas.  Anyone who has removed water damaged wallpaper or lifted soggy cardboard boxes and found black discolouration on the material, may very well have encountered Stachybotrys mould.  It seems to particularly like to feast on the glues used in wallpaper and paper tape adhesives.  That powdery black residue on water damaged books may also yield Stachybotrys.  Stachybotrys may contain up to fifty species though I suspect this is under review using molecular methods.

Stachybotrys has long been considered as non-pathogenic to humans or animals.  More recently, evidence has been gathering that, although not a source of topical or systematic infection, the mould may be responsible for toxicosis following inhalation of the spores.  It has been suspected as a cause of acute idiopathic pulmonary haemorrhage in infants, but its complete relation to human diseases is not yet fully understood.

The fungus has also been implicated in what has been termed ‘sick building syndrome’.  Buildings which have an overall moisture problem, or those that have had been damaged by flood waters may be prone to extensive Stachybotrys (and other) fungal invasion.  Wall coverings, ceiling tiles, and gypsum (sheet rock) wall boards and jute floorcoverings may all be contaminated.  Unless extensive renovations are undertaken, occupants may develop fatigue, headaches, chest tightness, mucous membrane irritation and pulmonary disease as a result of the fungal infestation.

Animals and horses in particular, may develop, what has been termed stachybotryotoxicosis after ingestion of feed (hay, etc.) heavily contaminated with Stachybotrys chartarum.  It is characterized as an irritation of the mouth, throat, and nose which may lead to dermal necrosis and possible shock.  Stachybotrys chartarum is known to produce a variety of toxins; macrocytic trichothecenes and related trichoverroids: roridin E and L-2; satratoxins F, G, and H; isosatratoxins F, G, and H; verrucarins B and J; and the trichoverroids, trichoverrols A and B and trichoverrins A and B.

According to the Center for Disease Control and Prevention, "The term 'toxic mould' is not accurate. While certain moulds are toxigenic, meaning they can produce toxins (specifically mycotoxins), the moulds themselves are not toxic, or poisonous.

Macroscopic Morphology:

Note:  Sadly I have no photos of Stachybotrys colonies to share with the reader.  A colleague provided swabs from blackened areas on damp washroom wallboard during a renovation.  Days later, a quick adhesive tape mount of preliminary growth revealed the Stachybotrys posted below.  Unfortunately it was quickly overgrown by other moulds present in the same sample and at a particularly busy time in the lab, I could not devote more time to its isolation.  My hope was to obtain the mould from some other source at a more convenient time, which sadly, never came.   'Google Images' brings up a number of images of both the colonies on mycological media as well as 'in-situ' -on walls etc.  Permissions could not be obtained to link to the photo which I felt best represented this mould's growth.

Stachybotrys exhibits rapid growth, maturing in three to four days.  It has a powdery to cottony texture.

The surface colouration is initially white but quickly becomes  black but may also exhibit pink or orange on the surface depending on the growth medium and species.

The reverse is also dark brown or black

Microscopic Morphology:

Stachybotrys has hyphae which are septate and hyaline when young but may darken with maturity.

Conidiophores may also be hyaline or may develop an olivaceous or dark pigmentation.  The conidiophores may be septate, simple or branched.  Conidiophores may show a rough-wall texture, particularly at the upper part, near the phialides.

Phialides (9-14 µm in length) may be hyaline, olivaceous to black in colour. They are ellipsoidal in shape and form in groups of 3 to 10 at the apex of the conidiophore
Phialides produce conidia singly and successively into a slime droplet that covers the phialides.

The conidia are 7 to 12 by 4 to 6 µm, black, unicellular, ellipsoidal in shape and smooth to rough-walled.  Conidia are borne in slimy masses at the apices of the phialides.

Stachybotrys species -phialides are 9-14 µm in length and are borne in slimy masses at the apices of the phialides.
  (1000X, LPCB, DMD-108)

Stachybotrys species -phialides are ellipsoidal in shape and form in groups of 3 to 10 at the apex of the conidiophore.  The phialides are 9-14 µm in length.
 (1000X, LPCB, DMD-108)
 Stachybotrys species -conidiophores may show a rough-wall texture, particularly at the upper part, near the phialides.
 (1000X, LPCB, DMD-108)

Stachybotrys species - conidiophores may be septate, simple or branched.  Here both septation and branching are clearly visible.
(1000X, LPCB, DMD-108)

 Stachybotrys species -conidia are 7 to 12 by 4 to 6 µm, black, unicellular, ellipsoidal in shape and smooth to rough-walled.  In this photo the slimy mass of conidia have been disrupted.
(1000X, LPCB, DMD-108)

 Stachybotrys species -This photo again shows a disrupted mass of ellipsoidal conidia.  The dark pigmentation obliterated fine detail.  Rough texture on the upper portion of the phialide is evident.
(1000X, LPCB, DMD-108)

Stachybotrys species -conidiophores may also be hyaline or may develop an olivaceous or dark pigmentation.  (1000X, LPCB, DMD-108)

 Stachybotrys species -hyphae are septate and hyaline when young but may darken with maturity.
 (1000X, LPCB, DMD-108)

Stachybotrys is highly cellulolytic which is why it enjoys growing on the various substrates already mentioned.  It grows quite readily in materials low in nitrogen.
Stachybotrys differs from Memnoniella by not producing conidia in chains
While both the above genera are environmental contaminants, they are seldom isolated in the clinical laboratory.
Stachybotrys grows well at 37ᵒC

One method of isolating or recovering Stachybotrys is to streak suspected conidia from the source onto dampened filter paper (eg. Whatman).  Incubate the paper at 25 to 30ᵒC for about a week and look for dark patches.  Transfer any growth to Sabouraud Dextrose Agar or Corn Meal Agar.  Some sources claim that the fungus grows better on less nutritious media and this feature may assist in isolating it from more fastidious, yet fast growing fungi.

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Saturday 28 November 2015

Saksenaea vasiformis complex

Saksenaea vasiformis complex - Zygomycete

Note:  I had been debating with myself whether to post this organism at all as I have so few photos to share.  The photos I am posting show only the most basic characteristic features that would clue you in that this is a Zygomycete.  Still, it may be of interest to some….and, what else am I going to do with the photos?

Until recently, Saksenaea vasiformis was recognized as the sole species within this genus.  Currently, three species are recognized[i] and can be differentiated by genetic, morphological and physiological characteristics.  Now recognized are; S. vasiformis which produces mainly cylindrical sporagniospores with rounded ends, S. erythrospora which has large sporangiophores and sporangia, which produce biconcave ellipsoidal sporangiospores, and S. oblongiaspora, characterized by oblong sporangiospores and its failure to grow at 42ᵒC.

Ecology:  Saksenaea species can be found worldwide, having been isolated from soils in India, Brazil, Panama, Honduras, as well as the United States.

Pathology:  Though found worldwide, Infections occur most commonly in tropical and sub-tropical climates.  Saksenaea species have been implicated in both human and animal disease.  Unlike many of the emerging fungal diseases, it appears that Saksenaea infects immunocompetent hosts more readily than those with underlying, and usually predisposing conditions.  The most common mode of infection is through some traumatic implantation of material containing the sporangiospores though the respiratory tract may be the route of infection with disseminated infections.  Clinical symptoms and presentation can be quite varied –from a slow, localized invasion to rapidly spreading disseminated infection.  The spectrum of infections described in the literature range from skin and soft tissue infection to bone (osteomyelitis) and rhino-orbito-cerebral involvement.  Skin and soft tissue infections may present with necrotizing fasciitis or cellulitis.  While infections remain localized, they may respond to a combination of necrotic tissue debridement and aggressive antifungal therapy, otherwise amputation may be the only recourse.  Overall the mortality rate as a result of infection is about 40%[ii].  Disseminated infections have a significantly higher associated mortality rate of about 75%, while rhino-orbito-cerebral infections have an estimated mortality rate of 83% based on published reports.  Thankfully, infections with Saksenaea species remain relatively uncommon.

 Saksenaea species characteristics (in general) follow

Macroscopic Morphology:
A rapidly expanding fluffy, spider-like colony on Sabouraud Dextrose Agar incubated at 30ᵒC.  The growth may fill the Petrie dish within 48 hours.  It may be a “lid-lifter” and growth attempt to escape through poorly sealed plates after 72 plus hours.  The colony appears off-white to greyish in colour.

 Saksenaea species - 48 hours growth on SAB (or SDA) at 30ᵒC (Nikon)

Saksenaea species -as above, but an oblique view (Nikon)

Saksenaea species - 4 days growth on SAB (or SDA) at 30ᵒC (Nikon) 

Microscopic Morphology:
Saksenaea produces broad hyaline, mostly aseptate, hyphae.  Simple and unbranched sporangiophores (24 – 64 µm in length), develop a flask-shaped sporangium (50 – 150 µm in length).  The base of the sporangium above the hemispherical collumella is rather broad or swollen in appearance but narrows down into a long neck towards the apex.  Smooth walled sporangiospores (1.5 – 2.0 X 3 – 4 µm) vary in shape depending on the species as described above in ‘Notes’.  When mature, the sporangiospores are released through the top of the sporangiophore.  Dichotomously branching rhizoids develop at the base of the sporangiophore.  If this Zygomycete has produced its typical fruiting structure (sporangium), the overall structure of Saksenaea is unique and easy to identify.  But there lies the problem…

It is rather difficult to induce Saksenaea to induce produce fruiting structures and subsequent sporulation.  Let’s back up a bit…

If you get a rapidly growing, fluffy colony which produces broad (wide) hyphae, there is a good chance you have a Zygomycete.  If you place the mould on relatively rich mycological media, such as Sabouraud Dextrose Agar or Potato Dextrose Agar, and don’t get fruiting structures produced, you may have either a Saksenaea species or Apophysomyces species.  Both are notoriously suborn when it comes to inducing sporulation on richer mycological media.  Media such as Czapek agar may produce better results.  If this fails and you have lots of patience, you can try the agar block – sterile water technique as outlined by Padhye & Ajello[iii].  While the experienced mycologist may be able to tell Apophysomyces from Saksenaea without inducing sporulation, it is valuable in further speciation when not resorting to other means of identification such as molecular.

Although I attempted the agar –sterile water technique described by Padhye & Ajello, I was not successful in inducing sporulation and therefore the distinctive flask-like sporangium was not produced, nor the spores within.  Features not produced cannot be photographed and I regret that I am unable to share them with you.  As I work in a clinical laboratory, we carry only basic media and time is limited –I regret that I could not continue to pursue this challenge.

Because the organism could not be induced to produce its fruiting structures and sporangiospores, it was impossible to use these morphological structures to determine the specific identification of this Saksenaea species.
Saksenaea species - so here is about all I could get.  The photo above shows the sporangiophore with the hemispherical shaped collumella at the apex (top).  From the inset photo you can see what the fungus would look like had it produced (or retained) the flask-like sporangium.
(400+10X, LPCB, DMD-108)

Important:  For the first time ever, I have posted a photo on this blog site which I have not personally taken.  I felt that I could not post the photos I had without showing what the mature and fully intact Saksenaea looked like.  The inset photo is shown here by the kind permission of Dr. David Ellis of the University of Adelaide's Mycology Website.  My photos are free to share, however I request that this photo not be shared as the inset photo is the property of Dr. Ellis.

Saksenaea species - line drawing of structures discussed

 Saksenaea species - again we see the hemispherically shaped collumella at the apex of the sporangiophore (arrows).  Sporangium and sporangiospores are absent.
(400X, LPCB, DMD-108) 

 Saksenaea species - as above
(400X, LPCB, DMD-108)

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

Saksenaea species - a clue that this is a zygomycete is the broad, almost aseptate hyphae that the mould produces.  The measurement seen within the large hyphae running through the photo reads 24.22 µm -that is wide!
  (400X, LPCB, DMD-108)

Saksenaea species - a close look at the hyphae in the previous photo.  Note that there is the septation at the center of the photo.  (1000+10X, LPCB, DMD-108)

 Saksenaea species - for the most part, the growth that I obtained, whether by slide culture or adhesive tape technique, appeared as above - just a tangled myceleum with no reproductive fruiting structures. (400X, LPCB, DMD-108)

So there you have it - the best I could do with the time and resources I had at hand.

[i]Molecular phylogeny and proposal of two new species of the emerging pathogenic fungus Saksenaea
Alvarez, E, Garcia-Hermoso, D, Sutton, DA, Cano, JF, Stchigel, AM. Hoinard, D, Fothergill, AW, Rinaldi, MG, Dromer, F, Guarro, J.  Microbiol 2010;48 (12):4410-16

[ii] Mucormycosis caused by unusual mucormycetes, non-Rhizopus, -Mucor, and Lichtheimia species
Gomes, MX, Lewis, RE, Kontoyiannis, DP, Clin Microbiol Rev. 2011; 24(2):411-445

[iii] Simple Method of Inducing Sporulation in Apophysomyces elegans and Saksenaea vasiformis
Arvind A. Padhye and Libero Ajello; Journ Clin Micro, Sept 1988; 1861 - 63

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