Saturday, 14 August 2010

Chaetomium Species

Chaetomium Species

Had some fun playing with this fungus in getting the ascoma (perithicium) to develop for these photographs. Still trying a few tricks so additional photos may be posted later.

In the clinical laboratory, media available is geared towards economical, rapid and efficient identification of the most common human pathogens. Unfortunately, resources (specialized media) are scarce if one wishes to experiment with ‘environmental’ organisms. Although generally considered to a saprophyte, these days virtually any organism can be responsible for disease in the imunocompromised host. Chaetomium has been implicated as agents of onychomycosis, peritonitis as well as having caused cutaneous lesions.

There are over 180 known species of Chaetomium, many of which are active in the breakdown of cellulose in the environment. Items such as paper or textiles in contact with soil, straw, dung etc decompose in part, due to the action of Chaetomium.

I believe the species I have here is Chaetomium globosum, one of the most common and widely distributed species of Chaetomium.

Pale yellow to a greyish-green depending on media and length of growth. Relatively rapid growth. Does not grow at 42 Celcius.

Hyaline septate hyphae. Ascoma (Perithecia) are spherical to ovoidal to obovoidal (175 - 200 µm in length) with numerous hairs, usually unbranched, flexsulose, undulating or coiled, septate, brownish in colour and up to 500 µm in length.
Asci are clavate (30 - 40 X 11 - 16 µm in size) containing eight brownish limoniform (in face view) ascospores 9 - 10 X 8 - 10 µm in size.

Photographs below were taken using sticky-tape preparations, slide cultures on SAB media as well as growing the fungus on somewhat nutritionally deficient Corn Meal Agar, partially covered with a cover slip to vary the atmospheric tension. Ascoma were best seen on the slide culture and at the edges of the cover slip after about 14 days of incubation.

Still haven’t managed to induce and photograph the asci and ascospores. From what I understand, the fruiting of Chaetomium in culture can be stimulated by the addition of cellulose in the form of filter paper, cloth or jute fiber. It seems that compounds excreted by the fungus Aspergillus fumigatus can stimulate fruiting as well. Sugar phosphates and phospho-glyceric acid, by-products produced by A. fumigatus have been shown to stimulate the production of asci and ascospores. Calcium may also have an effect on fruiting. Perhaps I’ll play around with a few of these to see if I can induce fruiting structures for future photographs.

Plate at right is Chaetomium on SAB after about 96 hours incubation at 30 C.

Chaetomium in slide culture growing on edge of corn meal agar as seen after about 14 days incubation at 30C (X100)

Chaetomium ascoma on Corn Meal Agar at about 10 -14 Days (x100)

Chaetomium ascoma

I just think they look cute! Only a microbiologist would understand...

Lacto Phenol Cotton Blue (LPCB) Tape Preparation

Chaetomium ascoma and ascospores ejected at top

Slide Culture (SAB) ~12 Days -x100

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Tuesday, 10 August 2010

Klebsiella pneumoniae Capsule

Klebsiella pneumoniae Capsules
Bacteria (Gram Negative Bacillus)

In a previous post I discussed the Quellung Reaction which is used to visualize capsules produced by the gram positive organism Streptococcus pneumoniae (pneumococcus), There I mentioned that organisms other than the pneumococcus are capable of producing a capsule that surrounds the bacterial cell. Bacteria strains of E.coli, Klebsiella pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa and Cryptococcus neoformans are capable of capsular production.

Bacterial capsules, often referred to as the Glycocalyx, are generally composed of polysaccharides with a high percentage of water, however other materials such as polypeptides and hyaluronic acid may be components.

Capsules may be beneficial to the cell because;
  • Capsules may provide a virulence factor by protecting the bacterial cell from destruction by phagocytes, thereby allowing it to survive and spread.
  • The high water content may protect the cell from dehydration, from hydrophobic agents such as detergents and their own parasitization by bacteriophages (viruses). The high water content also makes the capsule difficult to stain with conventional microbiological stains.
Although the capsular polysaccharides are difficult to stain directly using stains common to the microbiological lab, they may be visualized indirectly through negative staining(1). This may be done deliberately(2) or may be seen as an incidental effect through a routine staining process.

Below is a routine gram stain of a patient’s sputum specimen. On examination the presence of numerous coliform-like gram negative bacilli were noted. Occasionally, where conditions were just right, the dark red (gram negative) colour of the bacterial cell and the varying shades of the sputum can be seen separated by a small zone. This is the bacterial capsule and its presence in this case, along with the size of the bacterial cell, would provide immediate suspicion that this organism may be Klebsiella pneumoniae. Subsequent culture confirmed the initial suspicion of the organism responsible for this respiratory infection,

Here the capsule is visualized without using antibodies as with the Quellung reaction with Streptococcus pneumoniae. Antibodies against K. pneumoniae could be produced to enhance appearance of the capsule. These may be manufactured and utilized in research however they offer little to the diagnosis and management of K. pneumoniae in the clinical setting, The cost would preclude their use in routine diagnosis.

Streptococcus pneumoniae’s capsule may also be seen in the gram stain where conditions are right however, quick and confident diagnosis justifies obtaining and using Quellung antisera.

Gram Stain of Sputum Specimen Showing Capsules Surrounding a Gram Negative Bacillus

(1) Negative staining is a technique where you don’t stain what you wish to visualize but rather you stain everything around it thereby enhancing the contrast and making the object more visible.

(2) An example of deliberate negative staining - using Nigrosin (or India Ink) to visualized the capsule surrounding the yeast Cryptococcus neoformans. In a darkly stained field, the yeast cells would be seen as a natural or neutral colour, with a definite clear zone seen around the outside of the cellular wall.