Dr. Gislason's 2-Slide Culture of Blastomycosis

Quick Identification of Blastomycosis

The diagnosis of Blastomycosis is difficult to make. The technique described here would allow a physician or lab tech to take a sputum sample from patient and within a few hours make the diagnosis:

Sputum samples are usually a mix of saliva and mucous. I would cough up clumps of white mucous  My technique was to add one or two drops of distilled water and spread the white mucous on a glass slide. This was covered with a second slide.  The sputum specimen sandwiched between two glass slides becomes a fungal culture.

In this preparation the yeast cells would aggregate and metamorphose into a mycelia form at room temperature. All 2-slide cultures were stored at room temperature and examined at intervals.

The first event observed in the slide culture was the migration and aggregation of yeast cells. Within a few hours, the yeast cells were forming lines that became tubes in the central portions of the culture. At the periphery of the slide, yeast cells would aggregate along the edges to form single walls that grew toward each other and formed tubes toward the center. All other cells in the mucous would be trapped and digested in these dammed areas. At 24 hours hyphae were developed.

The migration and aggregation of yeast cells was helpful in their identification since infected mucous contains a dense population of mixed cells. Also mucous from the trachea and bronchi contains a variable mixture of inhaled particles, pollens and spores.

To view the fresh specimen, no staining is required. The microscope condenser should be low and the diaphragm closed to enhance contrast. I examine the slide first with low power lens (40X) and then at 160 X. The specimen then remains alive and has a depth of field. Here is an example of the 2-slide culture after 4 days at room temperature. A branching root-like growth is obvious. This, of course, is not a traditional fungal colony that grows in 3 dimensions on an agar medium, but a novel two-dimension growth pattern that occurs in a natural culture medium (sputum). In the longest-lived cultures, tangled mycelial growth appears and conidiophores develop. Further growth abnormalities were probably caused by itraconazole which I was taking when the samples were collected.

Closeup, contrast enhanced image

At 36 hours

1000x image of hyphus after 36 hours. Note double walls and and open end. It appears that digestive chemicals and enzymes are secreted by the tubes, digesting material from the adjacent area. As the hyphae develop, the slide is cleared of mucous and other cells.

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