|Fungi and Disease|
Dr. Gislason's 2-Slide Culture of Blastomycosis
Quick Identification of Fungal Infection in Sputum Samples
The diagnosis of fungal infections of the lung are 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:
I developed a nose, throat and lung infection in the summer of 2006. I realized that I had an infection that I did not recognize which would be difficult to diagnosis. I would cough up clumps of thick, white mucous. Sputum cultures were reported as negative and although I had many blood abnormalities, none pointed to a specific infection. Fortunately, I am a curious and have a lab with microscopy capabilities.
I discovered a simple culture technique: I added one or two drops of distilled water and spread the thick 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 I observed that 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.
Migration and aggregation of yeast cells
Within a few hours, the yeast cells clustered and formed 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 developing.
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. While you might see yeast cells in this dense aggregation, there became more obvious as they migrated away form the other cells and clustered.
To view the fresh, alive 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 sometimes develop days later. Further growth irregularities were probably caused by itraconazole which I was taking when the samples were collected.
After hours to days, I would divide some slides and examine them under higher power. I would stain some slides and examine others without staining. The following photomicrographs were of unstained specimens. Under the microscope the early stages (4hours) of yeast cell aggregation and transformation, looked like this:
Blastomycosis yeast cells transforming into mycelial form... hyphus creation at 6 hours.The yeast cells, elongate, flatten and eventually disappear, leaving behind relatively cellular chitin tubes that grow outward and branch. 2-slide culture image
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.
A profusion of fungi exists in the environment. Some fungi are able to cause an invasive infection in otherwise healthy individuals. Other fungi are opportunistic fungi that become invasive when immune defenses are compromised. Diagnosis of fungal infection is difficult. There are many problems when you try to connect a test result to a disease. Fungi are so abundant and there are so many varieties in every environment that it is seldom easy to pick just one cause among many. Fungi are inhaled and ingested. Foods always contain fungal spores and actively growing molds. Attempts to culture fungi often fail; only a small number grow in the culture media commonly used. Some new methods of detecting fungal DNA may be useful but development of reliable tests is slow and expensive.
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