Saturday, February 21, 2009

Challenges of live microscopy

I have just completed the observation of prostatic fluid, incubated at 37 C for 24 hours.
The object of this task, is to find any signs of apicompexia in the sample. The sample is much like surum, in that the contents contain many immune cells of haemotopoietic lineage. As mentioned in previous post, the cells are lymphocytes, monocytes, neutrophilic granulocytes, monocytic granulocytes, and mast cells.

The cells in the sample, were almost all denatured due to extremely active proteolytic proteins, defensins etc.
Evidence of parasitic infection, was proven at this time. There was active sporozoites present, with typical morphology. They were lancet-shaped, and banana-shaped, and some were deformed. The light reflection was very high, indicating tough or hard-like membrane. They had distinct, but low motiltiy.

Back to problems with live specimen. The sporozoites died quickly, from abrupt change in environment [presumably].
Several active life forms were found in specimen, which shared similar characteristics, and have me struggling for clear explanation.
Since the whole exercise is to find co-relationship between intestinal findings and prostate findings, my personal bias conflicts with my scientific objectivity. I am expecting to see direct morphological similarities, but this is not entirely the case.
This is what I observed:
Some of the orb-like opaque objects seen previously in\on macrophages of fresh specimen, have survived the lytic processes. Now they are maturing under glass, and "budding" exactly as yeast would. Some become very large, others are small, but both types will bud. If I had not seen this with intestinal oocsts, I would automatically consider yeast as my first guess.
There are no difference between looking at these, and looking at natural air bubbles, with the exception the live ones move. There is observable movement of small object inside the orb, moving back and forth within membrane. No other organelles are observable. When the object starts to bud from membrane, it retracts and moves about the membrane causing distortions. It is almost like something in an egg, trying to find a way out.
Other more advanced growth developments show the new bud, at one third size of mother cell, connected by a membrane. This is very similar to observation of intestinal oocysts.
The sporozoits, share same light frequencies so I am suspecting, the sporozoites emerge as singular unit from these "orbs". I don't have solid proof of this yet. I have seen these forms directly in live stool samples

There is something else. Small cell-like bodies emerged everywhere. They are dark, almost black and measure about 3 um [I am using phase contrast at full mag enhanced]; they look like another reproducing cell-type. There is obvious nuclear movement within cell, and it also produces bud, and orbiting granule, attached by small filament which is retracted back into cell. It has no light reflective qualities. I must question if it is residual host stem cell activity.
The UV absorbs some of the opaque orbs, not sure what this indicates [maybe tyrosine content].
I may attempt to place some images later.

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