Borrelia FITC stain experiment
Illumination provided by a 35w xenon HID with colour temp of 8000k. This provides lots of blue and some UV. The FITC specific filters are from a Nikon fluorescence microscope. A light-blue pre-filter is used above the lamp, the 450-490nM filter is beneath the condenser and the 520 long-pass filter is just above the objective lens.
Smears of peripheral blood or culture are fixed and stained in the normal way and viewed with a coverglass. This has the problem that inadequate removal of the stain generates considerable background fluorescence that illuminates imperfections in the coverglass which can be enough to interfere with observing.
Wet-drop staining of culture is done with around 0.2ml culture and 0.2ml stain, mixed and kept warm for 20 mins. Diluted to 2.5ml, mixed, centrifuged then drained, then repeat or use direct for wet-drop on a slide. I found that an extra wash-cycle barely reduced background fluorescence.
The conglomerations (perhaps biofilms?) are relatively scarce on the slide. Many appear misshapen, stretched and generally splattered in the production process. However, some have survived rough treatment indicating that something is holding them together quite strongly. The contents are irregular, with elongated and some spherical-looking components. I find it impossible to guess the nature/shape of the agents that form these groups. There does not appear to be a pattern.
The first picture shows some testing of fluorescence. An unstained slide from the same culture registered nothing with light-filtering. The 3 rows of pictures are of the same FITC stained wet-drop sample. When filtered for fluorescence detectable agents were sparce though very bright. The most commonly seen were the <1uM coccoids. This is a 2 year BSK culture which contained many of the spiral agents featured on previous pages.
This set of pictures is from a 6 month BSK culture of vienous blood, scale varies. This set used Koehler illumination with a brightfield condenser, which I think demonstrates the efficiency of the light-filters, though background fluorescence is more of a problem with this method.
The following experiment was to try quick detection in blood. 1ml veinous blood in an EDTA tube centrifuged and plasma transfered to a 2ml centrifuge tube. Centrifuged and top-part removed to leave around .2ml. Borrelia FITC stain (10:1 from aliquot strength) added as equal volume, kept warm and occasionally mixed for 15 minutes. Water added to make 2ml. Centrifuged and most liquid removed to leave around .2ml. Wet-drop slide then observed under fluorescence conditions.
Please see NEXT PAGE for FITC stained agents 'inside' red blood cells
Last update July 4th! 2012
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