BSK culture experiment introduction


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BSK Culture Experiment Introduction
BSK Culture Experiment Page 2
BSK Culture Experiment Page 3
BSK Culture Experiment Page 4
BSK Culture Experiment Page 5 Looking at coccoids attached to spiral agents
BSK Culture Experiment Page 6 Attempt at rapid culture - spiral forms produced in a week to 10 days
Biofilms Page 7 A phenomenon pre- or early treatment possibly following long infection
BSK Culture Experiment Page 8 FITC staining borrelia specific
BSK Culture Experiment Page 9 FITC staining borrelia specific high magnification and image processing
L-form Videos Blood and blood+BSK short culture time
Romanowsky Stain Thin film periferal blood smears stained with 'Diff-Quik' and viewed under darkfield
Borrelia Garinii FITC stain of culture FITC staining borrelia species specific
Borrelia Garinii FITC stain of blood FITC staining borrelia species specific

July 2009

These pages describe an experiment to see if spirochetes can be cultured from people with suspected borreliosis. I am not a scientist (my research field is psychotherapy) but I have been greatly helped by advice from a scientist with considerable laboratory experience.

Equipment:
Vickers Instruments M14/2 with darkfield modification
100x oil/iris plan objective and 15x compensating eyepiece
35w xenon HID lamp with 8,000k colour temperature (luminosity equivalent to 150w halogen)
Fuji F10 digital camera (sensitivity up to ISO1600)

Samples are from 5 people, all with long-term chronic illness, wide-ranging neuro and immune symptoms and sometimes severe disability. All have been diagnosed at some time with ME/CFS. 2 had negative and 1 equivocal ELISA tests for Lyme disease. All have either known tick bites or were very high risk for tick bite before becoming ill due to occupation and/or other activities. All sampled produced some spirochetes. The species of the spirochetes and any effect of their presence in donors blood is unknown.

Method:
1.5 to 2ml whole venous blood with EDTA
gravity-precipitated over several hours (not centrifuged)
most of the fluid part (serum/saline) pipetted off and discarded (see * below)
(removal of the topmost layer of the blood sample which includes most WBCs did not prevent spirochete culture)
various methods of lysing RBCs had disappointing results
various quantities of BSK 2 (with 6% rabbit serum) have been used from 2 to 10mls (it has been suggested by an expert that bovine serum might be better)
(all quantities of BSK appear to work, though some cultures are more productive for unknown reasons. *Some scientists have suggested using larger volumes of BSK because it dilutes any anti-spirochete components in the sample. Dr Mattman states that borrelia is a 'micro-aerophile' and thrives at a particular depth governed by oxygen absorption of the medium in a non-sealed container. I only use sealed tubes due to length of culture)
(latterly, Rifampicin added at 35 to 50 ug/ml)
(latterly, experiments adding zinc (16ug/ml), magnesium (100ug/ml), copper (0.8ug/ml) and chitosan (soluble chitin))
Incubated at room temperature 16 weeks
Fresh BSK added or bottom-most 1ml added to fresh BSK tube and incubated at 30C for a further 4 weeks
From >5 months spirochetes were often observed with more appearing over following months
(Attempts to accelerate the cultures have not been successful. Lysing, various incubation temperatures, pH experiments and different blood fractions have not achieved noticeable improvement in these experiments. Occasionally spirochetes have been observed after 8 weeks but these were scarce and very difficult to find. For some reason corkscrew-form spirochetes were very rarely observed when any erythrocytes remained, however decayed they may be. This is too bizarre for an observation but a distinct impression. Following many studies I have learned not to waste too much time examining samples before all the RBCs have dissolved.)*

*Update Dec 2011 - Dr Mattman notes in her book on Stealth Pathogens that borrelia grows in L-form in the presence of host cells - watch this space for future experiments with filtration*

Slide preparation:
approx 50ul with a 50mm coverslip
long sides of coverslip sealed with adhesive to prevent drying
if spirochetes are not observed immediately, re-examination after 24 and 48 hrs may prove interesting. (It often seems that changes due to slide preparation are a trigger for growth especially in younger cultures. Oxygen levels may be a factor, and perhaps light [i.e. a questing tick on a plant tip may have moved from shade to light], as well as temperature, pH etc. This does not alter the fact that in samples <5 months spirochetes were very rare.

Notes on microscopy:
I use comparatively low cost lenses that cannot match the quality of Olympus, Nikon etc. However, the equipment is capable of resolving detail down to around .2 microns with suitable subjects. Excellent illumination and the sensitive camera contribute to this. However, many observations suggest that there are spiral agents present that cannot be adequately visualized and this is not entirely due to their size. I suspect this may be due to the agent's having low density and reflectivity/refractivity, the latter probably being close to that of the medium. Therefore dark-field micrsocopy may be limited for observing these cultures.

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