BSK culture experiment introduction
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BSK Culture Experiment 2
BSK Culture Experiment 3
BSK Culture Experiment 4
BSK Culture Experiment 5
BSK Culture Experiment 6
BSK Culture Experiment 7
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)
(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.)
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 [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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BSK Culture Experiment 2
BSK Culture Experiment 3
BSK Culture Experiment 4
BSK Culture Experiment 5
BSK Culture Experiment 6
Updated November 2011