Biology Reference
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3. Methods
The methods described outline treatment (1) of cells with oxidants, (2) identi-
fication of DNA damage, (3) quantitation of DNA damage, (4) identification
of lipid damage, and (5) identification of lipid peroxidation intermediates.
3.1. Treatment of Borrelia Cells with Oxidants
1. Degas (1) BSK II liquid medium and (2) sterile side-arm and 25 mL serum bottles
overnight under an atmosphere of 5% CO 2 ,4%H 2 , and 91% N 2 to achieve 0%
oxygen.
2. Transfer 5 mL of anaerobic BSK II to a sealed 25-mL serum bottle and inoculate
with 5 × 10 5 cells of low-passage Borrelia . Incubate at 34 °C for 4-6 days or until
cells reach approximately5×10 7 cells/mL. Enumerate cells using a dark-field
microscope.
3. To expand the culture, transfer fresh anaerobic BSK II to a sealed side-arm bottle
and inoculate to a final concentration of1×10 5 cells/mL. Incubate at 34 °C for
2-3 days or until cells reach approximately5×10 7 cells/mL.
4. To test the sensitivity of Borrelia to oxidants, such as t -butyl peroxide, split the
culture into sterile, sealed side-arm bottles, and treat 1 bottle with 2 m Mt -butyl
peroxide. Incubate all bottles at 34 °C for 4 h.
5. During incubation, prepare P-BSK solid-plating medium and pour the 15-mL
bottom layer.
6. After the incubation, prepare four serial 10-fold dilutions of the cultures into fresh
BSK II.
7. For each dilution, add 100 μL of cells to 15 mL of the P-BSK-plating medium
and pour onto the previously prepared bottom layers. Allow to solidify at room
temperature.
8. Incubate in an anaerobic gas jar with an H 2 +CO 2 generator anaerobic envelope
at 34 °C for 7-14 days.
9. To determine sensitivity, count the number of colonies on treated plates versus
the number of colonies on untreated plates.
3.2. Identification of DNA Damage
This protocol is a quick way to determine whether DNA damage has
occurred. If DNA damage is present, DNA polymerase will not be able to extend
the reactions, and therefore, no product is generated. However, to quantitate
the damage, the next protocol should be used.
1. Treat cells as described above.
2. After the incubation, return the cultures to the anaerobic chamber, and transfer to
centrifuge bottles.
3. Harvest the cells at 3000 × g for 20 min at 4 °C. In the anaerobic chamber, remove
the supernatant and wash the pellets thrice in degassed HEPES/NaCl buffer.
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