3 weeks of incubation, growth should be present. Count the
colonies at this point. A significant reduction in CFU/mL will
indicate activity of test compound.
3.5 Lysing an NRP
Culture for Further
Analysis
1. Place an empty 50-mL conical centrifuge tube and a 50-mL
conical centrifuge tube of sterile PBS, both with their lids loose
inside the anaerobic cabinet for approximately 4 h.
2. Break wax seals on glass culture tubes inside the anaerobic
cabinet by scoring wax with a scalpel and break seal. Take due
care and caution whilst using a scalpel.
3. Pour culture into the deoxygenated, empty 50 mL conical
centrifuge tube.
4. Seal 50-mL conical centrifuge tube tightly with an excess of
parafilm (see Note 16).
5. Centrifuge at 3000 Â g, at 4
C for 10 min.
6. In the anaerobic cabinet, gently pour off the supernatant and
resuspend the pellet in equal volume of sterile, degassed PBS to
the original culture volume in step 3.
7. Repeat steps 5 and 6.
8. Centrifuge at 3000 Â g, at 4
C for 10 min.
9. In the anaerobic cabinet, resuspend the pellet in 500 μL of
deoxygenated sterile PBS with proteinase inhibitor cocktail
tablet and 500 μL of deoxygenated sterile PBS/Glass bead mix.
10. Bead beat for 30 s at 30 Hz and incubate on ice.
11. Immediately freeze at À20
C.
12. Defrost at room temperature.
13. Repeat steps 10 to 12 five times.
14. Cell lysate can then be stored on ice or at 4
C until required for
further analysis.
4 Notes
1. Do not autoclave ADC or OADC enrichment to sterilize.
Always use a filter as autoclaving will denature the enzymes in
the enrichment.
2. Screw caps should only be autoclaved twice as subsequent
rounds of autoclaving can degrade the oxygen seal of the cap.
This will be evident if you try to use this beyond this as if not
using paraffin wax methylene blue will re-colorize quickly after
exit from an anaerobic cabinet.
3. The maintenance of a hypoxic atmosphere in a glass tube or
96-well plate can be challenging. For glass tubes, consider
using high-grade paraffin wax (melting temperature > 70
C).
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Savannah E. R. Gibson et al.
colonies at this point. A significant reduction in CFU/mL will
indicate activity of test compound.
3.5 Lysing an NRP
Culture for Further
Analysis
1. Place an empty 50-mL conical centrifuge tube and a 50-mL
conical centrifuge tube of sterile PBS, both with their lids loose
inside the anaerobic cabinet for approximately 4 h.
2. Break wax seals on glass culture tubes inside the anaerobic
cabinet by scoring wax with a scalpel and break seal. Take due
care and caution whilst using a scalpel.
3. Pour culture into the deoxygenated, empty 50 mL conical
centrifuge tube.
4. Seal 50-mL conical centrifuge tube tightly with an excess of
parafilm (see Note 16).
5. Centrifuge at 3000 Â g, at 4
C for 10 min.
6. In the anaerobic cabinet, gently pour off the supernatant and
resuspend the pellet in equal volume of sterile, degassed PBS to
the original culture volume in step 3.
7. Repeat steps 5 and 6.
8. Centrifuge at 3000 Â g, at 4
C for 10 min.
9. In the anaerobic cabinet, resuspend the pellet in 500 μL of
deoxygenated sterile PBS with proteinase inhibitor cocktail
tablet and 500 μL of deoxygenated sterile PBS/Glass bead mix.
10. Bead beat for 30 s at 30 Hz and incubate on ice.
11. Immediately freeze at À20
C.
12. Defrost at room temperature.
13. Repeat steps 10 to 12 five times.
14. Cell lysate can then be stored on ice or at 4
C until required for
further analysis.
4 Notes
1. Do not autoclave ADC or OADC enrichment to sterilize.
Always use a filter as autoclaving will denature the enzymes in
the enrichment.
2. Screw caps should only be autoclaved twice as subsequent
rounds of autoclaving can degrade the oxygen seal of the cap.
This will be evident if you try to use this beyond this as if not
using paraffin wax methylene blue will re-colorize quickly after
exit from an anaerobic cabinet.
3. The maintenance of a hypoxic atmosphere in a glass tube or
96-well plate can be challenging. For glass tubes, consider
using high-grade paraffin wax (melting temperature > 70
C).
256
Savannah E. R. Gibson et al.