∗Count all bacteria within the center square millimeter
(1 mm  1 mm area), which is ruled into 25 groups of
16 small squares.
∗∗50,000 ¼ 50 (cell depth is 1/50) Â 1000
(1000 mm
3
¼ 1 mL)
3. Incubate bacteria (5 Â 10
7 to 5 Â 10
8 ) in NHS (final volume of
1 mL) for 60 min or 120 min at 37
C. A serum concentration
between 20 and 60% is generally adopted in the majority of
protocols assessing leptospiral serum resistance [4, 5, 13]. As a
control, incubate the same number of spirochetes in 20–60%
HI-NHS for 60 or 120 min at 37
C.
4. Assess leptospiral survival after each treatment by counting
viable bacteria as described above. Viable leptospires keep
their movement and helicoidal shape, while dead leptospires
lose their characteristic form as a result of fragmentation, as
mentioned above. Serum sensitivity is estimated by comparing
the counts in NHS-incubated samples to those incubated in
HI-NHS (100% survival). At least three independent experiments must be performed.
3.2 Acquisition
of Complement
Regulatory Proteins by
Leptospira
1. Prepare SDS polyacrylamide gel (1 mm thickness) prior to the
assay. Glass plates (10 cm  8 cm) must be cleaned with ethanol
before assembling casting stand. For running two 12%
SDS-PAGE, prepare 10 mL of the resolving (running) gel by
mixing 3.4 mL of ddH 2 O, 4 mL of 30% Acrylamide/Bis,
2.5 mL of resolving gel buffer, and 0.1 mL of 10% SDS. Add
0.1 mL of 10% APS and 0.004 mL of TEMED shortly before
pouring the gels. Pour 5 mL of the running gel into each set of
glass plates, add 0.1 mL of isopropanol to prevent dehydration,
and allow polymerizing for 40–60 min. Remove isopropanol
and place a filter paper between the glass plates to dry. For two
stacking gels, mix 2.7 mL of ddH 2 O, 0.67 mL of 30% Acrylamide/Bis, 0.5 mL of stacking gel buffer, and 0.04 mL of 10%
SDS. Add 0.04 mL of 10% APS and 0.004 mL of TEMED
shortly before pouring the gels. For each gel, pour approximately 1–2 mL over the running gel, and insert the comb.
Once polymerized, assemble the running unit, fill both the
upper and lower compartments with the running buffer, and
remove the comb.
2. Harvest leptospires by centrifugation at 5500 Â g for 25 min.
Discard supernatant, and wash the pelleted bacteria three times
in 20 mL of CFD.
3. Incubate 10
8 to 10
9 leptospires in NHS-EDTA for 60 min at
37
C with gentle agitation. For practical reasons, total volume
must not exceed 1 mL.
Complement Resistance Assays
193
(1 mm  1 mm area), which is ruled into 25 groups of
16 small squares.
∗∗50,000 ¼ 50 (cell depth is 1/50) Â 1000
(1000 mm
3
¼ 1 mL)
3. Incubate bacteria (5 Â 10
7 to 5 Â 10
8 ) in NHS (final volume of
1 mL) for 60 min or 120 min at 37
C. A serum concentration
between 20 and 60% is generally adopted in the majority of
protocols assessing leptospiral serum resistance [4, 5, 13]. As a
control, incubate the same number of spirochetes in 20–60%
HI-NHS for 60 or 120 min at 37
C.
4. Assess leptospiral survival after each treatment by counting
viable bacteria as described above. Viable leptospires keep
their movement and helicoidal shape, while dead leptospires
lose their characteristic form as a result of fragmentation, as
mentioned above. Serum sensitivity is estimated by comparing
the counts in NHS-incubated samples to those incubated in
HI-NHS (100% survival). At least three independent experiments must be performed.
3.2 Acquisition
of Complement
Regulatory Proteins by
Leptospira
1. Prepare SDS polyacrylamide gel (1 mm thickness) prior to the
assay. Glass plates (10 cm  8 cm) must be cleaned with ethanol
before assembling casting stand. For running two 12%
SDS-PAGE, prepare 10 mL of the resolving (running) gel by
mixing 3.4 mL of ddH 2 O, 4 mL of 30% Acrylamide/Bis,
2.5 mL of resolving gel buffer, and 0.1 mL of 10% SDS. Add
0.1 mL of 10% APS and 0.004 mL of TEMED shortly before
pouring the gels. Pour 5 mL of the running gel into each set of
glass plates, add 0.1 mL of isopropanol to prevent dehydration,
and allow polymerizing for 40–60 min. Remove isopropanol
and place a filter paper between the glass plates to dry. For two
stacking gels, mix 2.7 mL of ddH 2 O, 0.67 mL of 30% Acrylamide/Bis, 0.5 mL of stacking gel buffer, and 0.04 mL of 10%
SDS. Add 0.04 mL of 10% APS and 0.004 mL of TEMED
shortly before pouring the gels. For each gel, pour approximately 1–2 mL over the running gel, and insert the comb.
Once polymerized, assemble the running unit, fill both the
upper and lower compartments with the running buffer, and
remove the comb.
2. Harvest leptospires by centrifugation at 5500 Â g for 25 min.
Discard supernatant, and wash the pelleted bacteria three times
in 20 mL of CFD.
3. Incubate 10
8 to 10
9 leptospires in NHS-EDTA for 60 min at
37
C with gentle agitation. For practical reasons, total volume
must not exceed 1 mL.
Complement Resistance Assays
193