wrist and safety glasses. The silver nitrate wastes must be evacuated in a dedicated appropriate container and must never be
close to heating material. Once again, this note is not an
exhaustive review of the necessary precautions associated with
silver staining analysis, and consulting the data sheets associated with these chemicals is highly recommended.
4. To purify leptospiral LPS, one must consider the following
schedule (Fig. 4):
l
One to two weeks to expand the leptospiral culture.
l
Half a day to perform the centrifugation and obtain the
bacterial pellet.
l
Three hours to perform the hot water/phenol extraction.
l
One to two whole days of dialysis (timing can be reduced to
less than 24 h by frequent change of the water or increasing
the volumes used. Also, pre-heating the water that will be
used is also a good way to gain time).
l
Two days for the ultracentrifugations.
l
One night for the lyophilization of the LPS.
l
Two days for the silver staining analysis that required an
overnight incubation.
5. The duration of the bacterial culture incubation depends on
the growth rate of the strain. It is recommended to pellet the
bacteria in late exponential phase.
Fig. 4 Example schedule of the whole process of the LPS extraction, from the bacterial culture inoculation to
the silver staining analysis
62
Delphine Bonhomme and Catherine Werts