10 min at room temperature and under mild agitation
(70 rpm).
6. Wash thoroughly the gel with ultrapure water (three times
10 min in 50 mL of water).
7. Incubate the gel in the revelation solution for 1–5 min at room
temperature without agitation. Stop the reaction by discarding
the revelation solution and replacing it with water as soon as
the first bands of LPS become visible (see Notes 22 and 23).
4 Notes
1. The first dangerous step of the protocol is the centrifugation of
the bacterial cultures. Indeed, the bacterial pellet is composed
of around 10
12 bacteria, and hence there is a high risk of
contamination. Such step must be performed under a PSM to
ensure the purity of the bacterial pellet and to prevent contamination of the experimenter. Precaution measures also include
wearing safety lab coat and glasses and wearing two pairs of
gloves (including one that properly covers the wrist).
2. The extraction protocol involves the use of large volumes of
phenol, which is a highly toxic chemical component classified as
a CMR (carcinogenic, mutagenic and reprotoxic) substance.
Phenol is toxic by inhalation, toxic by ingestion, and toxic by
skin absorption, corrosive, and mutagen. Extra precautions
must be taken when manipulating:
l
Work exclusively under a functional chemical hood.
l
Wear a double pair of neoprene gloves.
l
Wear a cotton lab coat and safety glasses.
In case of contact with the skin, eyes, or mouth, rinse immediately with water and consult a physician in emergency. In
addition, all the phenol-contaminated wastes must be properly
discarded in the CMR trashes, away from any heat-inducing
material. Obviously, this note concerning the safety precautions linked to the usage of phenol is not exhaustive and one
must consult the chemical data sheet associated with the product before use.
3. The silver staining analysis of the LPS requires the use of
various chemicals that are toxic and corrosive. First of all, the
periodic acid used to oxidize the gel is an oxidizer and is highly
corrosive for the skin and eyes (always be reminded that acid
must be poured in water and not the other way around). The
silver nitrate solution presents the same harmful properties, but
in addition, it is explosive. Once again, the safety precautions
require wearing a cotton lab coat as well as gloves covering the
Purification of LPS from Leptospira
61
(70 rpm).
6. Wash thoroughly the gel with ultrapure water (three times
10 min in 50 mL of water).
7. Incubate the gel in the revelation solution for 1–5 min at room
temperature without agitation. Stop the reaction by discarding
the revelation solution and replacing it with water as soon as
the first bands of LPS become visible (see Notes 22 and 23).
4 Notes
1. The first dangerous step of the protocol is the centrifugation of
the bacterial cultures. Indeed, the bacterial pellet is composed
of around 10
12 bacteria, and hence there is a high risk of
contamination. Such step must be performed under a PSM to
ensure the purity of the bacterial pellet and to prevent contamination of the experimenter. Precaution measures also include
wearing safety lab coat and glasses and wearing two pairs of
gloves (including one that properly covers the wrist).
2. The extraction protocol involves the use of large volumes of
phenol, which is a highly toxic chemical component classified as
a CMR (carcinogenic, mutagenic and reprotoxic) substance.
Phenol is toxic by inhalation, toxic by ingestion, and toxic by
skin absorption, corrosive, and mutagen. Extra precautions
must be taken when manipulating:
l
Work exclusively under a functional chemical hood.
l
Wear a double pair of neoprene gloves.
l
Wear a cotton lab coat and safety glasses.
In case of contact with the skin, eyes, or mouth, rinse immediately with water and consult a physician in emergency. In
addition, all the phenol-contaminated wastes must be properly
discarded in the CMR trashes, away from any heat-inducing
material. Obviously, this note concerning the safety precautions linked to the usage of phenol is not exhaustive and one
must consult the chemical data sheet associated with the product before use.
3. The silver staining analysis of the LPS requires the use of
various chemicals that are toxic and corrosive. First of all, the
periodic acid used to oxidize the gel is an oxidizer and is highly
corrosive for the skin and eyes (always be reminded that acid
must be poured in water and not the other way around). The
silver nitrate solution presents the same harmful properties, but
in addition, it is explosive. Once again, the safety precautions
require wearing a cotton lab coat as well as gloves covering the
Purification of LPS from Leptospira
61