7. Inoculate tubes with 10
5 leptospires/10 ml media (10
4 leptospires/ml) by adding 100 μl of 10
6 leptospires/ml dilution from
step 5.
3.3 Preparation
of DMCs and Surgical
Implants
1. Prepare a workspace inside the BSC but first wiping down with
70% alcohol and then laying down a sterile 12-in. square drape
to use as a workspace.
2. Place the following in the BSC: at least one disposable, individually wrapped serological pipet for each DMC; an automatic
pipettor; one sterile 50-ml conical tube for each DMC; one
sterile pack containing only scissors and blunt-end forceps for
each strain being implanted; one sterile pack containing a
complete set of surgical instruments for each strain; scalpel
blades; sutures; applicator and clips; chlorhexidine/isopropanol swab sticks; gauze (see Note 5); and two pairs of gloves for
each DMC to be implanted plus 1–2 extra pairs of gloves.
3. Expose the workspace to UV light for at least 15 min.
3.3.1 Preparation
and Filling of DMC Tubing
1. Spray the outside of dilution tubes with 70% ethanol, wipe, and
place inside BSC.
2. Put on a pair of sterile surgical gloves (see Notes 8 and 9) and
loosen the caps of a sterile 50-ml conical tube and a Leptospira
culture tube. Open a 10-ml serological pipette package and
attach the pipette to automatic pipettor. Open the flask containing DMC tubing and place the bottle cap off to one side.
3. While still wearing gloves, carefully remove a strip of tubing
from its storage container using sterile blunt-end forceps. Use
your free hand to hold the tubing between index finger and
thumb. With the other hand, remove the cap from the 50-ml
conical tube containing diluted culture and transfer up to 10 ml
to the tubing using a 10-ml disposable serological pipette
(Fig. 2a). While continuing to hold the open end of the tubing,
flatten the unfilled tubing between the fingers of your free hand
to eliminate any air bubbles.
4. Gently twist the top end of the tubing to eliminate any remaining void volume and close off the top of the DMC (Fig. 2b).
Use your thumb and index finger to hold on to the twisted area
to prevent liquid from leaking out of the DMC while you tie off
the top. Tie off the open end using a simple overhand knot
(e.g., form a loop at the top of the DMC, passing the free end
of the tubing through the loop, and then, without releasing
your thumb and index finger, gently tighten the knot)
(Fig. 2c). Bring the knot as close as possible to the liquid to
ensure that the DMC is taut; this will help with maneuvering
the chamber into the peritoneal cavity during surgery.
Cultivation of Leptospira interrogans within DMCs
235
5 leptospires/10 ml media (10
4 leptospires/ml) by adding 100 μl of 10
6 leptospires/ml dilution from
step 5.
3.3 Preparation
of DMCs and Surgical
Implants
1. Prepare a workspace inside the BSC but first wiping down with
70% alcohol and then laying down a sterile 12-in. square drape
to use as a workspace.
2. Place the following in the BSC: at least one disposable, individually wrapped serological pipet for each DMC; an automatic
pipettor; one sterile 50-ml conical tube for each DMC; one
sterile pack containing only scissors and blunt-end forceps for
each strain being implanted; one sterile pack containing a
complete set of surgical instruments for each strain; scalpel
blades; sutures; applicator and clips; chlorhexidine/isopropanol swab sticks; gauze (see Note 5); and two pairs of gloves for
each DMC to be implanted plus 1–2 extra pairs of gloves.
3. Expose the workspace to UV light for at least 15 min.
3.3.1 Preparation
and Filling of DMC Tubing
1. Spray the outside of dilution tubes with 70% ethanol, wipe, and
place inside BSC.
2. Put on a pair of sterile surgical gloves (see Notes 8 and 9) and
loosen the caps of a sterile 50-ml conical tube and a Leptospira
culture tube. Open a 10-ml serological pipette package and
attach the pipette to automatic pipettor. Open the flask containing DMC tubing and place the bottle cap off to one side.
3. While still wearing gloves, carefully remove a strip of tubing
from its storage container using sterile blunt-end forceps. Use
your free hand to hold the tubing between index finger and
thumb. With the other hand, remove the cap from the 50-ml
conical tube containing diluted culture and transfer up to 10 ml
to the tubing using a 10-ml disposable serological pipette
(Fig. 2a). While continuing to hold the open end of the tubing,
flatten the unfilled tubing between the fingers of your free hand
to eliminate any air bubbles.
4. Gently twist the top end of the tubing to eliminate any remaining void volume and close off the top of the DMC (Fig. 2b).
Use your thumb and index finger to hold on to the twisted area
to prevent liquid from leaking out of the DMC while you tie off
the top. Tie off the open end using a simple overhand knot
(e.g., form a loop at the top of the DMC, passing the free end
of the tubing through the loop, and then, without releasing
your thumb and index finger, gently tighten the knot)
(Fig. 2c). Bring the knot as close as possible to the liquid to
ensure that the DMC is taut; this will help with maneuvering
the chamber into the peritoneal cavity during surgery.
Cultivation of Leptospira interrogans within DMCs
235