7. After 1 h, transfer liquid EMJH media from one tube equally
between at least three EMJH plates with 40 μg/mL of kanamycin (i.e., approximately 250–300 μL liquid media per plate).
Agitate to uniformly spread bacteria across plate using glass
beads.
8. Remove beads from plates.
9. To avoid desiccation, wrap plates in aluminum foil and place at
30
C for at least 7–10 days (Fig. 4). After this time period,
periodically check plates for colonies under aseptic conditions
for up to for 50 days. Leptospira colonies may appear on the
plates as small, medium, or large translucent circles within the
solid media (Fig. 6) (see Note 32).
3.4 Isolating Clones
from Solid Media
and Long-Term
Storage
1. Once colonies began to appear on plates, isolate them from
solid media by using a 1 mL filter pipette tip attached to a
micropipettor by piercing the media where the colony is present (i.e., a stab). The stab will collect a cylindrical column of
agar with the transconjugant Leptospira within it (Fig. 6) (see
Notes 33–35).
2. Transfer this isolated colony/stab into 3–5 mL of pre-warmed
EMJH liquid media containing 40 μg/mL of kanamycin within
15 mL conical tubes.
Fig. 5 15 mL conical with two membrane filters in 0.8 mL of EMJH liquid
medium (without kanamycin, without DAP). This will be incubated for 1 h at
30
C with agitation
Random Transposon Mutant Library
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