4. Incubate E. coli at 37
C with or without agitation until it
reaches OD 420 of 0.2–0.5, which correlates to
mid-exponential growth (typically 2–4 h) (see Note 18).
5. Check E. coli and Leptospira by dark-field microscopy to ensure
both samples demonstrate good motility, are not contaminated, and are in mid-exponential growth (e.g., approximately
2–3 Â 10
8 cells/mL) before proceeding to conjugation.
3.2 Conjugation:
Day 1
1. Set up filtration apparatus within the biosafety cabinet
(Fig. 1a). Apply a vacuum to the apparatus by using an in-line
or vacuum pump aspiration unit (see Notes 19 and 20).
2. Sandwich membrane filter between filter holder base with frit
and stopper and glass funnel. Hold this system in place using
the clamp to create a temporary seal between the glass pieces
(Fig. 1b).
3. Add 1:10 ratio of E. coli containing EMJH media to Leptospira
containing EMJH media by placing both into the filtration
apparatus via the 15 mL funnel (e.g., 0.5 mL E. coli to
4.5 mL of Leptospira) (see Notes 21 and 22).
4. Allow all media to flow through membrane filter into filtration
flask, depositing E. coli and Leptospira on the upper facing
surface of the membrane filter (see Note 23).
5. Once flow-through is complete and no bubbles/liquid remains
on membrane filter surface, remove clamp and funnel.
6. Remove membrane filter with deposited bacteria from glass frit
filter using forceps (Fig. 2) (see Note 24).
Fig. 1 (a) Complete filtration apparatus with membrane between frit and funnel.
(b) Demonstration of applying a membrane filter to the frit and stopper of the
filtration apparatus. Ensure aspiration is applied to system to hold membrane in
place and secure funnel to top with clamp
82
Christopher J. Pappas et al.
C with or without agitation until it
reaches OD 420 of 0.2–0.5, which correlates to
mid-exponential growth (typically 2–4 h) (see Note 18).
5. Check E. coli and Leptospira by dark-field microscopy to ensure
both samples demonstrate good motility, are not contaminated, and are in mid-exponential growth (e.g., approximately
2–3 Â 10
8 cells/mL) before proceeding to conjugation.
3.2 Conjugation:
Day 1
1. Set up filtration apparatus within the biosafety cabinet
(Fig. 1a). Apply a vacuum to the apparatus by using an in-line
or vacuum pump aspiration unit (see Notes 19 and 20).
2. Sandwich membrane filter between filter holder base with frit
and stopper and glass funnel. Hold this system in place using
the clamp to create a temporary seal between the glass pieces
(Fig. 1b).
3. Add 1:10 ratio of E. coli containing EMJH media to Leptospira
containing EMJH media by placing both into the filtration
apparatus via the 15 mL funnel (e.g., 0.5 mL E. coli to
4.5 mL of Leptospira) (see Notes 21 and 22).
4. Allow all media to flow through membrane filter into filtration
flask, depositing E. coli and Leptospira on the upper facing
surface of the membrane filter (see Note 23).
5. Once flow-through is complete and no bubbles/liquid remains
on membrane filter surface, remove clamp and funnel.
6. Remove membrane filter with deposited bacteria from glass frit
filter using forceps (Fig. 2) (see Note 24).
Fig. 1 (a) Complete filtration apparatus with membrane between frit and funnel.
(b) Demonstration of applying a membrane filter to the frit and stopper of the
filtration apparatus. Ensure aspiration is applied to system to hold membrane in
place and secure funnel to top with clamp
82
Christopher J. Pappas et al.