9. Once membrane filters have been placed, Parafilm Petri plate
and incubate for approximately 16–20 h (overnight) at 30
C
to allow conjugation to occur (Fig. 4) (see Note 28).
3.3 Conjugation:
Day 2
1. The next day, pre-warm to 30
C a sufficient supply of EMJH
liquid media (without kanamycin, without DAP) and EMJH
plates (with 40 μg/mL of kanamycin) for plating (see Note 29).
2. Transfer a pair of membrane filters deposited with bacteria into
a 15 mL conical tube with forceps (see Note 30).
3. Add 0.8 mL of pre-warmed EMJH (without kanamycin, without DAP) to the 15 mL conical tube. Using a handheld 1 mL
serological pipette as a tool, move each membrane filter one at a
time into the media.
4. Using the 1 mL serological pipette, create a gentle plunging/
up-and-down movement of the membrane filter into the media
to transfer the bacteria from the membrane filter to the EMJH
liquid media followed by briefly vortexing (Fig. 5) (see Note
31).
5. Repeat this procedure with additional pairs of membrane filters
into different 15 mL conical tubes until membrane filter supply
is exhausted (e.g., if you started with ten membrane filters, you
should now have five 15 mL conical tubes with membrane
filters).
6. Incubate 15 mL conical tubes for 1 h at 30
C with agitation.
Fig. 4 Right: Three conjugation plates wrapped in Parafilm (end of conjugation day 1). Left: aluminum foil
wrapped conjugation plates to be incubated for 10–50 days (end of conjugation day 2). Incubator is set to
30
C
84
Christopher J. Pappas et al.
and incubate for approximately 16–20 h (overnight) at 30
C
to allow conjugation to occur (Fig. 4) (see Note 28).
3.3 Conjugation:
Day 2
1. The next day, pre-warm to 30
C a sufficient supply of EMJH
liquid media (without kanamycin, without DAP) and EMJH
plates (with 40 μg/mL of kanamycin) for plating (see Note 29).
2. Transfer a pair of membrane filters deposited with bacteria into
a 15 mL conical tube with forceps (see Note 30).
3. Add 0.8 mL of pre-warmed EMJH (without kanamycin, without DAP) to the 15 mL conical tube. Using a handheld 1 mL
serological pipette as a tool, move each membrane filter one at a
time into the media.
4. Using the 1 mL serological pipette, create a gentle plunging/
up-and-down movement of the membrane filter into the media
to transfer the bacteria from the membrane filter to the EMJH
liquid media followed by briefly vortexing (Fig. 5) (see Note
31).
5. Repeat this procedure with additional pairs of membrane filters
into different 15 mL conical tubes until membrane filter supply
is exhausted (e.g., if you started with ten membrane filters, you
should now have five 15 mL conical tubes with membrane
filters).
6. Incubate 15 mL conical tubes for 1 h at 30
C with agitation.
Fig. 4 Right: Three conjugation plates wrapped in Parafilm (end of conjugation day 1). Left: aluminum foil
wrapped conjugation plates to be incubated for 10–50 days (end of conjugation day 2). Incubator is set to
30
C
84
Christopher J. Pappas et al.