19. To prevent tipping of filtration apparatus which would spill
media, we recommend attaching a clamp system to the filter
flask.
20. For a pump system, we find a vacuum pressure of 20–40 cmHg
works well. With a lower vacuum pressure, the filtration step
runs slowly. With higher vacuum pressure, the system may
create bubbles in the flow-through which can transfer into
the vacuum tubing.
21. Vary amounts since transformation efficiency will vary by density of E. coli and Leptospira (err on the side of more Leptospira). We typically vary between 3 and 8 mL of Leptospira and
vary the dilution ratio between 1:2 and 1:10. It is recommended to write down the volume of each added on the back
of the EMJH plate (e.g., Fig. 8) where membrane filters are
placed, since on conjugation day 2, two membrane filters will
be paired for the plating step. By doing this, it will help optimize volume vs. cell density for your lab.
22. Biosafety: While L. biflexa is considered non-infectious, if this
procedure is applied to pathogenic Leptospira, it is essential to
use prudent practices to avoid aspiration and contamination of
Leptospira, especially if using a vacuum pump. Risk of infection
in the laboratory by Leptospira is considered higher than average [45]. At least 67 laboratory-acquired infections and ten
deaths have been reported for pathogenic Leptospira [46]. This
is typically by direct or indirect contact with cultures or from
the urine of an infected animal. Therefore, it is imperative to
use an in-line filter when using this filtration technique with
Fig. 8 Plate showing four membrane filters, as well as two circular cloudy
patches (arrows) where membrane filters were removed. These cloudy
patches are Leptospira that have traversed through the membrane filter during
the 18-h incubation period into the EMJH solid media
90
Christopher J. Pappas et al.
media, we recommend attaching a clamp system to the filter
flask.
20. For a pump system, we find a vacuum pressure of 20–40 cmHg
works well. With a lower vacuum pressure, the filtration step
runs slowly. With higher vacuum pressure, the system may
create bubbles in the flow-through which can transfer into
the vacuum tubing.
21. Vary amounts since transformation efficiency will vary by density of E. coli and Leptospira (err on the side of more Leptospira). We typically vary between 3 and 8 mL of Leptospira and
vary the dilution ratio between 1:2 and 1:10. It is recommended to write down the volume of each added on the back
of the EMJH plate (e.g., Fig. 8) where membrane filters are
placed, since on conjugation day 2, two membrane filters will
be paired for the plating step. By doing this, it will help optimize volume vs. cell density for your lab.
22. Biosafety: While L. biflexa is considered non-infectious, if this
procedure is applied to pathogenic Leptospira, it is essential to
use prudent practices to avoid aspiration and contamination of
Leptospira, especially if using a vacuum pump. Risk of infection
in the laboratory by Leptospira is considered higher than average [45]. At least 67 laboratory-acquired infections and ten
deaths have been reported for pathogenic Leptospira [46]. This
is typically by direct or indirect contact with cultures or from
the urine of an infected animal. Therefore, it is imperative to
use an in-line filter when using this filtration technique with
Fig. 8 Plate showing four membrane filters, as well as two circular cloudy
patches (arrows) where membrane filters were removed. These cloudy
patches are Leptospira that have traversed through the membrane filter during
the 18-h incubation period into the EMJH solid media
90
Christopher J. Pappas et al.