pathogenic Leptospira, to clean all tubing by chemical or autoclave sterilization methods regularly, and to wear relevant personal protective equipment.
23. Depending on concentration of bacteria and volume, this can
take between 2 and 25 min to complete. Patience is key since
disturbing the setup can result in bacteria bypassing the membrane filter if funnel is removed before all media has completed
flow-through.
24. The membrane filter can carry static cling and as a result may
jump as it is being removed from glass frit filter. To avoid the
membrane filter from becoming contaminated by falling to the
cabinet surface, we place the funnel at a 45
angle on the glass
frit filter surface (Fig. 2). This gives purchase to the membrane
filter as it is removed with forceps and reduces risk of
static jump.
25. Ensure no bubbles remain between membrane filter and
EMJH plate to optimize transfer of nutrients to bacteria.
26. If spaced properly, up to seven membrane filters can fit on one
EMJH plate (Fig. 3).
27. We usually prepare at least 50 mL of Leptospira-containing
EMJH media disbursed between two 50 mL conicals. This
gives us sufficient volume to complete between seven and
nine conjugations (i.e., create seven to nine membrane filters
containing E. coli and Leptospira) on average.
28. Clean glassware and tubing used for conjugation day 1 by
chemical disinfection, rinse thoroughly with ultrapure water,
and allow to dry. Place in autoclave sleeves and autoclave for
future use.
29. For every two membrane filters (pairs), 0.8 mL of EMJH liquid
and three EMJH solid media plates are required.
30. When transferring, you may notice a cloudy circular patch on
the EMJH plate where the membrane filter was located. This is
Leptospira that has migrated through the membrane filter
overnight. While there will still be sufficient Leptospira on the
membrane, this is one reason why using a 0.1 μm filter pore
and not exceeding a 20-h incubation period for conjugation to
occur on EMJH solid media are best practices (Fig. 8).
31. Place the residual liquid media (i.e., ~10 μL) in the serological
pipette onto a microscope slide, and visualize by dark-field
microscopy. E. coli and Leptospira should be visualized in
close proximity and in certain instances appearing attached
(i.e., undergoing conjugation). This will allow you to verify
amount of bacteria present and their motility. This will aid you
in determining the likelihood that generation of transconjugants was successful (Fig. 9).
Random Transposon Mutant Library
91
23. Depending on concentration of bacteria and volume, this can
take between 2 and 25 min to complete. Patience is key since
disturbing the setup can result in bacteria bypassing the membrane filter if funnel is removed before all media has completed
flow-through.
24. The membrane filter can carry static cling and as a result may
jump as it is being removed from glass frit filter. To avoid the
membrane filter from becoming contaminated by falling to the
cabinet surface, we place the funnel at a 45
angle on the glass
frit filter surface (Fig. 2). This gives purchase to the membrane
filter as it is removed with forceps and reduces risk of
static jump.
25. Ensure no bubbles remain between membrane filter and
EMJH plate to optimize transfer of nutrients to bacteria.
26. If spaced properly, up to seven membrane filters can fit on one
EMJH plate (Fig. 3).
27. We usually prepare at least 50 mL of Leptospira-containing
EMJH media disbursed between two 50 mL conicals. This
gives us sufficient volume to complete between seven and
nine conjugations (i.e., create seven to nine membrane filters
containing E. coli and Leptospira) on average.
28. Clean glassware and tubing used for conjugation day 1 by
chemical disinfection, rinse thoroughly with ultrapure water,
and allow to dry. Place in autoclave sleeves and autoclave for
future use.
29. For every two membrane filters (pairs), 0.8 mL of EMJH liquid
and three EMJH solid media plates are required.
30. When transferring, you may notice a cloudy circular patch on
the EMJH plate where the membrane filter was located. This is
Leptospira that has migrated through the membrane filter
overnight. While there will still be sufficient Leptospira on the
membrane, this is one reason why using a 0.1 μm filter pore
and not exceeding a 20-h incubation period for conjugation to
occur on EMJH solid media are best practices (Fig. 8).
31. Place the residual liquid media (i.e., ~10 μL) in the serological
pipette onto a microscope slide, and visualize by dark-field
microscopy. E. coli and Leptospira should be visualized in
close proximity and in certain instances appearing attached
(i.e., undergoing conjugation). This will allow you to verify
amount of bacteria present and their motility. This will aid you
in determining the likelihood that generation of transconjugants was successful (Fig. 9).
Random Transposon Mutant Library
91