32. Since Leptospira transconjugants may be motile, it is important
to check periodically to ensure colonies remain clonal.
33. Only a small fraction of the colony is required for growth in
EMJH (approx. 10–50 μL of volume of the agar containing the
colony or one stab into solid media).
34. Some colonies will appear faintly translucent as they spread in
the media. To aid in removal, we typically use a black sheet of
paper placed below the Petri plate to more easily visualize the
colonies during removal.
35. Continue to monitor plates for up to 50 days, since some
mutants may grow slowly or be motility-deficient which will
delay the appearance of a colony. If a plate develops mold,
discard the plate according to your standard biohazard disposal
protocol.
36. If Leptospira culture is contaminated with E. coli or another
microbe which may occasionally occur, the contaminating bacteria can be removed by syringe filtration. Take a clean 15 mL
conical and place 1–3 mL of fresh EMJH liquid media into
it. Filter into this fresh media the contaminated sample by
using a 5 mL syringe outfitted with 0.22 μm syringe filter.
Leptospira should transit through this filter while other
microbes will not. Allow Leptospira to grow for a minimum
of 5–7 days at 30
C with or without agitation. Likelihood of
Fig. 9 Microscopy of E. coli β2163 and L. biflexa potentially undergoing conjugation. In instances where E. coli and Leptospira are overlapped (e.g., arrows),
cells are monitored to determine if those move together. If so, E. coli will appear
bound to cell membrane of Leptospira (visualized as E. coli traversing longitudinally along the length of the leptospire). (Image captured with Zeiss Axio Lab A.1
(EC PLAN-Neofluar 20Â/0.5 NA) with attached Axiocam 105 color CMOS
camera)
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Christopher J. Pappas et al.
to check periodically to ensure colonies remain clonal.
33. Only a small fraction of the colony is required for growth in
EMJH (approx. 10–50 μL of volume of the agar containing the
colony or one stab into solid media).
34. Some colonies will appear faintly translucent as they spread in
the media. To aid in removal, we typically use a black sheet of
paper placed below the Petri plate to more easily visualize the
colonies during removal.
35. Continue to monitor plates for up to 50 days, since some
mutants may grow slowly or be motility-deficient which will
delay the appearance of a colony. If a plate develops mold,
discard the plate according to your standard biohazard disposal
protocol.
36. If Leptospira culture is contaminated with E. coli or another
microbe which may occasionally occur, the contaminating bacteria can be removed by syringe filtration. Take a clean 15 mL
conical and place 1–3 mL of fresh EMJH liquid media into
it. Filter into this fresh media the contaminated sample by
using a 5 mL syringe outfitted with 0.22 μm syringe filter.
Leptospira should transit through this filter while other
microbes will not. Allow Leptospira to grow for a minimum
of 5–7 days at 30
C with or without agitation. Likelihood of
Fig. 9 Microscopy of E. coli β2163 and L. biflexa potentially undergoing conjugation. In instances where E. coli and Leptospira are overlapped (e.g., arrows),
cells are monitored to determine if those move together. If so, E. coli will appear
bound to cell membrane of Leptospira (visualized as E. coli traversing longitudinally along the length of the leptospire). (Image captured with Zeiss Axio Lab A.1
(EC PLAN-Neofluar 20Â/0.5 NA) with attached Axiocam 105 color CMOS
camera)
92
Christopher J. Pappas et al.