56. Make sure vials stay upright to prevent leaking of radiorespirometry media from the glass vial.
57. A higher setting will create more bubbles and make it more
difficult to work with. The top of the chamber might be warm
or hot to the touch so care should be taken when removing the
chamber from the homogenizer.
58. Incubate BHI media and TSA plates at 37
C for 4 days and the
L-J slant at 37
C for 2 weeks. BHI: Check for turbidity; TSA:
Check growth and record growth characteristics; L-J Slants:
Check for growth. Positive contamination is represented by
orange/white colonies. Note: L-J slant may turn orange with
age and may display a false positive.
59. Placing a large stir bar under the beaker while pouring will
make it easier to pour the beaker contents without pouring the
stir bar too.
60. Supernatant can be used as starting material for extraction of
PGL-1.
61. When rinsing tubes, pour ~15 mL of buffer to one tube, vortex
to ensure that the pellet is resuspended, and transfer to the next
tube. Continue vortexing and transferring until all the tubes
have been rinsed. One more rinse is done to make sure that
particles from the pellet are not on the sides of the tube. If the
pellet is difficult to resuspend, more buffer maybe used.
62. If the tissue size is small and/or if the first upper did not look
very cloudy (indicating that there is low amount of cells) then
taking a second upper is not necessary as it will give a very low
yield with a higher tissue contamination. Continue with the
other steps for the first upper.
63. This secondary wash is to make sure that the PEG/Dextran is
completely washed out of the cell pellet.
64. Follow this formula for each upper:
0.362 Â ______(OD540) Â 100(dilution) ¼ _________mg/
mL.
0.362 Â ______(OD540) Â 200(dilution) ¼ _________mg/
mL.
Average ¼ ________mg/mL.
_________mg/mL Â _______mL ¼ ___________mg.
65. The final pellet color can range from white to dark brown. The
pigment contamination scale is a number assigned indicating
color #1 ¼ white/cream to #5 ¼ dark brown. Only pigment
contamination number 1–2 pass QC. Tissue contamination is
assigned to each tube and is based on the slide and the presence
of tissue particles (see Fig. 10). Tissue particles usually look like
little blue spots. First upper is usually 1% tissue contamination,
Culturing Mycobacteria
55
57. A higher setting will create more bubbles and make it more
difficult to work with. The top of the chamber might be warm
or hot to the touch so care should be taken when removing the
chamber from the homogenizer.
58. Incubate BHI media and TSA plates at 37
C for 4 days and the
L-J slant at 37
C for 2 weeks. BHI: Check for turbidity; TSA:
Check growth and record growth characteristics; L-J Slants:
Check for growth. Positive contamination is represented by
orange/white colonies. Note: L-J slant may turn orange with
age and may display a false positive.
59. Placing a large stir bar under the beaker while pouring will
make it easier to pour the beaker contents without pouring the
stir bar too.
60. Supernatant can be used as starting material for extraction of
PGL-1.
61. When rinsing tubes, pour ~15 mL of buffer to one tube, vortex
to ensure that the pellet is resuspended, and transfer to the next
tube. Continue vortexing and transferring until all the tubes
have been rinsed. One more rinse is done to make sure that
particles from the pellet are not on the sides of the tube. If the
pellet is difficult to resuspend, more buffer maybe used.
62. If the tissue size is small and/or if the first upper did not look
very cloudy (indicating that there is low amount of cells) then
taking a second upper is not necessary as it will give a very low
yield with a higher tissue contamination. Continue with the
other steps for the first upper.
63. This secondary wash is to make sure that the PEG/Dextran is
completely washed out of the cell pellet.
64. Follow this formula for each upper:
0.362 Â ______(OD540) Â 100(dilution) ¼ _________mg/
mL.
0.362 Â ______(OD540) Â 200(dilution) ¼ _________mg/
mL.
Average ¼ ________mg/mL.
_________mg/mL Â _______mL ¼ ___________mg.
65. The final pellet color can range from white to dark brown. The
pigment contamination scale is a number assigned indicating
color #1 ¼ white/cream to #5 ¼ dark brown. Only pigment
contamination number 1–2 pass QC. Tissue contamination is
assigned to each tube and is based on the slide and the presence
of tissue particles (see Fig. 10). Tissue particles usually look like
little blue spots. First upper is usually 1% tissue contamination,
Culturing Mycobacteria
55