27. Mix by swirling beaker gently and pour into sterile separatory
funnel. Allow mixture to separate for 1 h at room temperature
with minimal disturbance.
28. Slowly drain lower phase into beaker. Wait 5–10 min for layers
to resettle.
29. Using a 25 or 50 mL sterile pipet (depending on volume),
remove top phase and transfer to new sterile 50 mL Oakridge
tubes ~25 mL per tube. This will be called the first upper.
30. Take the beaker where the funnel was drained and add 6%
PEG/8% dextran/0.01 M phosphate saline buffer to the
same final volume as in step 26.
31. Repeat the previous three steps. This top layer removed at this
step will be considered the second upper (see Note 62).
32. Place 20–25 mL of 0.2% Tween 80 solution in each tube,
balance and centrifuge at 27,000 Â g for 30 min at 4
C.
Discard supernatant into biohazard waste.
33. Resuspend pellet in buffered water, combining all first upper
pellets into one tube and all the second upper pellets into a
different tube (see Note 61). Keep all the rinses with its
corresponding upper tube, do not mix the rinse liquid with
different upper tubes.
34. Balance and centrifuge tubes at 10,000 Â g for 10 min at 4
C.
Discard supernatant into biohazard waste (see Note 63).
35. Resuspend pellet in buffered water and centrifuge balanced
tubes at 10,000 Â g for 10 min at 4
C. Discard supernatant
into biohazard waste (see Note 63).
36. Resuspend pellets with 1–5 mL buffered water and transfer to a
cryotube. Rinse Oakridge tube and transfer to cryotube.
37. Perform a sterility test with a small amount of cell suspension in
the cryotube as done in step 6.
38. Prepare a microscope slide by adding 5 μL of each upper pellet
and spreading the sample evenly with the pipet tip and let the
slide rest in the hood until dry. Perform an acid-fast stain and
take a picture, making sure no other microorganisms are visible
indicating contamination or other cellular debris (see Subheading 3.3).
39. Prepare 1:100 and 1:200 dilutions of sample: buffered water
dilution in a total volume of 2 mL for each upper collected in
glass test tubes and take an optical density reading at a wavelength of 540 nm.
40. Record readings and use the formula (see Note 64) to calculate
the concentration of cells in the cryovial. Also, record the final
pellet color, pigment contamination, and tissue contamination
(see Note 65).
Culturing Mycobacteria
45
funnel. Allow mixture to separate for 1 h at room temperature
with minimal disturbance.
28. Slowly drain lower phase into beaker. Wait 5–10 min for layers
to resettle.
29. Using a 25 or 50 mL sterile pipet (depending on volume),
remove top phase and transfer to new sterile 50 mL Oakridge
tubes ~25 mL per tube. This will be called the first upper.
30. Take the beaker where the funnel was drained and add 6%
PEG/8% dextran/0.01 M phosphate saline buffer to the
same final volume as in step 26.
31. Repeat the previous three steps. This top layer removed at this
step will be considered the second upper (see Note 62).
32. Place 20–25 mL of 0.2% Tween 80 solution in each tube,
balance and centrifuge at 27,000 Â g for 30 min at 4
C.
Discard supernatant into biohazard waste.
33. Resuspend pellet in buffered water, combining all first upper
pellets into one tube and all the second upper pellets into a
different tube (see Note 61). Keep all the rinses with its
corresponding upper tube, do not mix the rinse liquid with
different upper tubes.
34. Balance and centrifuge tubes at 10,000 Â g for 10 min at 4
C.
Discard supernatant into biohazard waste (see Note 63).
35. Resuspend pellet in buffered water and centrifuge balanced
tubes at 10,000 Â g for 10 min at 4
C. Discard supernatant
into biohazard waste (see Note 63).
36. Resuspend pellets with 1–5 mL buffered water and transfer to a
cryotube. Rinse Oakridge tube and transfer to cryotube.
37. Perform a sterility test with a small amount of cell suspension in
the cryotube as done in step 6.
38. Prepare a microscope slide by adding 5 μL of each upper pellet
and spreading the sample evenly with the pipet tip and let the
slide rest in the hood until dry. Perform an acid-fast stain and
take a picture, making sure no other microorganisms are visible
indicating contamination or other cellular debris (see Subheading 3.3).
39. Prepare 1:100 and 1:200 dilutions of sample: buffered water
dilution in a total volume of 2 mL for each upper collected in
glass test tubes and take an optical density reading at a wavelength of 540 nm.
40. Record readings and use the formula (see Note 64) to calculate
the concentration of cells in the cryovial. Also, record the final
pellet color, pigment contamination, and tissue contamination
(see Note 65).
Culturing Mycobacteria
45