26. This dose has been approved by Colorado State University to
inactivate pellets (5–75 g of wet cell paste) in a JL Shepherd
Model 81-14 sealed source Cs-137 irradiator. Proper dosing
studies (D 10 studies) should be carried out for different sample
types and sources.
27. Until inactivation has been confirmed, the cell pellet is to be
treated as dangerous goods, risk group 3 organism.
28. Cells can be stored at À20
C until Alamar Blue is performed.
Cells not used in Alamar Blue should be kept at À20
C for
storage. Efforts should be made to reduce freeze–thaw, if
possible.
29. Ideally, the live M. tuberculosis control should correspond to
the same strain as the sample being tested.
30. This blank tube will serve as a sentinel for potential contamination and will also be used as a blank during spectrophotometer
readings.
31. The addition of Alamar Blue reagent to all neat (OD 600 ¼ 1.0)
samples may turn samples pink (indicating enzymatic activity)
due to the number of bacterial cells present, even if they are
inactivated. Thus, adding Alamar Blue to these sample wells
may be omitted.
32. Move the microplate spectrophotometer into a biosafety cabinet if the positive control cells will be exposed to the environment, such as if the 96-well plate lid needs to be removed to
read the plate.
33. When setting up the microplate spectrophotometer, select dual
wavelength. Set 600 nm as the reference and 570 nm as the
measurement.
34. The incubation and plate reading time points provided here
were established for M. tuberculosis. Mycobacteria with more
rapid replication rates, for example, M. smegmatis need to be
read at earlier time points (M. smegmatis can generally be read
at 4, 8, and 12 h time points). Optimum time points should be
determined for all mycobacteria other than M. tuberculosis
empirically.
35. It is important that all O-rings are free of debris or tears;
visually inspect O-rings, fittings, and tubing for integrity, replacing as needed.
36. Consult the manufacture’s user manual for stirred ultrafiltration cell unit assembly instructions specific to the unit model.
37. PTFE tape should be used on all threaded male fittings. For
best results, wrap the tape around the male end in a manner
opposite the direction required for tightening the fitting. Use a
wrench to ensure a tight seal.
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Megan Lucas et al.
inactivate pellets (5–75 g of wet cell paste) in a JL Shepherd
Model 81-14 sealed source Cs-137 irradiator. Proper dosing
studies (D 10 studies) should be carried out for different sample
types and sources.
27. Until inactivation has been confirmed, the cell pellet is to be
treated as dangerous goods, risk group 3 organism.
28. Cells can be stored at À20
C until Alamar Blue is performed.
Cells not used in Alamar Blue should be kept at À20
C for
storage. Efforts should be made to reduce freeze–thaw, if
possible.
29. Ideally, the live M. tuberculosis control should correspond to
the same strain as the sample being tested.
30. This blank tube will serve as a sentinel for potential contamination and will also be used as a blank during spectrophotometer
readings.
31. The addition of Alamar Blue reagent to all neat (OD 600 ¼ 1.0)
samples may turn samples pink (indicating enzymatic activity)
due to the number of bacterial cells present, even if they are
inactivated. Thus, adding Alamar Blue to these sample wells
may be omitted.
32. Move the microplate spectrophotometer into a biosafety cabinet if the positive control cells will be exposed to the environment, such as if the 96-well plate lid needs to be removed to
read the plate.
33. When setting up the microplate spectrophotometer, select dual
wavelength. Set 600 nm as the reference and 570 nm as the
measurement.
34. The incubation and plate reading time points provided here
were established for M. tuberculosis. Mycobacteria with more
rapid replication rates, for example, M. smegmatis need to be
read at earlier time points (M. smegmatis can generally be read
at 4, 8, and 12 h time points). Optimum time points should be
determined for all mycobacteria other than M. tuberculosis
empirically.
35. It is important that all O-rings are free of debris or tears;
visually inspect O-rings, fittings, and tubing for integrity, replacing as needed.
36. Consult the manufacture’s user manual for stirred ultrafiltration cell unit assembly instructions specific to the unit model.
37. PTFE tape should be used on all threaded male fittings. For
best results, wrap the tape around the male end in a manner
opposite the direction required for tightening the fitting. Use a
wrench to ensure a tight seal.
102
Megan Lucas et al.