20. Calculate the M. leprae/mL using the following formula for
individual dilutions:
¼ Count1 þ Count2 þ Count3
ð
Þ Ä 60
ð
Þ Â Microscope Constant
 Dilution Factor:
21. Calculate the mean of the dilutions to get a more accurate
M. leprae/mL count.
3.7 Small-Scale
Culture
of M. tuberculosis
1. Thaw frozen stock or rehydrate lyophilized stock according to
manufacturer’s instructions.
2. Transfer contents (up to 1.5 mL) to 6 mL of 7H9 + ADC broth
(see Subheading 2.7, item 3). Pipette up and down slowly to
mix without aerosolizing.
3. Transfer 0.1 mL each to selective media (Middlebrook 7H10
+OADC, see Subheading 2.7, item 4) and nonselective media
(usually TSA) and streak for isolation (see Note 26).
4. Incubate according to species recommendations (see Note 27).
3.8 Large-Scale
Growth
of M. tuberculosis
for Research Purposes
For an overall description of this protocol, please see the diagram in
Fig. 8. The production of mycobacterial cells and culture filtrate
protein (CFP) in a large-scale format is necessary for the generation
of raw material used to generate reagents and standards for research
conducted by the scientific community at large.
1. Thaw a 1 mL frozen stock of M. tuberculosis.
2. Pipette 200 μL of the stock onto a large 7H11 + OADC agar
plate and streak to grow as a lawn with a sterile bent
plastic loop.
Fig. 8 Diagram of large-scale culture.
*
An extra plate and extra Fernbach are optional but recommended as
back-up if needed. FB Fernbach, RB Roller bottle, CFP culture filtrate protein
Culturing Mycobacteria
35
individual dilutions:
¼ Count1 þ Count2 þ Count3
ð
Þ Ä 60
ð
Þ Â Microscope Constant
 Dilution Factor:
21. Calculate the mean of the dilutions to get a more accurate
M. leprae/mL count.
3.7 Small-Scale
Culture
of M. tuberculosis
1. Thaw frozen stock or rehydrate lyophilized stock according to
manufacturer’s instructions.
2. Transfer contents (up to 1.5 mL) to 6 mL of 7H9 + ADC broth
(see Subheading 2.7, item 3). Pipette up and down slowly to
mix without aerosolizing.
3. Transfer 0.1 mL each to selective media (Middlebrook 7H10
+OADC, see Subheading 2.7, item 4) and nonselective media
(usually TSA) and streak for isolation (see Note 26).
4. Incubate according to species recommendations (see Note 27).
3.8 Large-Scale
Growth
of M. tuberculosis
for Research Purposes
For an overall description of this protocol, please see the diagram in
Fig. 8. The production of mycobacterial cells and culture filtrate
protein (CFP) in a large-scale format is necessary for the generation
of raw material used to generate reagents and standards for research
conducted by the scientific community at large.
1. Thaw a 1 mL frozen stock of M. tuberculosis.
2. Pipette 200 μL of the stock onto a large 7H11 + OADC agar
plate and streak to grow as a lawn with a sterile bent
plastic loop.
Fig. 8 Diagram of large-scale culture.
*
An extra plate and extra Fernbach are optional but recommended as
back-up if needed. FB Fernbach, RB Roller bottle, CFP culture filtrate protein
Culturing Mycobacteria
35