2.5 Measuring
Growth Using
Alamar Blue
1. 96-well clear flat-bottom, sterile black microtiter plates with
lids (see Note 6).
2. Sterile basins.
3. Alamar Blue solution, e.g., ThermoFisher (see Note 7).
4. Colorimeter/Fluorimeter, e.g., CLARIOstar Plus, BMG
LABTECH.
5. Sealable plastic bags, e.g., Ziploc bags.
2.6 Measuring
Growth Using
Fluorescence of M.
tuberculosis pMSP12::
GFP
1. 96-well, flat-bottom, sterile black microtiter plates with lids.
2. Sterile basins.
3. Fluorimeter, e.g., CLARIOstar Plus, BMG LABTECH.
4. Sealable plastic bags, e.g., Ziploc bags.
2.7 Measuring
Growth on Solid
Medium
1. Sterile basins.
2. Alamar Blue solution, e.g., ThermoFisher.
3. Sealable plastic bags, e.g., Ziploc bags.
4. 1–10 μL or 2–20 μL Multichannel Pipette.
5. 20 μL sterile filter tips (RAININ).
3 Methods
3.1 Preparation of
Inoculum
1. In a 60 mL square bottle seed 10 mL of medium (see Notes
8 and 9) with 200 μL frozen stock of M. tuberculosis (see
Note 1).
2. Incubate at 37
C at 15 rpm for ~7 days (10–12 days for
growth for 7H9/butyrate/Tyloxapol) until the culture reaches
an OD 650 of 0.2–0.4.
3. Subculture cells 1:100 in medium (see Note 10) and incubate
for 4–5 days until the culture reaches an OD 650 of ~0.2.
4. Dilute the cells 1:1000 in medium for liquid MIC or 1:200 for
agar MIC with the following exceptions: dilute cells 1:10 for
liquid MIC in 7H9/butyrate/Tyloxapol medium; and 1:10 for
liquid MIC in 7H9/butyrate/nitrite/Tyloxapol medium (see
Note 10).
5. Transfer the cell suspension to a sterile basin (see Note 11).
3.2 Preparation of
Plates for Liquid MIC
Determination
1. Dispense 40 μL compound stock solution in column 1 of a
96-well polypropylene plate (see Notes 12–15).
2. Dispense 20 μL of DMSO to columns 2–12.
3. Transfer 20 μL of compound from column 1 into column
2. Mix by pipetting up and down 6 times. Continue to make
MIC Determination for Mycobacterium Tuberculosis
601
Growth Using
Alamar Blue
1. 96-well clear flat-bottom, sterile black microtiter plates with
lids (see Note 6).
2. Sterile basins.
3. Alamar Blue solution, e.g., ThermoFisher (see Note 7).
4. Colorimeter/Fluorimeter, e.g., CLARIOstar Plus, BMG
LABTECH.
5. Sealable plastic bags, e.g., Ziploc bags.
2.6 Measuring
Growth Using
Fluorescence of M.
tuberculosis pMSP12::
GFP
1. 96-well, flat-bottom, sterile black microtiter plates with lids.
2. Sterile basins.
3. Fluorimeter, e.g., CLARIOstar Plus, BMG LABTECH.
4. Sealable plastic bags, e.g., Ziploc bags.
2.7 Measuring
Growth on Solid
Medium
1. Sterile basins.
2. Alamar Blue solution, e.g., ThermoFisher.
3. Sealable plastic bags, e.g., Ziploc bags.
4. 1–10 μL or 2–20 μL Multichannel Pipette.
5. 20 μL sterile filter tips (RAININ).
3 Methods
3.1 Preparation of
Inoculum
1. In a 60 mL square bottle seed 10 mL of medium (see Notes
8 and 9) with 200 μL frozen stock of M. tuberculosis (see
Note 1).
2. Incubate at 37
C at 15 rpm for ~7 days (10–12 days for
growth for 7H9/butyrate/Tyloxapol) until the culture reaches
an OD 650 of 0.2–0.4.
3. Subculture cells 1:100 in medium (see Note 10) and incubate
for 4–5 days until the culture reaches an OD 650 of ~0.2.
4. Dilute the cells 1:1000 in medium for liquid MIC or 1:200 for
agar MIC with the following exceptions: dilute cells 1:10 for
liquid MIC in 7H9/butyrate/Tyloxapol medium; and 1:10 for
liquid MIC in 7H9/butyrate/nitrite/Tyloxapol medium (see
Note 10).
5. Transfer the cell suspension to a sterile basin (see Note 11).
3.2 Preparation of
Plates for Liquid MIC
Determination
1. Dispense 40 μL compound stock solution in column 1 of a
96-well polypropylene plate (see Notes 12–15).
2. Dispense 20 μL of DMSO to columns 2–12.
3. Transfer 20 μL of compound from column 1 into column
2. Mix by pipetting up and down 6 times. Continue to make
MIC Determination for Mycobacterium Tuberculosis
601