a twofold serial dilution by transferring 20 μL of the dilution
from column 2 into 3 and then into subsequent columns
ending with column 11.
4. Prepare a similar dilution series with the control drugs (see
Notes 4 and 16).
5. Using a 20 μL multichannel pipette, transfer 2 μL of the drug
dilution to the microtiter assay plate with clean sterile tips for
each row.
3.3 Preparation of
Plates for Solid
Medium MIC
Determination
1. Prepare compound/drug dilutions as described in steps 1–5 of
Subheading 3.2.
2. Using a 1000 μL multichannel set at 200 μL, transfer warm
7H11/OADC into the wells of the microtiter plate taking care
to avoid formation of bubbles (see Note 17).
3. Dry the plates with lids on for 24 h inside the biosafety cabinet.
3.4 Measuring
Growth Using an
Inverted Mirror
1. Using a multichannel pipette, add 200 μL of cells prepared in
step 5 of Subheading 3.1 to the 96-well U-bottom microtiter
plates containing the 2 μL of compound (see step 5 in Subheading 3.2). Use sterile tips for each row. Mix and transfer
100 μL of the cell/compound mix to a sterile microtiter plate
as a technical repeat (see Notes 11 and 15).
2. Replace the lids, put the plates in sealable bags and incubate for
7 days at 37
C.
3. Record growth by placing the plates above an enlarging
inverted mirror (see Note 18).
4. Return the plates to the sealed bags and incubate at 37
C for
an additional 7 days.
5. Record growth at the end of week 2 (see step 3 in Subheading
3.4).
6. The MIC is the lowest concentration of compound that results
in full inhibition of growth (see Notes 19 and 20).
3.5 Measuring
Growth Using
Alamar Blue
1. Using a multichannel pipette, add 200 μL of cells (from Subheading 3.1) to the 96-well clear flat-bottom black microtiter
plates containing 2 μL of compound (from Subheading 3.2).
Use sterile tips for each row.
2. Mix and transfer 100 μL of the cell/compound mix to a sterile
microtiter plate as a technical repeat (see Notes 11 and 15).
Label duplicate pates as “Week 1.”
3. Prepare a second set of plates for the week 2 (see Note 20).
Label duplicate plates as “Week 2.”
4. Incubate plates in sealed bags at 37
C for 7 days (week 1) or
14 days (week 2).
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Qinglan Wang and Helena I. M. Boshoff
from column 2 into 3 and then into subsequent columns
ending with column 11.
4. Prepare a similar dilution series with the control drugs (see
Notes 4 and 16).
5. Using a 20 μL multichannel pipette, transfer 2 μL of the drug
dilution to the microtiter assay plate with clean sterile tips for
each row.
3.3 Preparation of
Plates for Solid
Medium MIC
Determination
1. Prepare compound/drug dilutions as described in steps 1–5 of
Subheading 3.2.
2. Using a 1000 μL multichannel set at 200 μL, transfer warm
7H11/OADC into the wells of the microtiter plate taking care
to avoid formation of bubbles (see Note 17).
3. Dry the plates with lids on for 24 h inside the biosafety cabinet.
3.4 Measuring
Growth Using an
Inverted Mirror
1. Using a multichannel pipette, add 200 μL of cells prepared in
step 5 of Subheading 3.1 to the 96-well U-bottom microtiter
plates containing the 2 μL of compound (see step 5 in Subheading 3.2). Use sterile tips for each row. Mix and transfer
100 μL of the cell/compound mix to a sterile microtiter plate
as a technical repeat (see Notes 11 and 15).
2. Replace the lids, put the plates in sealable bags and incubate for
7 days at 37
C.
3. Record growth by placing the plates above an enlarging
inverted mirror (see Note 18).
4. Return the plates to the sealed bags and incubate at 37
C for
an additional 7 days.
5. Record growth at the end of week 2 (see step 3 in Subheading
3.4).
6. The MIC is the lowest concentration of compound that results
in full inhibition of growth (see Notes 19 and 20).
3.5 Measuring
Growth Using
Alamar Blue
1. Using a multichannel pipette, add 200 μL of cells (from Subheading 3.1) to the 96-well clear flat-bottom black microtiter
plates containing 2 μL of compound (from Subheading 3.2).
Use sterile tips for each row.
2. Mix and transfer 100 μL of the cell/compound mix to a sterile
microtiter plate as a technical repeat (see Notes 11 and 15).
Label duplicate pates as “Week 1.”
3. Prepare a second set of plates for the week 2 (see Note 20).
Label duplicate plates as “Week 2.”
4. Incubate plates in sealed bags at 37
C for 7 days (week 1) or
14 days (week 2).
602
Qinglan Wang and Helena I. M. Boshoff