BACKGROUND
Microplate based Alamar Blue Assay (MABA) is a sensitive, practical and inexpensive assay method of cell viability in which fluorescence reduction assay of alamar blue, a resazurin (a dark blue dye and nonfluoroscent in oxidized form, available as a sterile and liquid reagent commercially), is used in a microplate format.1
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MABA utilizes four weeks incubation in microplates along with the dye which reduces to resorufin, becomes pink colour and fluoroscent in nature as a result of cellular metabolism.2
During drugs development against Mycobacterium tuberculosis, the activity against non replicating organism is one of the important factor to determine. So, the objective of this experiment is to assay cell viability by MABA method.
REQUIREMENTS
Middlebrook 7H11 solid medium: Combine 8.4 g of 7H11 Middlebrook agar powder (Becton Dickinson), 360 ml of H2O, and 4 ml of 50 % v/v glycerol. Autoclave for 15 min at 121 °C. Add 40 ml OADC when the temperature falls below 50 °C.
7H9-OADC medium: Combine 1.88 g of 7H9 Middle brook broth base (Becton Dickinson), 360 ml of H2O, 1 ml of 20 % w/v Tween 80, 1.6 ml of 50 % v/v glycerol. Autoclave for 15 min 121 °C. When the temperature falls below 50 °C, add 40 ml oleic acid, albumin, dextrose, and catalase supplement (OADC, Becton Dickinson).
- Sterile 96-well clear plates
- Nephelo culture flask
- Alamar Blue
PROCEDURE
Prepare inoculum of M. tuberculosis in 200 ml of 7H9 broth in a 500 ml Nephelo flask and into individual wells of a microplate and controls are incubated in medium only.1,2 Incubate at 37°C in 5% CO2 95% humidified atmosphere. Incubate for 7 days and check contamination. If fungal colonies appear in blood agar, dispose the batch.
Mix in a ratio of 8:5 Alamar Blue (or 0.6 mM resazurin dye) and 20% Tween 80 (for 1 plate, 2 ml of Alamar Blue (or 0.6 mM resazurin dye).2 Add 20 µl of stock alamar blue to each well. Incubate the plate for more than 20 days and measure the emitted fluorescence periodically at excitation 530 nm and emission 590 nm.
MIC90 is defined as the lowest concentration effecting a 90% inhibition of fluorescence relative to the untreated bacterial control.2
90% inhibition = 0.1× (Bacterial Control – Background)
CONCLUSION
MABA is an useful method for assaying drugs susceptibility for non replicating M. tuberculosis.
REFERENCES
- Mendoza-Aguilar M, Almaguer-Villagrán L, Jiménez-Arellanes A, Arce-Paredes P, Cid-Gutiérrez JL, Rojas-Espinosa O. The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs. J Infect Chemother. 2012;18(5):652-61.
- Cho S, Lee HS, Franzblau S. Microplate Alamar Blue Assay (MABA) and Low Oxygen Recovery Assay (LORA) for Mycobacterium tuberculosis. Mycobacteria Protocols. 2015;1285:281–92.
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