Fluorescent BODIPY-FL vancomycin staining

Protocol for using Fluorescent BODIPY^TM FL Vancomycin dye (Invitrogen, Catalog number V34850) to label peptidoglycan (PG) in Mycobacterium tuberculosis. 

The fluorescent vancomycin dye binds to the terminal dipeptide D-alanine-D-alanine found on the peptidoglycan stem peptide periplasmic precursor lipid II and consequently indicates the sites of new peptidoglycan synthesis. 

1. Grow a Mycobacterium tuberculosis H37Rv culture in middlebrook 7h9 to O.D₆₀₀ 0.6 at 37 C with shaking at a 100 rpm.

2. Havest 1 ml of cells into a 1.5 ml tube by centrifugation at 13 000 rpm for 5 minutes and discard the supernatant 

3. Wash the cells with 500 μl 1X PBS (pH 7.4) by centrifugation at 13 000 rpm for 5 minutes, discard the  supernatant and resuspend the cells in 500 μl of 1X PBS. 

4. Add, 1.25 μl of vancomycin (200 micrograms/ml, Sigma aldrich catalog number V2002) and 2.5 μl of fluorescent BODIPY^TM FL vancomycin (100 micrograms, Invitrogen, Catalog number V34850) to the cells and incubate at 37 C with shaking at a 100 rpm for 1.5 hours.

5. Wash cells with 500 μl of 1X PBS three times by centrifugation at 13 000 rpm for 5 minutes, discard the supernatant and fix cells overnight in 250 μl of 2.5% glutaraldehyde (Sigma aldrich, Catalog number A17876.AP) at room temperature.

6. Fixed cells were centrifuged at 13 000 rpm for 2 min and resuspended in 1X PBS before spotting onto 1% agarose on glass slide and covered with a cover slip.

7. Images of the cells were captured using the Nikon Eclipse Ti-E fluorescence microscope, in the pre-configured FITC channel (excitation 504 nm, emission 598 nm; 2 s exposure).

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