HADA Hydrochloride (HCC-Amino-D-alanine hydrochloride) is a blue (λem~450 nm) fluorescent D-amino acid (FDAA). FDAAs are efficiently incorporated into the peptidoglycans (PGs) of diverse bacterial species at the sites of PG biosynthesis. HADA Hydrochloride can be used in specific and covalent probing of bacterial growth with minimal perturbation.

FDAA labeling can be completed in as little as 30 seconds for rapidly growing species like Escherichia coli. Despite HADA Hydrochloride being dimmer and less photostable compared to FDL or TDL, it consistently and effectively labels the PG of a wide range of bacterial species, usually without requiring significant optimization[1].

HADA labeled PG site experiment of E. coli cells
I. Solution preparation
Mother solution preparation: Prepare 50 mM (100×) stock solution with DMSO; (it is recommended to store at -20 ℃ or -80 ℃ in the dark after aliquoting)
II. Operation steps
1. Inoculate cells from -80°C bacterial cryopreservation solution into sterile liquid culture medium and culture cells using optimal species-specific conditions until exponential growth is achieved.
2. Add 10 μL of 50 mM HADA DMSO stock solution to the culture tube.
3. Dilute the culture to OD600 = 0.05 in fresh LB;
4. Add 990 μL of the culture to the culture tube containing FDA to obtain a final LB solution of [HADA] = 500 μM and [DMSO] = 1%, and incubate the culture at 37°C with shaking;
5. When the culture reaches OD600 = 0.4 – 0.6, pellet the cells (1 min, maximum speed in a microcentrifuge, room temperature);
6. Resuspend the cells in 1 mL 70% (vol/vol) ice-cold EtOH and fix the cells on ice for 10 – 15 min.