PHA granule staining: Heat-fixed smears of bacterial cells were stained with the Nile blue A
(Sigma, UK, CAS 3625-57-8) dissolved in 1%H2S04 at 55°C for 10 min. After being stained, the
slides were washed with tap water to remove excess stain and with 8% aqueous acetic acid for 1
min. The stained smear was washed and blotted dry with bibulous paper, remoistened with tap
water, and covered with a no. 1 glass cover slip. The cover slip is necessary, as standard
immersion oil will extract some of the fluorescent dye and obscure the field with a general yellow
fluorescence. The cover slip thus protects the stained cells from immersion oil.