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Chapter 5
II.
III.
IV.
A colony from the plate LB was picked off the plate and diluted into
10ml LB broth without salt (LB w/o NaCl).
The LB broth w/o NaCl consisted of bacto-tryptone 10g/L, yeast
extract 5g/L.
The overnight culture of Salmonella was diluted in LB broth without
NaCl to an optical density of 0·2 using the handheld OD meter
(Siemens-Microscan Turbidity Meter).
V. Thirty micro-litres of the bacterial suspension broth was transferred
to each well in a 96-well polystyrene microtitre plate containing
100 μl LB w/o NaCl.
VI. The plates were incubated at 22 ± 2°C (monitored room
temperature) or at 37°C for 2 and 7 days.
VII.
VIII.
IX.
During biofilm development over 7 days, the media was aspirated
from each well every 48 hours and replaced with fresh sterile
media (130 μl) using a multi-well pipette with sterile tips.
After 7 days of incubation, the plates were emptied and gently
washed once with sterile distilled water.
The biofilm was stained by adding 130 μl of 1% crystal violet (stock
concentration) and incubated for 30 min at room temperature.
X. The plates were gently washed three times with sterile distilled
water to remove excess dye.
XI. One hundred and thirty micro-litres of an ethanol/acetone (70:30
v/v) solution was added to each well to resuspend the bound crystal
violet dye.
XII.
The plates were incubated for an additional 10 minutes at room
temperature before OD 590 was measured.
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