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Chapter 5
V. The plates were filled in 2 batches of 12 with 130µl of the bacterial
suspension (0.2OD).
VI.
VII.
VIII.
IX.
Six plates were incubated at room temperature (measured as
~22°C) or at 37°C for 48 and 168 hours.
After 48 hours the 48 hour test plates were treated with
disinfectant as described below.
After 48 hours incubation the spent media was extracted from each
well in the 168 hour test plates and replaced with sterile LB w/o NaCl
media. After the 168 hours had elapsed the plates were treated
with disinfectants as described below.
To ensure all cells in each well were treated with disinfectant the
plates were treated with 200 µl of each disinfectant for the
assigned contact times of 10, 45 and 90 minutes with the
disinfectant agent.
X. After the time had elapsed the wells were drained of the
disinfectant agent.
XI.
XII.
XIII.
XIV.
XV.
The disinfectant treatment was directly followed by the addition of
200 µl of the corresponding neutralizing agent for 30 minutes.
One hundred and thirty micro-litres of the sterile media was added
to each well of the microtitre plate.
The plates were re-incubated at 22°C and 37°C accordingly.
After 24 hour incubation the media was extracted from each well
and placed into the corresponding wells of a sterile microtitre plate
to examine the turbidity the broth.
Increased turbidity indicated that cells in biofilm had survived
disinfectant exposure and were able to grow in the fresh broth.
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