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Chapter 4
4.2. Methods
4.2.1. Broth Minimum Inhibitory Concentration Method
I. The test organisms were stored at -20°C on glycerol protect beads
between experiments.
II.
III.
IV.
All 13 strains described in chapter 2 were used to establish the MIC.
Before each experiment the test strain was defrosted on the bench
and streaked on Tryptic Soy Agar (TSA) and Xylose Lysine
Deoxychocolate (XLD) plates which were incubated for 24 hours at
37°C.
The three disinfectants were made to above the working
concentration on each day of experiment in order to assess a range
of concentrations. Sodium hypochlorite (2000mg/L), sodium
hydroxide (4M) and benzalkonium chloride (0.08%) solution were
prepared using sterile H 2 0 and filter sterilized compounds.
V. Doubling dilutions were performed to achieve the test
concentrations.
VI.
Concentrations assessed were
o Sodium hypochlorite (1000mg/L, 500mg/L, 250mg/L,
125mg/L, 75mg/L)
o Sodium hydroxide (2M, 1M, 0.5M, 0.25M, 0.125M)
VII. Benzalkonium chloride (0.04%, 0.02%, 0.01%, 0.005%,
0.0025%)Isolated colonies were picked off the TSA plate and made
to a 0.5 McFarland suspension (~1.5-5x10 8 CFU/ml) in sterile water
and gently shaken.
VIII.
1ml of the suspension was added to 9ml of sterile Tryptic Soy Broth
(TSB) and vortexed vigorously.
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