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Chapter 4
V. The coupons were released from the CDC biofilm reactor and
aseptically placed into individual wells of a 24 well microtiter plate.
VI.
VII.
After 10, 45 and 90 minutes contact time with 1ml of each of the
disinfectant at the test concentration the solution was aspirated out
of the plate and the coupons were then immersed in 1ml of the
appropriate neutralizing agent for 30 minutes (see Figure 4.1).
Difco D/E Neutralizing broth (39g/L) (Becton Dickenson) was used
to neutralize sodium hydroxide and benzalkonium chloride and
sodium thiosulfate (11.2g/L) to neutralize sodium hypochlorite.
VIII. The coupons were separated into the groups and assessed for cell
reduction and cell recovery.
o Cell Reduction
o Cell Recovery
IX. The 1 st group (cell reduction) of coupons were aseptically placed in
capped glass universal containers with 10ml of PBS and sonicated
using the conditions outlined in chapter 2 (section 2.5.1).
X. The universals (containing the coupon) were vortexed at high speed
to break down biofilm clumps and serial dilutions were made of the
PBS solution (coupons were taken out of the glass containers for
method described in chapter 2 as vortexing the surface in glass
containers was not possible for steel or glass coupons).
XI. The plate count method was performed as previously described in
chapter 2 (section 2.5.1)
XII. The Mean Log 10 Density was used to calculate the Log 10 reduction.
XIII. Log 10 reduction = Mean Log 10 untreated control coupons–Mean
Log 10 treated coupons as previously described (ASTM Standard
E2871-12) [125].
XIV. Each run performed twice using 1 coupon for each contact time and
disinfectant from each run.
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