Page 116 - Journal of Special Operations Medicine - Summer 2016

often more robust than their environmental counterparts Investigation with a constant temperature of 25°C and
and given that there can be great variability in the stabil- limited sunlight for 2 weeks. It was found that only the
8
ity of individual isolates. In the current study, three com- distilled water reservoir maintained viable organisms.
mon cleaning methods—bleach treatment, baking soda No organisms could be recovered from the reservoir con-
treatment, and proprietary CAMELBAK Cleaning Tabs taining the Honolulu municipal water. Given the results
(CamelBak Products LLC, Petaluma, CA)—were evalu- of this preliminary study, distilled water was chosen for
ated for the ability to remove E. coli contamination from all further inoculation and cleaning experiments.
hydration pack water reservoirs.
Reservoir Inoculation and Storage
Sixty-one hydration water reservoirs were filled with
Materials and Methods 500mL of distilled water and inoculated with 1mL of
phosphate-buffered saline containing an E. coli clini-
Bacterial Isolate Used in This Study cal isolate at an optical density of 1. All reservoirs were
A clinical isolate of E. coli was obtained from the Tripler capped and stored in a covered, open-air shed for 4
Army Medical Center (TAMC) Department of Pathol- weeks at TAMC to simulate the temperature and hu-
ogy. This isolate was maintained on tryptone soy agar midity of field conditions. The average high temperature
supplemented with 5% sheep blood (TSA/blood) at during the 4-week period was 29.4°C/84.92°F during
37°C without CO , and fresh plates were streaked prior the day. After 4 weeks, reservoirs were gently mixed and
2
to inoculation. All E. coli growth experiments were per- 100μL of water was collected from each reservoir. Col-
formed on TSA/blood agar. lected water samples were immediately plated on TSA/
blood agar and incubated overnight at 37°C.
Hydration Pack Water Reservoirs
A total of 66 new and unused surplus hydration pack Water Reservoir Cleaning
water reservoirs were obtained from the military supply A total of three cleaning methods were evaluated.
detachment at Schofield Barracks, HI. Each water reser- Twenty reservoirs were used for each treatment method.
voir had a capacity of 3L (100oz) and was composed of For the bleach cleaning method, the reservoirs were
polyether-thermoplastic polyurethane (polyether-TPU). first emptied and then refilled with 1L of hot tap wa-
ter (43°C/110°F) and 30mL of bleach (Clorox Regular-
Water Source Bleach ). The reservoirs containing bleach solutions
®
The goal of this study was to evaluate the capacity of were vigorously agitated for 30 seconds and allowed to
three common cleaning methodologies with respect to lay flat for 30 minutes. The bleach solutions were emp-
the removal of E. coli contamination from a hydration tied out and then refilled with 500mL of distilled water.
pack water reservoir. A preliminary study was conducted Reservoirs were agitated for 30 seconds and a 100μL
to compare bacterial growth and survival in a nutrient- sample was collected. The water samples were immedi-
limited, closed environment using water obtained from ately plated on TSA/blood agars.
the Honolulu County Municipal Water System and
distilled-deionized water obtained from the Department The same procedure was used for the baking soda (Arm
®
of Clinical Investigation at TAMC (Lab Pure Clinical and Hammer Pure Baking Soda ) treatment, with the ex-
Laboratory Reagent Water System by Water Solutions ception of substituting 30mL of baking soda and 1L of
Inc., Honolulu HI). Water from the Honolulu County hot tap water for bleach. The proprietary CAMELBAK
Municipal Water System had a total dissolved solute Cleaning Tablet treatment was performed as described
™
level of 202ppm (HM Digital TDS-EEZ Handheld Me- for the bleach and the baking soda with the exception
ter, HM Digital Inc., Culver City, CA), and the distilled- that a single tablet was used instead of the bleach/bak-
deionized water had a total dissolved solute reading of ing soda and the incubation time was 5 minutes instead
0ppm, indicative of an efficient purification process. of 30 minutes as suggested by the manufacturer.

Two water reservoirs were filled with 500mL of the mu- Data Analysis
nicipal water and distilled/deionized water, respectively. E. coli was identified by colony morphology. Colony
Both reservoirs were inoculated with 1mL of E. coli sus- counts before and after treatment were tabulated in
pended in phosphate-buffered saline at an optical density Micro soft Excel. Fisher’s exact test was used to deter-
®
(OD) of 1 at 600 nm (Cary 60 UV-Vis spectrophotom- mine the statistical significance of the tabulated results.
eter; Agilent Technologies, Santa Clara, CA). A 1OD
solution of E. coli is approximately equal to a concen- Results
tration of 500 million bacterial cells/mL, also known as
colony-forming units (CFUs). The reservoirs were incu- A total of 60 water reservoirs were used for experimen-
bated in a closed room at TAMC Department of Clinical tal cleaning treatments. Each was filled with 500mL of

102 Journal of Special Operations Medicine Volume 16, Edition 2/Summer 2016