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a rk D
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i
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n cm
etals
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s :1
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a ci ll
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p
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ai
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sted p
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s ch
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al i
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tio n
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LI ST O F F IG U R ES
F
i g u r e1 Co m p a r i s o n o f c u l t u r a b l e m i c r o b e c o n c e n t r a t i o n r e d u c t i o n s f r o m
6
r e p l i c a t e t r i a l s f o r t h e0 2
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m g/
l
m e t a l d o s e s i n s y n t h e t i c a n d o r g a n i cw
a t e r f o rb
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c o l i, a n dm s
-2
co l i p h a g e
(
P
a n e l sA
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]
E
a c h e r r o r bar i s c o n s t r u c t e d u si n g a9 5
%
c o n f i d e n ce i n t e r v a l o f t h eM E A N
1 8
F
i g u r e2 Co m p a r i s o n o f
L
o gi o r e d u c t i o n s f r o m6
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w
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CE R E U S,E
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c o l i p h a g e(
Pa n e l s
A
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.Ea c h e r r o r
b a r i s c o n st r u c t e d u s i n g a
9 5
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c o n f i d e n c e i n t e r v a l o f t h e m e a n2 0
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i gur e3 F
i r s t o r d e r m o d e l f i t o fE
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m e t a l s do se
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F
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i g u r e7 H
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(
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M E TAL S d o s e2 8
F
i gu r e9 O H T P
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m g/
L
]
m e t a l s d o s e
2 9
F
i g u r e1 0 O H T P
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M E T A L S d o s e
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i g u r e1 1
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M E T A L S D O S E
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F
i g u r e1 2 O H T P
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M E TAL S d o s e
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F
i g u r e1 3 O H T P
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F
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L
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O
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T
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o d e l p a r a m e t e r s a n d st a t i st i c a l s i g n i f i c a n c e r e s u l t s o f b a s e l i n e p a r a m e t r i c a n a l y s i s o fM E T A L I O N D O S E C O N D I T I O N A T
0 2
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a b l e1 0 M
o d e l p a r a m e t e r s a n d s t a t i s t i c a l s i g n i f i c a n c e o f p a r a m e t r i cj
o i n t c o n fid e n c e s p r e a dA N A L Y S I S O F M E T A L I O N D O S E CO N D I T I O N A T
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o d e l p a r a m e t e r s a n d s t a t i st i c a l s i g n i f i c a n c e r e s u l t s o f ba s e l i n e pa r a m e t r i c a n a l y s i s o fn a t u r a l o r g a n i c m a t t e r
w
a t e r c o n d i t i o n a t e a c h d i s i n f e c t a n t d o s e2 3
T
ab l e1 2 M
o d e l p a r a m e t e r s a n d s t a t i st i c a l s i g n i f i can c e o f p a r a m e t r i cj
o i n t c o n f i d e n c e s p r e a da n a l y s i s o f n a t u r a l o r g a n i c m a t t e r
w
a t e r c o n d i t i o n a t e a c h d i s i n f e c t a n t d o s e2 4
T
a b l e1 3
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D A T A S E T
2 4
T
a b l e1 4
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a t s o n m o d e lp a r a m et e r s a n d r e s u l t s f o r s t a t i s t i c a l s i g n i f i c a n c e f r o m z e r o o f t h e
S L O P E O F t h e l o g-l i n e a r R E G R E S S I O N L I N E
1
I
N
T R
O
D U
CT I
O
N
E
nte ri
cd i
s e a s eh
a s ap
a rti
c ul
a rl y h
a r sh i m p
a cti
n th
ed
e v el
op i
ng w
o rl d w h
e r e a c c e s s to cl
e a nw
ate ri
s n otr e a
d i ly
a v ai l
ab l
eE
sti m
ate sf
r om
th
eW
o rl d H
e al
th O
rg
a ni
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o np
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e ath
sf
r om
a n n u al g l
ob
al d i
a r rh
e al
d i
s e a s e at tw
ob i l l i
o n c a s e s e v e ry y
e a r,w i
th 1 5 m i U i
o n ch il d d
e ath
sp
e ry
e a r r e s ul
ti
ng
(W
H O 2 0 0 9
)
E
v e nw h
e r ew
ate rd i
strib
u ti
o n sy
stem
s a r ep
r e s e nt, th
ey m
ay b
ep
o o rly
m
ai
ntai
n ed
a nd
th i
sh i g h li g h
ts th
e n e ed
f
o rw
ate rd i
si
nf
e cti
o nm
eth
od
s th
atc a nb
e ef f
e cti
v ely
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n th
eh
om
eT h
ep
ote nti
al
e xi
sts top
r o vid
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v e, r eli
ab l
e, a c c ep
tab l
e,l
ow
-c o st a n
d
th
u s a c c e s si b l
ed
ri
nk i
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w
ate rd i
si
nf
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o nm
eth
od
th
at c a nb
e u ti l i
z ed i
n th
eh
om
ei
nd
e v el
op i
ng
c o u ntri
e sI
o ni
z ed
c op p
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zi
n cm
etal
sm
ay p
r o vi d
e o n e s u ch
al
te r n ati
v em
eth
od
T h
e a nti
m
i
c r ob i
al p
r op
e rti
e s of
c op p
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zi
n ch
a v eb
e e n e x am
i
n ed i
nd
ep
e nd
e ntly
a nd i
n c om b i
n ati
o nw i
th
oth
e rh
e a vy m
etal
s s u ch
a s si l
v e r,b
ut at tem p
ts to u s e c op p
e r a nd
zi
n ci
o n si
n c om b i
n ati
o n a r el im i
ted
A
m
o r e sy
stem
ati
c a nd
c om
p l
ete u nd
e r sta nd i
ng
of
th
e ef f
e cti
v e n e s s a nd
m
e ch
a ni
sm
of d i
si
nf
e cti
o n of
th
e s e tw
om
etal
si
n c om
b i
n ati
o nm
ay l
e ad
to a n o v el
d
ri
nk i
ng w
ate rd i
si
nf
e cti
o n te ch
ni q
u ew i
th
ap p l i
c ati
o n si
n th
ed
e v el
op i
ng w
o rld
,d
u ri
ng d i
s a ste r r el i
ef
op
e r ati
o n s, o r e v e ni
n th
em
od
em m
i li
ta ry
1
.1 O B JE
C
T
I VE
S
T h
eg
o al h
e r e,w i
th f
u rth
e r r e s e a r ch
a nd
r e s o u r c e s,i
s to e v e ntu all y d
e v el
op
a n af f
o rd
ab l
e, ef f
e cti
v e, s u stai
n ab l
e, a c c ep
tab l
e,p
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em
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s al
td i
si
nf
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r od
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at c a nb
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oy
ed
a nd
s ub
s eq
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th
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e al
th
ef f
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ep
r op
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m
eth
od w
o ul d
n ot r eq
ui
r efi l
tr ati
o n,m
ai
nte n a n c e of
ap
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a n e ntd
e vi
c e, a nd w
o uld
n ot af f
e ct th
ep
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atab i li
ty
o r od
o r of
th
ed i
si
nf
e cted w
ate rT h
ep
ri m
a ry
ob
j
e cti
v ei
s to te st th
e ef f
i c a cy
of d i
s s ol
v ed i
o ni
c c op p
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zi
n c o n th
ei
n a cti
v ati
o n of
th
em i
c r o o rg
a ni
sm
s,E
s ch
e ri
ch i
a c ol i
,B
a ci l l
u s c e r e u s, a nd
M
S
-
2 b
a cte ri
op h
ag
eT h
e s e c o nd
a ry
ob
j
e cti
v ei
s to ch
a r a cte ri
z e th
e r ed
u cti
o nk i
n eti
c s of
th
e s em i
c r o o rg
a ni
sm
sb y
th
e c om b i
n ed
m
etal d i
si
nf
e cta nti
n o rd
e r toq
u a nti f y
th
e ap p l i
c ab i l i
ty
a nd f
e a sib i li
ty
of
th
em
etal f
o rd i
si
nf
e cti
o n of
sto r ed d
ri
nk i
ng w
ate rA b
od y
of
r e s e a r ch
sh
ow
s e vi d
e n c e of
th
ek i l l i
ng
c ap
a ci
ty
of
ei
th
e rd i
s c r ete ap p l i
c ati
o n s of
c op p
e ri
o n s o rc o
m b i
n ed
c op p
e r a nd
sil
v e ri
o n s o nb
a cte ri
aR
e c e nt r e s e a r ch
c o nd
u cted
atT h
eU
ni
v e r si
ty
of N
o rth
C
a r ol i
n a atC h
ap
el H i l l
e x am i
n e s th
ed i
si
nf
e cti
o n c ap
a ci
ty
of
a c om
b i
n ati
o n of
c op p
e r a nd
zi
n ci
o n s(
H i
s e r2 0 0 9
,K
om
a nd
u r2 0 11
)
B
ui l d i
ng
o n th i
s af
o r em
e nti
o n ed
ap p
r o a ch
, th i
s stud y w i l l
ch
a r a cte ri
z e th
ed i
si
nf
e cti
o n c ap
a ci
ty
of d i f f
e r e nt c o n c e ntr ati
o n s of
c om
b i
n ed i
o ni
c c op p
e r a nd
zi
n ci
o n s o n th
r e em i
c r ob i
al
o rg
a ni
sm
si
n tw
o te stw
ate r s, o n ew i
th
a nd
o n ew i
th
o ut n atu r al w
ate r o rg
a ni
c c o nte ntT h
e r e sp
o n s e of
th
e te stm
i
c r o o rg
a ni
sm
s to th
ed i
si
nf
e cta ntw i l l b
eq
u a nti fi
ed
o v e r ti m
e u si
ng
a sta nd
a rd b
atch
e xp
o s u r e sy
stem
1
.2
A P P R O A CH
1
.2
.1 E
xp
e
ri
m
e
nt
a
l D
e s
i g
nT h
e e xp
e ri
m
e ntal
d
e si g
nw
a s am
od i fi
c ati
o n of
a sta nd
a rd b
atch
p
r oto c ol
o ri g i
n all y d
e v el
op
ed b y
K
om
a nd
u r(
2 0 1 1
)
a nd A
rm
str o ng
(
2 0 1 1
)
i
nw h i
ch
c om
b i
n ed
c op p
e r a nd
zi
n c s al
tsw
e r e u s ed
at v a ry i
ng
c o n c e ntr ati
o n si
n te stw
ate r ag
ai
n stE
s ch
eri
ch i
a c ol i b
a cte ri
ai
n al
ab
-c r e ated
"sy
nth
eti
c"w
ate r o v e rdi f f
e r e nt c o nta ct tim
e sT h i
s stud y
a ug
m
e nts th
ei
rw
o rk b y
em p l
oy i
ng
t wod i f f
e r e nt c o n c e n tr ati
o n s of
c om b i
n ed
c op p
e r a nd
zi
n c s al
tsi
nb
atch d i
si
nf
e cta nt e xp
e ri m
e nts c a r ri
ed
o uti
n tri p l i
c ate u si
ng
tw
o ty p
e s of
te stw
ate rT h
e te stw
ate r sw
e r ei
n o c ul
ated w i
th
th
r e ef
e c al i
nd i
c ato r o rg
a ni
sm
s, n am
ely E
s ch
eri
ch i
a c ol i
.B
a ci l l
u s c ere u s, a nd
M
S
-
2 b
a cte ri
op h
ag
e to e v al
u ate th
e a nti
m
i
c r ob i
al
ef f
e ct of
th
e c op p
e r a nd
zi
n c s al
ts o v e rd i f f
e r e ntc o n ta ct ti m
e sf
o r up
to6 h
o u r sT h
e e xp
e ri m
e ntal
s etup i
n cl
ud
ed
n eg
ati
v e c o ntr ol
v e s s el
s of
te stw
ate r s sp i k
ed w i
th m i
c r o o rg
a ni
sm
s al
o n eto o
b
s e r v e th
e stab i l i
ty
of
th
e te stm
i
c r ob
e s a nd i
n cl
ud
ed
oth
e r v e s s el
s of 10
% (
0 2 m g/ L
)
a nd 1 0 0
%
(
2
m g / L
)
m
etal d
o s e s ad d
ed
tob
oth
sy
nth
eti
c(
H E P E S
-b
uf f
e r ed
)
a nd
n atu r al
o rg
a ni
c(
H E P E S
-b
uf f
e r ed w
ate rp l
u s n atu r al l
ak
ew
ate r)
w
ate r sp ik
ed w i
th
te stm i
c r o o rg
a ni
sm
sT h
ed
u r ati
o n of
th
ed i
si
nf
e cti
o n e xp
e ri m
e ntw
a s6 h
o u r s,w i
th
s am
p l
e s tak
e n ati
n te r v al
s of 0
,2 0
,60
,18 0
, a nd 3 6 0 m i
n ute sS
am
p l
e sf
r om
e a ch
te st v e s s el w
e r ei
m
m
ed i
ately
n e utr al i
z ed
u si
ng
th
e ch
el
ati
ng
ag
e ntE D T A
to s eq
u e ste r th
em
etal i
o n sE
a ch
s am
p l
ew
a s th
e ndi l
uted
s e ri
al ly
te n-f
ol d
to1
:1
,
0 0 0
C h
el
ated
,d i l
uted
s am p l
e sw
e r e a s s ay
ed f
o r te sts a
m
p l
e s treated w i
th
th
e tw
od i f f
erent c o n c en tr ati
o n s of
m
etal i
o ns o ve r tim
e, th
e r ate and
extent of
m i
c r ob i
al
r ed
u cti
o n s o v e r ti m
ew
e r eq
u a nti fi
ed
a nd
e x am i
n ed
o n th
eb
a si
s of
d i f f
e r e ntm
od
el
sf
o rd i
si
nf
e cti
o nk i
n eti
c sI
nd
u cti
v ely
c o up l
ed p l
a sm
am
a s s sp
e ctr om
etry
(
I C P
-M
S
)
w
a s u s ed
tom
e a s u r e th
e c o n c e ntr ati
o n of
c op p
e r a nd
zi
n cm
etal i
o n sb
oth at ti m
e z e r o a nd
at th
e c o n cl
u si
o n of
the e xp
e ri
m
e nt aft
e r si
xh
o u r sh
ad
el
ap
s ed
.1
.2
.2
M
et
al I
o nD
osa
g
eT h
ed
o s e of
c op p
e ri
o n si
n te stw
ate rw
a si
nit
i
all y d
ete rm
i
n ed b
a s ed
o nW
o rl
d
H
e al
th O
rg
a ni
z ati
o n a nd
th
eU
ni
ted
S
tate sE
n vi
r o nm
e ntal P
r ote cti
o nA g
e n cy
(
U S
E P A
)
p
ub l i
sh
ed g
ui d
el i
n e sf
o rd
ri
nk i
ng w
ate rq
u ali
ty
A d
ail y d
ri
nk i
ng w
ate r c op p
e ri
ntak
e v al
u e of 2
a nd 1 3 m g/ L w
a s e stab li
sh
ed by
th
eW
H O
a nd
th
eU S
E P A
, r e sp
e cti
v el y
,w h i
ch m
ak
e s al l
ow
a n c ef
o r th
e ad d i
ti
o n al p
ote nti
al i
ntak
e of
c op p
e rf
r om
oth
e r s o u r c e s s u ch
a sf
o od
o r n utrit
i
o n al
s upp l
em
e nt(W
H O 2 0 0 8
,U S E P A
2 0 0 8
)
T h
ed
ri
nk i
ng w
ate ri
ntak
eg
ui d
el i
n ef
o r c op p
e r a s s u r e st h
at them
a xi
mu
m
d
ai ly i
ntak
ef
r om
al l
c opp
e r s o u rce s of 10
m
i ll i g
r am
sp
e rd
ay i
s n ot e x c e ed
ed
a nd w il l
n otl
e ad
to ad
v e r s eh
e alt
h
ef f
e cts(W
H O 2 0 0 4
)
T h
eW
H O
a nd U S
E P A
g
ui d
el i
n e sf
o rd
ail y i
ntak
e of
zi
n ci
sh i gh
e r th
a n c op p
e r, at15
-2 2
a n
d 7 4
-2 6 m g / L
, r e sp
e cti
v ely
( W
H O
2 0 0 8
,U S E P A 2 0 0 8
)
A lt
h
o ug h
th i
sm
a xi
m
um d
ai l y d
ri
nk i
ng w
ate ri
ntak
ef
o r zi
n ci
sm
u ch h ig h
e r tha nf
o r c op p
e r, zi
n c c o n c e ntr ati
o n s e x c e ed i
ng 3 m g / L p
r od
u c e u np
al
atab l
ed
ri
nk i
ng w
ate r( W
H O 2 00 3
)
F
o r th
e af
o r em
e nti
o n ed
r e a s o n s, th
e c op p
e r a nd
zi
n ci
o nd
o s e c o n c e n tr ati
o n s u s ed h
e r ew
e r e0 2m g / L d
e si g
n ated
a st
h
e10
%
d
o s e, a nd 2 m g/ L d
e si g
n ated
a sthe10 0
%
d
o s ef
o r e a ch
m
etal
.1
.2
.3 T
e st
W
at
e r sT h
e tw
ob
uf f
e r ed
te stw
ate r sw
e r ed
e si g
n ated
a s "sy
n theti
c" a nd
"o rg
a ni
c. "T h
e sy
ntheti
cw
ate rw
a sd
ei
o ni
z ed
, a uto cl
a v ed w
ate r atr o om
tem p
e r atu r e(
2 5
°C
)
c o ntai
ni
ng 10 g / L
(
0
.04 2
M
)
H E P E S b
uf f
e r atp H
7 2
±0 3 p H
u ni
tsT h
e o rg
a ni
cw
ate rw
a sp
r ep
a r ed i
n the s am
ew
ay
a s th
e sy
nth
eti
cb
uti
n cl
ud
ed
th
e ad d it
i
o n of 10 m i l l ig
r am
s of
s ol id d
eh y d
r ate c om b i
n ed w it
h 10 m i ll i l it
e r s of
sto ck
n atu r al l
ak
ew
ate rp
e rl it
e r of
te st s ol
uti
o n to a ch i
e v e am
e a s u r ed T
otal O
rg
a ni
cC
a rb
o n(
T O C
)
c o n c e ntr ati
o n of 11 8
m
g/ L i
n th
e o rg
a ni
c te stw
ate rT h
e n atu r al l
ak
ew
ate rh
ad
am
e a s u r ed E l
e ctri
c al C
o nd
u cti
vi
ty
(
E C
)
of 6 4 0 |
iS /
cm
(
c o r r e sp
o nd i
ng
to aT
otal D i
s s ol
v ed S
oli d
s(
T D S
)
c o n c e ntr ati
o n of 3 2 p p m
)
S
e v e r al
stud i
e s r ep
o rt th
atp H
i
s a ni
m
p
o rta ntf
a cto ri
n ef fi
c a cy
of
c op p
e r a nd
si l
v e ri
n c o ntr ol l i
ng L
eg i
o n el l
ab
a cte ri
ai
nw
ate rT h
ati
s, atp H
ab
o v e8
, th
e ef f
e cti
v e n e s s of
c op p
e ri
o n sd
e c r e a s e s(
L i
n19 9 6
,K im
2 0 0 3
,B l
a n c2 0 0 5
)
T h
e r ef
o r e,m
ai
ntai
ni
ng
ap H
of 7 2
±0 3 p H
u ni
tsi
n the e xp
e ri m
e ntal d
e si g
nw
a s e s s e nti
al
top
o s si b l y m
a xi m i
z e a nti b
a cte ri
al
a cti
vi
ty
.1
.2
.4 P l
at i
ng A
s s ay
sA
sta nd
a rd
sp
r e ad p l
ate c ul
tu r e a s s ay
u si
ng
a n o v el
c ult
u r em
ed i
um
,d
e s c ri b
ed b
el
ow
,w
a sd
e v el
op
ed f
o r thep l
ating
a nd
c o u nti
ng
of E
c ol i
a nd B
c ere u s c ol
o ny
-f
o r
m i
ng
u nit
s(
C P U
)
M
S
-2 b
a cte r