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AN

APPROACH

TO

THE

DISINFECTANT

MENSURATION

OF

PEN ETRA BILITY

By
M A NLEY

A

MANDEL

THESIS

Su bmi tte d , to t h e S c h o o l of G r a d u a t e S t u d ie s of M i c h i g a n
S t a t e C o l l e g e of A g r i c u l t u r e an d A p p li e d S c ie n c e
in p a r t i a l f u l f i l l m e n t of the r e q u i r e m e n t s
f o r the d e g r e e of

DOCTOR

OF

PHILOSOPHY

D e p a r t m e n t of B a c t e r i o l o g y

1952

To m y M o t h e r

and F a t h e r

ACKNOWLEDGMENTS

I extend my m o s t sin c e re
M allm ann.

H is guid ance

work m ad e the

gratitude

to P r o f e s s o r

W. L.

and c o u n s e l d u r in g the c o u r s e

of t h i s

s u c c e s s f u l c o m p l e t i o n of the w o r k p o s s i b l e .

e n c o u r a g e m e n t a n d p r o v i s i o n of i d e a s ,
were

assistance

His

and m a te r ie l

invaluable.
The a u th o r w ish e s

D elap p e f o r h i s p a t i e n c e
problem s
of t h i s

to e x p r e s s

d u r in g n u m e r o u s

discussions

of the

m anuscript.

of P r o f e s s o r H. J .
I am

m y a p p r e c i a t i o n of the e f f o r t s

S t a f s e t h in the c o r r e c t i o n of th is

i n d e b t e d to M r .

of e q u i p m e n t ;

nations;
b u m in ;

to M r . I. P .

e x a m i n e d h e r e i n and f o r h is a i d in the p r e p a r a t i o n

I a l s o w i s h to e x p r e s s

item s

his gratitu d e

to M r .

I. L . D a h l j e l m f o r the us e

of m a n y

R. W. G r i m e s f o r n i t r o g e n d e t e r m i ­

to D r . K. B. M c C a ll f o r
a n d to M r .

m anuscript.

sam ples

of h u m a n

serum

al­

E. J . S w a r t h o u t f o r t e c h n i c a l a s s i s t a n c e .

I w i s h to e x t e n d m y a p p r e c i a t i o n to the A l u m n i of M i c h ­
i g an S t at e

C o l l e g e f o r t h e i r f i n a n c i a l a id t h r o u g h the MSC

F un d.

iv
In f a i r n e s s

to a l l who have a id e d m e , l e t it be u n d e r ­

st o o d t h a t a n y e r r o r s
t h o s e of the a u t h o r .

of o m i s s i o n o r c o m m i s s i o n a r e

entirely

M a n le y M andel
c a n d i d a te f o r the d e g r e e of
D o c to r of P h i lo s o p h y

F i n a l e x a m i n a t i o n , M a r c h 24,
D issertation:

1952

An A p p r o a c h to the M e n s u r a t i o n of D is i n f e c ta n t
Penetrability

O u tlin e of St u d i e s
M a jo r
M i n or

subject:
subject:

Bacteriology
B iochem istry

B iographical Item s
Born, July

10,

1923, in P h i l a d e l p h i a , P e n n s y l v a n i a

U n d e r g r a d u a t e S t u d i e s , B r o o k ly n C olle g e ,

1939-43

G r a d u a t e S t u d i e s , M i ch ig an State C o lle g e ,
U n i v e r s i t y of C a l i f o r n i a , 1948-50

1947,

Experience:

1950-52;

L a b o r a t o r y a s s i s t a n t a t B r o o k ly n C olleg e ,
1946-47; G u e s t I n v e s t i g a t o r at H a s k in s L a b ­
o r a t o r i e s , 1946, 1947-48; T e a ch in g A s s i s t a n t
a t U n i v e r s i t y of C a l i f o r n i a a t Dav is, 1948-50;
R e s e a r c h F e l l o w a t M ic h ig a n State Colle ge ,
1950-52.

M e m b e r of S o c i e t y of A m e r i c a n B a c t e r i o l o g i s t s , Societ y
of the S i g m a Xi, C a l i f o r n i a A c a d e m y of S c i e n c e s

TABLE

OF

CONTENTS

Page
I N T R O D U C T I O N .........................................................................
HISTORICAL

REVIEW

1

..........................................................................

10

The D e v e l o p m e n t of M ethod s f o r M e a s u r i n g the
P e n e t r a t i v e P o w e r s of D i s i n f e c t a n t s ..............................
C o l l o i d a l I o d i n e .........................................................................
M A TERIALS

AND

10
19

M E T H O D S ...........................................................

26

The I n h i b i t i o n of S p o r u l a ti o n of C o c c id ia l
O o c y s t s ........................................................................................................
The

"P enetube"

T e c h n ic

.

.

.

.

.

.

.

.

.

.

28

The D i s i n f e c t i o n

of C h ick e n F e c e s ....................................

Special V e s s e l s

Three

AND

33

C om partm ent

T e c h n i c s .................................................................................................
EXPERIMENTAL

26

R E S U L T S ....................................................

E x p e r i m e n t s U s i n g O o c y s t s of I£.t e n e l l a
E x p e r i m e n t s wi th the " P e n e t u b e " T e c h n ic

.

.
.

.

.
.

34
36
36

.

37

I n d i c a t o r o r g a n i s m s ...................................................................

37

The e f f e c t of

pH v a r i a t i o n ..................................................

39

The e f f e c t of

tem perature

41

v a r i a t i o n ............................

Vll
Page
T h e e f f e c t of v a r i a t i o n of d i s i n f e c t a n t
concentration

............................................................................

T he v a r i a t i o n of s t a t i o n a r y p h a s e
The

rate

E xperim ents

.

48

of d i f f u s i o n ....................................................................

52

o n the

com ponents .

44

D i s i n f e c t i o n of C h i c k e n

F e c e s ................................................................................................
T h e v a r i a t i o n of d i s i n f e c t a n t c o n c e n t r a t i o n
The

rate

of d i s i n f e c t i o n of c h i c k e n f e c e s

The ch a n g e s

53
.

.

53

.

.

58

in th e b a c t e r i a l f l o r a of the

f e c e s following d is in fe c tio n

.

.

.

.

.

.

.

61

D I S C U S S I O N ................................................................................................

63

The M e a s u r e m e n t of P e n e t r a b i l i t y ...........................

63

T e c h n i c ..........................................................

66

The

"P enetub e"

The

D isinfection P r o c e s s

a s A f f e c t e d by

P e n e t r a b i l i t y ..................................................................................
T he P e n e t r a t i v e P o w e r

of C o llo id a l Iodine

.

70
.

.

79

S U M M A R Y ..........................................................................................
B I B L I O G R A P H Y ..........................................................................................................

84

INTRO DU CTION

L o g ic is a n i n t e l l e c t u a l t r e a d ­
m i l l w h ic h t a k e s the m i n d r o u n d
a n d r o u n d in the s a m e c o u r s e ,
p e r p e t u a l l y r e s t r a i n i n g it f r o m
n e w a v e n u e s of a d v e n t u r e , a nd
leading it now here.
R o b e r t C.
M iller
In t h e e v a l u a t i o n of s u b s t a n c e s u s e d f o r o r
for disinfection p r o c e s s e s ,

a l a r g e n u m b e r of t e s t i n g p r o c e d u r e s

h av e b e e n d e v e l o p e d in the p a s t h a lf c e n t u r y .
developm ents

i s b e y o n d the

say that m ethods

recom m ended

A r e v i e w of s u c h

s c o p e of t h i s e f f o r t ;

su ff ic e i t to

h a v e b e e n d e v i s e d w hic h a r e a d e q u a t e f o r

the d e t e r m i n a t i o n of the e f f i c a c y of a c o m p o u n d in k il li n g t e s t
o rganism s

s u s p e n d e d in an a q u e o u s m e d i u m o r

lim ite d conditions.
form

The u s e of s u c h t e s t s

under special

w ill i m m e d i a t e l y i n ­

the i n v e s t i g a t o r if a c o m p o u n d is w ith o u t v a lu e

infectant,

as a d is ­

i n a s m u c h a s i t m a y be a s s u m e d t h a t a c o m p o u n d f a i l ­

ing to k i l l m i c r o o r g a n i s m s

u n d e r the

of the P h e n o l C o e f f i c i e n t t e s t (a s
p e c t e d to b e a s a t i s f a c t o r y

rath er

id e a liz e d conditio ns

an e x a m p le )

c a n n o t be e x ­

d i s i n f e c t a n t u n d e r the m o r e

r

c o n d i t i o n s o b t a i n i n g in p r a c t i c a l s i t u a t i o n s .

rigorous

2
Com pounds w hich have been

sho w n to be of h ig h g e r m i ­

c i d a l a c t i v i t y b y t h e P h e n o l C o e f f i c i e n t t e s t m a y o f te n be d e m ­
o n s t r a t e d to b e
wh en o r g a n i c
T h is

ineffective

compounds a re

illustrates

stance

o r of c o n s i d e r a b l y l e s s e n e d a c t i v i t y
a d d e d to the t e s t s y s t e m

t h e f a c t t h a t the v a l u e of a d i s i n f e c t i n g

c a n n o t b e p r e d i c t e d by one t e s t a l o n e .

(3) r e c o m m e n d t h a t a

"p ro file”

made em ploying an a r r a y
dem onstrate
terio sta sis,
organic
m irable,

a g e rm ic id e 's
its

m atter

(1, 2).

This

perform ance
tissue

and its p e n e tra b ility .*

a l t h o u g h i t m a y be

c l u s i o n of t i s s u e

S a ll e a n d C a t l i n

e v a l u a t i o n of a d i s i n f e c t a n t be

of t e s t s .

s p e e d of a c t i o n ,

sub­

array

is d e s i g n e d to

in the a b s e n c e
toxicity,
This

of b a c -

i n a c t i v a t i o n by

v ie w p o in t i s a d ­

s o m e w h a t e x a g g e r a t e d by th e i n ­

toxicity te s ts

(a p r o t e a n t a s k in e x e c u t i o n !) .

The c h i e f c u r r e n t t r e n d in c h e m i c a l d i s i n f e c t i o n is t o w a r d e n ­
vironm ental

sanitation

(4) a n d the c l i n i c a l u s e of d i s i n f e c t a n t s

f o r wound t r e a t m e n t h a s l a r g e l y b e e n
s u lf a d r u g s

a nd the a n t i b i o t i c s

(4),

s u p p l a n t e d by u s e

It i s ,

how ever,

of the

the l a s t

* " P e n e t r a b i l i t y " is u s e d in the a n t i t h e t i c a l s e n s e of
the d i c t i o n a r y d e f i n i t i o n a n d , a s s u c h , i s e q u i v a l e n t in m e a n i n g
to " t h e p e n e t r a t i v e p o w e r of a s u b s t a n c e " o r " t h e d i f f u s i b i l i t y
of a s u b s t a n c e . "
T h i s d e f i n i t i o n is w a r r a n t e d by r e a s o n of
usage and co n v en ien ce.

3
criterion

t h a t of p e n e t r a b i l i t y

upon w hich I w ish to p lac e

m ajor em phasis.
P re s e n t practices

in s a n it a ti o n e m p h a s i z e the i m p o r t a n c e

of the r i g o r o u s c l e a n i n g of s u r f a c e s p r i o r to any a t t e m p t s
d i s in f e c t io n .

at

T h i s e m p h a s i s is fully j u s t i f i e d and the following

r e m a r k s p e r t a i n only to the p r o v i s i o n of an ad eq ua te

m a r g i n of

s a f et y in the d i s i n f e c t i o n of the r e s i d u a l d e b r i s on s u r f a c e s
s u b j e c t e d to t r e a t m e n t with c h e m i c a l d i s i n f e c t a n t s a f t e r ad e qu a t e
c l ean i n g of the

surface.

It s ho u ld be o bv io us th a t u n l e s s a c h e m i c a l d i s i n f e c t a n t
is ca p ab l e of p e n e t r a t i n g an o r g a n i c o r p r o t e i n a c e o u s m a t r i x
to r e a c h o r g a n i s m s e m b e d d e d t h e r e i n , it will be of doubtful
value a s an e n v i r o n m e n t a l o r c l i n i c a l d i s i n f e c t a n t in situ a ti o n s
where o r g a n is m s

are

so p r o t e c t e d .

F o r th o se d i s i n f e c t a n t s

whose m od e of a c t i o n in killing m i c r o b e s is o t h e r than by p u r e l y
surface

a c t i v i t y p h e n o m e n a , the c e l l wall and m e m b r a n e of the

o r g a n i s m to be k i l l e d a c t s a s a b a r r i e r to the p e n e t r a t i o n of
the d i s i n f e c t a n t ,

a nd it is a p p a r e n t th a t s u c h com po un ds a s do

k ill m u s t have the p o w e r to p e n e t r a t e
or le s s e r

th e s e b a r r i e r s

to a g r e a t e r

e x t e n t in a d d i ti on to p o s s e s s i n g c h a r a c t e r i s t i c s

c a u s e t h e m to be l e t h a l once within the c y t o p l a s m .

which

Speed of

4
d is i n f e c ti o n t e s t s
two e f f e c t s ;

c a n give us in f o r m a t i o n c o n c e r n in g the s u m of

the r a t e of d is i n f e c ti o n will be a function of the e x ­

t r i n s i c m o b i l i t y of the d i s i n f e c t a n t a c r o s s the c e ll wall o r m e m ­
brane b a r r i e r

and of the i n t r i n s i c

r a t e of r e a c t i o n of the d i s i n ­

f e c ta n t with s u c h c e n t e r s of a c ti v it y within the o r g a n i s m a s m a y
be r e s p o n s i b l e f o r th e u l t i m a t e killing a c tio n .

F a i l u r e to give a

high r a t e of k i l l m a y , of c o u r s e , be a t t r i b u t a b l e to the influe nc e
of e i t h e r f a c t o r in the p a r t i c u l a r t e s t s y s t e m .

It is c e r t a i n

that, in a s p e e d of d i s i n f e c t i o n t r i a l , if a com pound c a n a c c o m ­
p li s h the d e s t r u c t i o n of life of the o r g a n i s m s

in the r e a c t i o n

v e s s e l in a v e r y s h o r t t i m e i n t e r v a l , then the t im e n e c e s s a r y
f o r the c o m p o u n d to have p e n e t r a t e d the o r g a n i s m s

in su ffi c i e n t

c o n c e n t r a t i o n to be l e t h a l in s u b s e q u e n t r e a c t i o n s with the c yt o­
p l a s m of the o r g a n i s m s m u s t be s o m e w h a t l e s s than the t ota l
ti m e inv olv e d.

T h i s t i m e f o r p e n e t r a t i o n of the c o m pou n d to

the ad e q u a t e c o n c e n t r a t i o n c a n n o t be g r e a t e r th a n the to ta l t i m e
i n t e r v a l i n v o l v e d in the t e s t .

If t h i s t i m e i n t e r v a l is a c c e p t e d

a s the m a x i m u m v a lu e f o r p e n e t r a t i o n to have o c c u r r e d within,
th en s p e e d of d i s i n f e c t i o n t e s t s

ca n be c o n s i d e r e d to m e a s u r e

the o v e r —
a l l e f f i c a c y of a c o m po und with the p e n e t r a b i l i t y of
the co m p o u n d c o n s i d e r e d a s one of the l i m i t a t i o n s of the r a t e of

5
kill.

T h e s e t e s t s , h o w e v e r , ca nno t divulge i n f o r m a tio n about

the e x a c t r a t e of p e n e t r a t i o n and, thus, w he re p e n e t r a b i l i t y alone
is to be m e a s u r e d , o t h e r m e a n s of m e a s u r i n g p e n e t r a t i o n of c o l ­
loid b a r r i e r s

m u s t be r e s o r t e d to.

A t t h i s po int, the t e r m p e n e t r a b i l i t y m u s t be defined and
I cho ose to do so a s f o l lo w s.

The p e n e t r a b i l i t y of a c h e m ic a l

d i s in f e c t a n t is the a b i l i t y of a d is in f e c t a n t a t som e o r ig in a l c o n ­
c e n t r a t i o n in the e n v i r o n m e n t to e n t e r a co lloid b a r r i e r and r e ­
sult in a c o n c e n t r a t i o n s u f f ic ie n t to kill o r g a n i s m s at this site
in so m e m i n i m u m t i m e i n t e r v a l .

T h is m ay be a l t e r n a t e l y t ake n

to m ea n th a t p e n e t r a b i l i t y is a fu nction of the r a t e
d i s in f e c ta n t wi ll t r a v e r s e

a l i n e a r d is ta n c e

at which the

th ro u g h a colloid a l

b a r r i e r w hic h m a y o r m a y not have the c a p a c ity to e n t e r into
c h e m i c a l o r p h y s i c a l c o m b in a tio n with the p e n e t r a t i n g

s u b s ta n c e .

This i s c l e a r l y a s t a t e m e n t of the r a t e of diffusion of a su b ­
s t an ce w ith the a d d e d q u a l ific atio n th a t the s t a t i o n a r y p h a se of
the s y s t e m m a y e n t e r into c h e m i c a l o r p h y s ic a l co m b in a tio n
with the m i g r a t i n g

substance.

It then follows that when one

c o n s i d e r s p e n e t r a b i l i t y , one m u s t c o n s i d e r the n a t u r e of both
the diffusing s u b s t a n c e a n d the s t a t i o n a r y p h a s e , the c o n c e n t r a ­
tion of the diffusing s u b s t a n c e and of the e l e m e n t s of the

6
stationary phase

s u s c e p t i b l e of c o m b in a tio n with this

substance.

The co n d i t i o n s of t e m p e r a t u r e , p r e s s u r e , pH (due to the effe ct
on ionic s p e c i e s of the c o m p o n e n ts of both p ha ses )

and the

p r e s e n c e of a d d i t i o n a l ions p r e s e n t in e i t h e r ph ase

will l i k e ­

w ise a f f ec t the r a t e a nd e x te n t of the p e n e t r a t i o n .
If a d e q u a t e i n f o r m a t i o n c o n c e rn in g the diffusion c o e f fi ­
c ie n t s of s u b s t a n c e s u s e d as d is in f e c ta n ts v e r s u s the a p p r o p r i a t e
s t a t i o n a r y p h a s e s l i k e l y to be e n c o u n t e r e d in a p p lic a tio n of d i s ­
i n f e c tan t s w e r e a v a i l a b l e , it would be a m a t t e r of s im p le c a l ­
cu latio n to p r e d i c t p e n e t r a b i l i t y .

U n fo rtu n a tel y , this conditi on

does n o t e x i s t f o r a n y c o m b in a tio n of e i t h e r p r a c t i c a l o r t h e ­
o r e t i c a l i n t e r e s t , a s a b r i e f e x a m i n a ti o n of the I n te r n a t io n a l
C ritical Tables

(5) wi ll d i s c l o s e .

Therefore,

e m p i r i c a l m e t hod s

f o r the d e t e r m i n a t i o n of p e n e t r a b i l i t y m u s t be em ploy ed.
Many e m p i r i c a l t e s t s (to be d e s c r i b e d m o r e c o m p l e t e l y
in the s u c c e e d i n g se ctio n) have b e e n d e v i s e d f o r the o s t e n s i b l e
d e t e r m i n a t i o n of p e n e t r a b i l i t y employing
tionary p h ases.

1' n o n r e p r o d u c i b l e 11 s t a ­

An e x a m p le of one suc h tech nic is a m od i fi e d

a g a r cup p r o c e d u r e w h e r e i n the d is in f e c t a n t is s e a le d in a sac
m ad e of a l e n g t h of c h i c k e n in te s tin e tie d at the a m p u ta te d ends
and the whole e m b e d d e d in a b a c t e r i a - s e e d e d n u t r i e n t a g a r

7
p l a t e , d i f f u s i o n of the
the i n t e s t i n e

d i s i n f e c t a n t th r o u g h the t i s s u e l a y e r s

r e s u l t i n g in a g r e a t e r o r l e s s e r

of

zone of in h ib i t i o n

of the b a c t e r i a l g r o w t h in the

surrounding m edium .

Such t e s t s ,

e m p lo y i n g t i s s u e s a s b a r r i e r s

to the p e n e t r a t i o n of a d i s i n f e c ­

t a n t , f a i l in s i m p l i c i t y of e x e c u t i o n and c o m p a r a t i v e va lue b e ­
c a u s e of th e e s s e n t i a l n o n r e p r o d u c i b i l i t y of the
T h is type of t e s t i s i n a d e q u a t e in y e t a n o t h e r
i n v e s t i g a t o r ‘ s c h o i c e of p r i m a r y

stationary phase.
r e s p e c t , a s the

stationary phases

is d i s t i n c t l y

l i m i t e d and e x p e r i m e n t a l v a r i a t i o n of the c o n c e n t r a t i o n and c o n ­
d itio n of t h i s p h a s e i s n o t f l e x i b l e .
If, t h e n , a t i s s u e b a r r i e r be o m i t t e d and only a b a c t e r i a seed ed n u t r i e n t a g a r gel p h a s e u tiliz e d , a s e r io u s
rem ains

in t h a t g r o w t h of the o r g a n i s m s

d ic a to r s is p r o c e e d in g

in a l o g a r i t h m i c

o b je c tio n

u s e d a s b io lo g i c a l i n ­
o r d e r while the diffu­

sio n of th e d i s i n f e c t a n t c a n n o t e x c e e d t h a t of a f i r s t o r d e r
a c ti o n r a t e .

Hence,

still

re­

the a d v a n c in g g e r m i c i d a l c o n c e n t r a t i o n of

the d i s i n f e c t a n t e n c o u n t e r s i n c r e a s i n g l y g r e a t e r n u m b e r s
t e r i a in v a r y i n g p h y s i o l o g i c a l s t a t e s .
when d i f f e r e n t o r g a n i s m s

are

at identical c o n cen tratio n s.

of b a c ­

T h is f a c t o r is m o s t e v i d e n t

t e s t e d a g a i n s t the
The d if f e r in g

s a m e c o m po un d

d i a m e t e r s of the z on e s

of in h ib i ti o n o b v i o u s l y c a n n o t be due to a v a r y in g diffusion r a t e

8
(a c o n s t a n t f o r i d e n t i c a l c o n d it io n s ) ,
a p p e a r to b e c a u s e d by d if f e r in g
to the c o m p o u n d .

s u p e r f i c i a l l y th e n th is would

s u s c e p t i b i l i t i e s of the o r g a n i s m s

A c t u a l l y the p h e n o m e n o n m a y be

m o s t e n t i r e l y to t h e d i f f e r e n t g r o w t h r a t e s

of the o r g a n i s m s and

p a r t l y to d i f f e r e n t s u s c e p t i b i l i t i e s of the o r g a n i s m s
f e c t a n t.

This c r itic is m

to d e t e r m i n e

sh o u ld l i k e w i s e

the a n t i b i o t i c

related al­

to the d i s i n ­

e x te n d to t h o s e

s p e c t r a by the

same

attem pts

rationale.

A

f u r t h e r l i m i t a t i o n of t h i s m e t h o d i s t h a t the i n v e s t i g a t i o n is
l i m i t e d , by the r e q u i r e m e n t t h a t g r o w t h of the i n d i c a t o r o r g a n ­
ism m u s t take pla c e

in the

su sp e n d in g

m enstruum ,

to the p h y s i ­

o l o g ic a l r a n g e of t e m p e r a t u r e , pH a nd s a l t c o n c e n t r a t i o n , a nd
the e l i m i n a t i o n of b a c t e r i o s t a s i s

is qu ite d if f ic u lt when not i m ­

possible.
T he p r o b l e m

t h u s p r e s e n t e d is to ev olve

for m e a s u r i n g the r a t e s
in r e p r o d u c i b l e

s y s t e m s of the e x p e r i m e n t e r Ls d e s ig n ,

to h a v e a v a i l a b l e

te ch ni c

of d iff u si o n of d i s i n f e c t a n t c o m p o u n d s

none of the a b o v e c r i t i c i s m s p e r t a i n .
sirable

some

w here

F u r t h e r , it would be d e ­

a s e c o n d te c h n ic u ti li z in g a c o m p l e t e l y

d i f f e r e n t m o d u s o p e r a n d i w h e r e b y c h e c k in g of r e s u l t s cou l d be
pos s i b l e .

9
T h is h a s b e e n the a u t h o r ' s ob je c tive , but the d e g r e e to
w hich he h a s b e e n s u c c e s s f u l in attaining this end m u s t r e s t
upon the a c c e p t a n c e a nd e x te n s io n of h is l a b o r s by independe nt
inve s t i g a t o r s .

HISTORICAL

REVIEW

The D e v e l o p m e n t of Methods f o r M e a s u r i n g the
P e n e t r a t i v e P o w e r s of D i s in f e c ta n ts

The m e t h o d o l o g i e s w hich have be e n de v elo p ed f o r m e a ­
su r in g the p e n e t r a t i v e p o w e r s of d i s i n f e c t a n t s

m ay be c o n s i d e r e d

u n d e r f o u r h e a d i n g s , o r g a n i z e d a s to the type of b a r r i e r p r i ­
m a r i l y i n v o l v e d in the t e s t .

These c la s s e s

are:

(1) th o s e in

which the o r g a n i s m s to be k il le d a r e p r o t e c t e d by a b a r r i e r
the f o r m of a d h e r e n t p a r t i c l e s of o r g a n ic m a t t e r ;
which the b a r r i e r
sue);

is a " n a t u r a l 11 t i s s u e

(3) t h o s e in which the b a r r i e r

in

(2) thos e in

(e.g., an e p i d e r m a l t i s ­

is an a r t i f i c i a l c o lloi d a l

m a t r i x s u c h a s an a g a r i f i e d n u t r i e n t solution;

and (4) tho s e in

w hich the c e l l w a l l o r m e m b r a n e of a p a r t i c u l a r o r g a n i s m

serves

a s the only b a r r i e r to e n t r a n c e of the d is in f e c t a n t.
Un d e r the f i r s t heading, this m ethod olog y was in tr o d u c e d
by V i n c e n t (6) in 1897.
disinfecta nts

T his i n v e s t i g a t o r stu d ie d the act ion of

in u s e a t t h a t tim e upon f e c a l m a t e r i a l and d e m o n ­

s t r a t e d t h a t c h e m i c a l a g e n ts w e r e l a r g e l y ineffective in the d i s ­
i n f ect io n of s u c h m a t t e r .

The e n c l o s u r e of b a c t e r i a in the o r ­

g an ic m a t t e r of the f e c e s

s e r v e d as an effective p r o t e c t i v e

d evice a g a i n s t the a c t i o n of the c h e m i c a l a ge nts em ployed.

This

s a m e m eth o do lo g y was l a t e r em ploye d by Mallm ann and Ch a n dl e r
(7) in an i n v e s t i g a t i o n d e s ign ed to find a di si n fe c ta n t which could
d e s t r o y all the b a c t e r i a p r e s e n t in f e c e s while not a d v e r s e l y a f­
fecting c o c c i d i a .

In th is study, it was r e c o g n iz e d that the finely

d ivided p a r t i c l e s of the a vian f e c e s
t r a t i o n of the d i s i n f e c t a n t .

s e r v e d as a b a r r i e r to p e n e ­

As a r e s u l t a n t of this e x p e r im e n t a l

p r o c e d u r e , i t was found t h a t Iodine S u sp e n so id (Merck) was the
only d i s i n f e c t a n t of the many th a t w e r e t e s t e d which would ra pidly
s t e r i l i z e the a v i a n f e c e s u n d e r the t e s t conditions.

A high d e ­

g r e e of p e n e t r a b i l i t y was a s c r i b e d to this p r e p a r a t i o n , about
which m o r e will be s a i d below.
Jo h n s (8) d e s c r i b e d a m eth o d f o r d e te r m in in g

sanitiz ing

e f f i c ie n c ie s of c o m p ou nd s which em ploye d an o r g a n i s m - s e e d e d
sk i m m ilk d r i e d in a f i l m on s t e r i l e g l a s s

s l id e s .

These p r e ­

p a r e d s l i d e s w e r e d ipp ed in the solution to be te s t e d , r i n s e d ,
p la c e d in s t e r i l e P e t r i d i s h e s and c o v e r e d with a n u t r i e n t a g a r .
The r e s u l t i n g d i mi n u t io n in n u m b e r s of colonies a p p e ar in g ex­
p r e s s e d the e f f i c i e n c y of the t e s t compound in p e n e tr a ti n g the
m ilk so lid p a r t i c l e s and in killing the e m be dde d o r g a n i s m s .

12
The s e c o n d c a t e g o r y of m e th o d s, w here n a t u r a l b a r r i e r s
have b e e n u s e d in d e m o n s tr a ti n g p e n e t r a b i l i t y of d isin fe c ta n t s ,
m ain ly h a s b e e n u s e d f o r the study of com pounds design ed f or
use a s a n t i s e p t i c s in p r e s u r g i c a l p r e p a r a t i o n s .

Seelig and

Gould (9) i m p l a n t e d c a p s u l e s containing iodine o r alcohol in
living a n i m a l sk in t i s s u e to d e t e r m i n e the o sm o tic b e h a v io r of
th ese co m p o u n d s in p e n e t r a t in g the living t i s s u e .

N y iri and

Ja n n itt e (10) p l a c e d v a r i o u s iodine p r e p a r a t i o n s on the sound
skin of dogs and r a b b i t s to d e m o n s t r a t e the p e n e tr a ti o n of the
skin by iodine.

Only i o d i d e -c o n tain in g p r e p a r a t i o n s a p p e a r e d

to p e n e t r a t e to a ny d e g r e e .

K a r n s (11) and K a r n s , C r e t c h e r and

Beal (12) u s e d guin ea pig skin as the b a r r i e r to d e m o n s tr a te
th at i o d id e - c o n t a i n i n g and aqueous p r e p a r a t i o n s of iodine we re
s u p e r i o r in p e n e t r a b i l i t y to t i n c t u r e s and non iodide-con tainin g
p r e p a r a t i o n s of iodin e.

E s s e n t i a l l y the s a m e r e s u l t s w ere ob­

tai n ed by B i s k i n d (13) e mploying fro g e p i d e r m i s .

A n d e rso n and

M al lm an n (14) s t u d i e d the p e n e tr a ti o n of living r a b b it skin, u s ­
ing a m o d i f ic a t i o n of the ti s s u e i r r i t a n t d e te r m in a t io n technic
d e v ise d by E t c h e l l s and F a b ia n (15), and the p e n e tr a ti o n of r a b b i t
skin r e m o v e d f r o m

sacrificed anim als.

The ' ‘d e a d 11 skin in the

l a t t e r t e s t was e m p l o y e d as a b a r r i e r betw een the t e s t disi n fe c t a n t

and a sali n e s u s p e n s i o n of b a c t e r i a .

The r a t e of b a c t e r i a l death

in the l a t t e r i n s t a n c e c o r r e l a t e d with the r e s u l t s obtained in the
t e s t s emplo y in g the " l i v i n g "

skin and with the a fo re m e n tio n e d

r e s u l t s of K a r n s e t al. on iodine p r e p a r a t i o n s .

In the sa m e

p ub licatio n, A n d e r s o n and M allm an n ex am in ed the s a m e s e r i e s
of d isin f e c t i n g a g e n t s f o r t h e i r ability to p e n e t r a t e e x c ise d adult
ch icken c e c a .

The w a s h e d c e c a w e re filled with the compound

to be t e s te d , the o pe n end tie d off with t h r e a d and e a c h ce c u m
su sp en d ed in a b r o t h c u l t u r e of the t e s t o r g a n i s m .

The rap i di t y

with which d e a t h of the o r g a n i s m s in the c u lt u r e m e d iu m en ­
sued i n d i c a t e d the r a t e of p e n e t r a t i o n throug h the t i s s u e of the
ce cu m by the d i s i n f e c t a n t .

Again, as in the p r e v i o u s ly cited

p u b li c a ti on s , the i o d i d e - c o n tain in g iodine p r e p a r a t i o n was su p e ­
r i o r to o t h e r iodine p r e p a r a t i o n s , and iodine in the c o n c e n t r a ­
tion em p lo y e d s u p e r i o r to all o th e r d is in f e c ta n ts

studied.

The t h i r d c l a s s of m etho dolog ies can be c o n s i d e r e d to
be an i n v e r s i o n of the c l a s s i c a l auxa nog raph ic te c h n ic s deve l ­
oped by B e i j e r i n c k (16) in 1889 fo r the study of n u tr iti o n a l
r e s p o n s e s of m i c r o b e s to o rg a n ic com pounds.

In an auxanogra phic

t ech n i c, a s the n a m e i m p l i e s , the t e s t w r i t e s its own a n s w e r

a

p u tative i n h i b i t o r of b a c t e r i a l growth p la c e d on a b a c t e r i a —se e de d

14
n u t r i e n t a g a r p l a t e i n d i c a t e s its activ ity by the p r e s e n c e o r a b­
sence of a zone of inhibition at the site of p la c e m e n t.

In this

m an n e r , K e n d a l l and E d w a r d s (17) p r e p a r e d a g a r c y li n d e r s
s e ed ed with b a c t e r i a .

A f t e r c o n ta c t with a solution of a d i s i n ­

f ectan t a c o r e was r e m o v e d and the ex te nt of inhibition of grow t h
in t h is c o r e

r e p r e s e n t e d the p e n e t r a ti o n of the d isinfe ctan t.

In

1918, C a r n o t and Dumon t (18) a s c e r t a i n e d diffusion of d i s in f e c ­
tants in the h o r i z o n t a l plane by p o uring se e d e d a g a r p la te s and
placing the d i s i n f e c t a n t in a p e r f o r a t e d g l a s s cup e m be dd ed in
the c e n t e r of the p l a t e .

The p e n e t r a b i l i t y of the dis infe cta n t

was r e p r e s e n t e d a s the d i a m e t e r of the su b se qu e n t zone of in­
hibition.

Th is t e c h n i c , with m in o r m odification, was l a t e r r e c ­

o m m en d e d by Ruehle and B r e w e r (19) fo r d e te r m in in g the c o m ­
p a r a t i v e p e n e t r a b i l i t y of d is in f e c ta n ts and a n t i s e p t i c s .

This

t e s t is g e n e r a l l y r e f e r r e d to as the " F D A a g a r - c u p p r o c e d u r e "
and i s p r o b a b l y the m o s t widely u s e d t e s t f o r p e n e tr a ti v e po w e rs
of d i s i n f e c t a n t s

in th o se r a r e

in s ta n c e s when p e n e tr a b i li ty is

c o n s i d e r e d in the e v a l u a t i on of a d isin fe c tan t.
pro ced u re has

This testing

s in c e b e e n ad a p te d f o r s p e c i a li z e d app lica tio n s .

A n d e r so n and M a l l m a n n (14) d e m o n s t r a t e d that volatile sub­
s ta n c e s c o ul d not be a s s a y e d by the FDA p r o c e d u r e b e c a u s e of

15
d i st il lati o n of the d i s i n f e c ta n t f r o m the c e n t e r cup to the su rf a c e
of the p l a t e wi th c o n s e q u e n t e r r o n e o u s i m p r e s s i o n s of high a c ­
tivity.

T h i s o b j e c t i o n was m e t by co v erin g the a g a r s u rf a c e

with a l a y e r of p a r a f f i n .

To a c c o m p l i s h the m e a s u r e m e n t of

the r a t e s of d iffusio n of iodine p r e p a r a t i o n s , the b a c t e r i a in the
a g a r w e r e r e p l a c e d with s t a r c h - i o d i d e

solution and the ex te nt

of the blue zone wa s r e c o r d e d a t f r e q u e n t i n t e r v a l s .
organism s were

Where

s e e d e d in the a g a r , and a s e r i e s of disinfe ct ing

agents t e s t e d by t h i s a d a p te d te c h n ic , the r a t e s of diffusion w e re
not in t h e m s e l v e s d e t e r m i n a b l e , only the fin a l e x te n t of the zones
of in hibition w e r e

recorded.

by A n d e r s o n a nd M a l l m a n n ,

This technic was f u r t h e r m od ifie d
so th a t the d is in f e c ta n t was e n c l os e d

in a sac ma d e of a s e c t i o n of chicken in te st in e and the l a t t e r
b u r i e d in the n u t r i e n t a g a r .

The p r e s e n c e

of a zone of inhi bi ­

tion about the s e c t i o n of in te s tin e ind ica ted p e n e t r a t i o n of the
t i s s u e , the a u t h o r s d i s c l a i m in g any im p o r ta n c e a s c r i b a b l e to the
size of the zone of inh ibition.

This m ethod, which has b e e n d i s ­

c u s s e d a t l e n g t h in the I ntrodu ction, is a c tu a lly m o r e c lo se l y
r e l a t e d to the s e c o n d g r o up of m e tho do logies than to this t h i r d
group, in w h ic h i t h a s b e e n included b e c a u s e of its develop­
m en t al h i s t o r y .

16
The f o u r t h c a t e g o r y of m ethod olog ical a p p r o a c h e s to the
p r o b l e m of p e n e t r a b i l i t y c o m p r i s e s those m ethod s in which
so me p a r t i c u l a r
a m in ed .

usually l a r g e

m i c r o o r g a n i s m has been e x­

In 1924, C h a n d le r (20) r e p o r t e d that aqueous

of iodine w e r e of high v e r m i c i d a l activity.

solutions

This a u th o r st a t e d

his b e lie f t h a t n e m a t o d e eggs and e n c ap s u late l a r v a e w ere

re­

s i s t a n t to m o s t f o r m s of d i s in fe c ta n ts b e c a u s e th e s e d is i n f e c ­
tan ts

. . i m m e d i a t e l y coagulate the album inous coatings of

n em ato d e e g gs and l a r v a e fo rm in g , th e re b y , an im p e rv io u s m e m ­
b r a n e which p r e v e n t s the f u r t h e r p e n e t r a t i o n of the c h e m ic a l . .
C h a n d le r b e l i e v e d t h a t iodine did not do this and thus was c a p ­
able of p e n e t r a t i n g the v ita ls of the o r g a n i s m s and cau sing t h e i r
death.
In 1930, K n a y s i (21, 23, 24, 25) and K naysi and Gordon
(22) p u b l i s h e d a s e r i e s of p a p e r s dealing with the kinetic a s p e c t s
of d is in f e c t i o n .

K n a y s i and Gordon s u b s c r i b e d to the view that

the d e a t h r a t e of b a c t e r i a obtained in stud ies on c h e m ic a l d i s ­
in fectio n was a f unc tion of the d is tr ib u ti o n of v a r ia ti o n in r e ­
s is t a n c e a mo n g the b a c t e r i a l population.

T his view was at

v a r i a n c e with t h o s e p r o po un de d by Chick (26) and by Rahn (27)
who held the r a t e of d e ath to be an e x p r e s s i o n of the ra te of

17
c h e m i c a l c o m b i n a t i o n in m o n o m o le c u la r and p o ly m o le c u la r r e ­
a c t io n s .

K n a y s i s t r e s s e d p e n e t r a b i l i t y as the lim iting f a c t o r

in the r a t e of d e a t h and stated:

1‘If a poison is to kill . . .

a

cell, it m u s t f i r s t p e n e t r a t e the s y s t e m of m e m b r a n e s and come
into c o n t a c t with the p r o t o p l a s m . 11

He c l a im e d to d e m o n s t r a t e ,

u sing S a c c h a r o m y c e s c e r e v i s i a e , that individual c e ll s v a r i e d in
t h e i r p e n e t r a b i l i t y to iodine and that th e r e w e re a p p a r e n t r e ­
gions of the c e l l wall and m e m b r a n e s p o s s e s s i n g h ig h e r p e r m e ­
ab ility to iodine t han o t h e r a r e a s .

K naysi a ls o d e m o n s t r a t e d

t h at iodine was t a k e n up f r o m solution by y e a s t c e lls in a c c o r ­
dance with an a d s o r p t i o n i s o t h e r m .
F i s h (28), in 1931, d e m o n s t r a t e d the e x t r e m e

resistance

of o o c y s t s of E i m e r i a t e n e ll a to p h y s ic a l and c h e m ic a l age nt s .
A n d e r s o n and M a l l m a n n (14) a tt r i b u te d this high r e s i s t a n c e of
the c o c c i d i a l o o c y s t s to the p r e s e n c e of a heavy c ell wall which
a c t e d as a b a r r i e r to p e n e tr a ti o n of c h e m ic a l agents.
in v e s t i g a t o r s t h e r e f o r e

The s e

us ed su s p e n si o n s of the c y s ts of E .

t e n e l la f o r d e t e r m i n i n g the p e n e tr a ti v e power of c h e m ic al d i s ­
in f e c tan t s .

The r a t e s of p e n e t r a ti o n of d isin fe c ta n ts w ere m e a ­

s u r e d by the p e r c e n t a g e

inhibition of s p o ru la tio n of the ooc ys ts

following e x p o s u r e to the d is in f e c ta n ts .

They found an iodine

18
h y d r o so l to be the only su b sta n c e effective in killing the o o c y s t s
and r e l a t e d t h is to a s u pposed high p e n e t r a ti v e power of the
preparation.

A n d e r s o n and M allm ann (14) a lso s e le c te d stro ng yl i d

la r v a e a s o r g a n i s m s
in f e c tan t s .

s u i te d f o r o b se rv in g the p e n e tr a ti o n of d i s ­

This o r g a n i s m is p r o t e c t e d by a waxy cuticle and

was shown to be r e s i s t a n t to the action of m o s t disin fe c ta n t s
ex cep t the h a l o g e n s

c h lo rin e and iodine.

Chlorine was o b s e r v e d

to d ig e s t the o u t e r c o v e rin g of the la r v a e and su bse qu en tly m o v e ­
m e n ts of the l a r v a e c e a s e d ; iodine c a u s e d a ra p id c e s s a t i o n of
m o v e m e n t s of the l a r v a e , with no p r i o r digestion of the cu ti cle
observed.
A n d e r s o n and M a llm ann (14) a ls o d e s c r i b e d a speed of
disinfection t e s t e mplo ying M ic r o c o c c u s pyogenes v a r . a u r e u s
as the t e s t o r g a n i s m .

This t e s t was a r a te of disinfe ction d e ­

t e r m i n a t i o n in the a b s e n c e of organic m a t t e r with sam plin g c on­
ducted in the s h o r t e s t p o s s ib le in t e r v a l s .

The t e s t was r e p o r t e d

to m e a s u r e p e n e t r a b i l i t y of d is in fe c ta n ts and gave s i m i l a r r e ­
su lts with the c o mp ou nd s t e s t e d as did the t e s t s employing c o c cid i al o o c y s t s and s t r o n g ylid l a r v a e .
In 1947, M a l l m a n n (29) upon a d d r e s s in g the National A s ­
so c iati o n of I n s e c t i c i d e and Disinfectant M a n u f a c tu r e r s ,

stressed

19
the i m p o r t a n c e of a d i s i n f e c t a n t having the p r o p e r t y of p e n e t r a n c e
into o r g a n i c b a r r i e r s

and e m p h a s i z e d the n e g l e c t of this c h a r a c ­

t e r in the l a b o r a t o r y e v a l u a tio n of p r o s p e c t i v e d i s in f e c ta n ts .
difficulty of p e r f o r m i n g
s t a t e m e n t,

The

s u c h ev a lu atio n s was highlighted by his

" A t p r e s e n t t h e r e a r e no good ro utine l a b o r a t o r y

p r o c e d u r e s ( fo r d e t e r m i n i n g p e n e tr a b il it y ) th a t yie ld s a t i s f a c t o r y
resu lts."

Th is a u t h o r is in c o m p le te a c c o r d with M a llm a n n in

t ha t r e s p e c t a nd would go so f a r as to s ta te th a t in his opinion
t h e r e do not a p p e a r to be any r e s e a r c h p r o c e d u r e s f o r the d e ­
t e r m i n a t i o n of the p e n e t r a t i v e p o w e r s of d is in f e c t a n t s which have
been e s t a b l i s h e d on a sound b a s i s .

C o l lo ida l Iodine

A m m a n (30), in 1910, o b s e r v e d th a t c e r t a i n solutions of
iodine e x h i b i t e d a T y n d a l l effec t and thus concluded that s u s ­
pe n d ed iodine p a r t i c l e s of fine size could e x is t.

B o r d i e r and

Roy (31) o b t a i n e d a f a i r l y stab le h y d r o s o l of iodine in w a t e r by
the d ilu t io n with w a t e r of a s a t u r a t e d alc o h o lic
in the p r e s e n c e of g e l a t i n as a s t a b i l i z e r .

solution of iodine

In 1924, C ha nd le r

(20) r e p o r t e d the p r e p a r a t i o n of a " h y p e r a c t i v e "
aration.

iodine p r e p ­

C h a n d l e r a nd M i l l e r (32) d e s c r i b e d the p r e p a r a t i o n

20
and p h y s i c a l p r o p e r t i e s of th is 1’h y p e r a c t i v e 11 iodine and r e d e s i g ­
n a t e d it a s Co l l o i d a l I odine.

The p r e p a r a t i o n was m ad e by a c i d i ­

f i c a t io n with h y d r o c h l o r i c a c id of an o s te n s i b l e
p oio dite a t 0 C in the p r e s e n c e
s t a b il izin g a g e n t.
p r e p a r a t i o n in fu ll .

sodium iodohy-

of gum a r a b i c as a p r o te c t i v e

B e n n e tt (33) h as d e s c r i b e d the d e ta ils of
T h i s c o llo id a l s u s p e n s i o n of iodine has

b e en m a r k e t e d a s Iodine S u sp e n s o id (Merck) and as Iodine V e r ­
m i ci d e ( M e r c k ) .
The c h ie f i n t e r e s t s in the use of C olloidal Iodine as a
d i s i n f e c t a n t a nd a s a t h e r a p e u t i c a g e n t lie in th a t it c onta i ns
n e i t h e r io d i d e s n o r a l c o h o ls and s t ill is a v a ila b le in c o n c e n t r a t e d
f o r m , in t h a t it h a s b e e n shown to be of low toxicity, in i t s value
a s a v e r m i f u g e , and in the f a c t th a t it is the only p r e p a r a t i o n to
the a u t h o r ' s kno wle dg e f o r which the sp ec ific p r o p e r t y of high
p enetrability has been claim ed.
In 1924, C h a n d l e r (20) r e p o r t e d th a t " h y p e r a c t i v e "

iodine

in a c a s t o r o i l v e h i c l e was effective in the t r e a t m e n t in the fie l d
of a s c a r i d a nd h e t e r a k i d infe c tion s of p o u lt r y , a s c a r i d and hook­
w o r m i n f e c t i o n s of fo x e s and a s c a r i d infections of swine.
M e r c k p r e p a r a t i o n s w e r e u s e d in s u b s e q u e n t s tu d ie s .

The

Cha nd l e r

and F e r g u s o n (34) d e m o n s t r a t e d one h u n d r e d p e r cent efficiency

Z1

f o r the p r e p a r a t i o n s
w o r m s of p o u l t r y .

in the t r e a t m e n t of t a p e w o r m s and rou nd ­
A l d e r m a n (35) c o n f i r m e d t h e s e findings.

C h a n d ler (36) r e p o r t e d k il ling the s to m a c h w o r m s in 97 of 98
lam bs

s u b j e c t e d to t r e a t m e n t with Colloidal Iodine.

W e is n e r

(37) d e m o n s t r a t e d t h a t Co l loid al Iodine was of value in the c on­
t r o l of c o c c i d i o s i s in p o u l t r y and r a b b i t s , and C h a n d l e r (38) d e m ­
o n s t r a t e d the a b i l i t y of t h i s p r e p a r a t i o n to k ill s p o r u l a t e d and
u n s p o r u l a t e d o o c y s t s of E i m e r i a t e n e l la and r e c o m m e n d e d its
use a s a d i s i n f e c t a n t in p o u l t r y p r a c t i c e .

W ilson (39) r e p o r t e d

the d e s t r u c t i o n of H a b r o n e m a spp. in the s t o m a c h and up p e r in­
t e s t i n e of the h o r s e , a nd the r e m o v a l of l a r g e l a r v a e of a G a s t e r o p h i l u s s p . , a lth ou gh s m a l l l a r v a e w e r e not a f f e c te d by the
iodine t h e r a p y .
S ta f s e t h (40) d e t e r m i n e d t h a t Iodine V e r m i c id e (Merck)
w as ca p ab l e m v i t r o of k i llin g a s c a r i d s
o n c h o s p h e r e s of t a p e w o r m s .

and the p r o g l o tt i d s and

He d e m o n s t r a t e d , how ev er, th at

the s c o l i c e s of the t a p e w o r m s infecting the c h ick e n w ere n e i t h e r
d i sl o d g e d n o r k i l l e d by the t r e a t m e n t with Colloidal Iodine.

Sub­

seq u e n t to the r e m o v a l of the s t r o b i l a e of the t a p e w o r m s by the
Iodine V e r m i c i d e

d o s a g e , S ta f s e th d e m o n s t r a t e d th a t the d e v e l ­

o p m e n t of the p a r a s i t e s c on tinu ed.

Two to t h r e e weeks were

th en n e c e s s a r y f o r the d e v e lo p m e n t of m a t u r e R a i ll ie ti n a c e s t i cil lu s in c h i c k e n s .

It i s a p p a r e n t then, th a t the high effic i e n c y

of iodine p r e p a r a t i o n s

in a p p a r e n t l y c u rin g t a p e w o r m infect ions

in p o u l tr y , m a y have b e e n only a c o n c o m it a n c e of the tech ni c
of a d m i n i s t e r i n g a c h e c k dose a few days a f t e r the in itial t h e r a ­
p eu t ic d ose and adjudging the infe ction t e r m i n a t e d if no ova n or
p r o g lo t t i d s w e r e found in the f e c e s .

As S ta fs e th po inted out,

C o l lo id al Iodine c a n s t i l l be r e c o m m e n d e d as a v e rm if u g e w he re
the o b jec t i v e i s to b r e a k the cycle of d e v e lo p m e n t of the p a r a s i t e
for a lim ited tim e.

At the p r e s e n t , h o w ev er,

Colloidal Iodine

is not r e p o r t e d a s the d ru g of choice fo r any of the w o r m o r
p r o to z o a n infections of a n i m a l s o r m an.
As m e n t i o n e d a b ove, M allm a n n and C h a n d le r (7) r e p o r t e d
the e f f e c t i v e n e s s of C o l l oidal Iodine in d isin fe c tin g chicken fe c e s ,
a c c o m p l is h i n g

s t e r i l i z a t i o n (sic) of the f e c e s in ten m in u te s

while no o t h e r d i s i n f e c t a n t t e s t e d could a c c o m p l i s h this fe a t
in m o r e e x t e n d e d p e r i o d s of ti m e .

A n d e r s o n (41) d e t e r m i n e d

the P h e n o l C o e f f i c i e n t of Colloidal Iodine to be ide n tic a l to tha t
of L u g o l 's Solution and to T in c t u r e of Iodine.

A n d e r s o n and

M a l lm a n n (14) a nd M a l l m a n n (29) a s c r i b e d the h i g h e r ac tivi t y
of the Co l l o i d a l Iodine to g r e a t e r p e n e t r a t i v e pow er of this

23
preparation.

A n d e r s o n a nd M a llm a n n (14) c o m p a r e d the p e n e t r a ­

b i l it y of the t h r e e

io dine p r e p a r a t i o n s by m e a n s of the p a r a f f i n -

c o v e r e d a g a r cup p l a t e m e t h o d and found th a t Co lloid al Iodine
in five p e r c e n t c o n c e n t r a t i o n gave a l e s s e r

zone of inhibitio n

than did the L u g o l ' s Solution o r the T i n c t u r e of Iodine in like
concentrations.

S i m i l a r r e s u l t s w e r e o b ta in e d usin g the te c h ­

n i c s em p lo y i n g c h i c k e n i n t e s t i n e s o r c e c a a s b a r r i e r s .
o r d e r of e f f e c t i v e n e s s in t h e s e c a s e s w as:

(1) L u g o l ’s Solution,

(2) T i n c t u r e of Io d in e , a n d (3) C olloidal Iodine.
w a s m o s t e f f e c t i v e in the

The

Co lloid al Iodine

s p e e d of d i s in f e c ti o n t e s t , in the in­

hib i tio n of s p o r u l a t i o n of c o c c i d ia l o o c y s ts and in the d e s t r u c ­
tion of s t r o n g y l i d l a r v a e .
disparate
ologies.

It w a s a p p a r e n t th at ex ce e d in g ly

r e s u l t s w e r e o b t a in e d by t h e s e two g r o u p s of m e th o d ­
A n d e r s o n a nd M a ll m a n n b e l i e v e d th a t the s e ttlin g of

the c o l lo i d p a r t i c l e s

in the f i r s t g r o u p of e x p e r i m e n t s

c ited

m a y h ave p r e v e n t e d e s t a b l i s h m e n t of a u n if o r m m a x i m u m c o n c e n ta ti o n of io din e in th e s olution, and that the
t i o n s a c t u a l l y m a y have h a d a g r e a t e r
c o l lo i d a l p r e p a r a t i o n .
separate

This

"diffusibility"

"diffusibility"

an d d i s t i n c t f r o m p e n e t r a b i l i t y

b elieves untenable.

s o lv a te d p r e p a r a ­
than the

they b e li e v e d to be
an a r g u m e n t this a u t h o r

It m u s t be noted th a t in t h e i r e x p e r i m e n t s

24
the h y d r o g e n io n c o n c e n t r a t i o n s of the p r e p a r a t i o n s
a d j u s t e d to l i k e v a l u e s n o r r e c o r d e d ,
were

stu dy in g the p r o p e r t i e s

w e r e not

i n a s m u c h as t h e s e a u t h o r s

of the p r e p a r a t i o n s

as g e n e r a l l y

o b t a in e d a nd the pH of e a c h p r e p a r a t i o n was of no i n t r i n s i c
i n t e r e s t to t h e m .

G e r s h e n f e l d and W itlin (42) have d e m o n s t r a t e d

the a cu te d e p e n d e n c e of the b a c t e r i c i d a l e f f ic ie n c y of iodine upon
the pH a t w h i c h t e s t s a r e
of the t h r e e

co nd uc ted.

iodine p r e p a r a t i o n s

The d i f f e r e n c e in a c t i v i t i e s

a s d e t e r m i n e d by the

s p e e d of

d i s i n f e c t i o n t e s t , the i n h i bitio n of s p o r u l a t i o n of c o c c i d i a l o o c y s t s
and the kill in g of s t r o n g y l i d l a r v a e

m a y be a r e f l e c t i o n of dif­

f e r i n g pH v a l u e s in e a c h p r e p a r a t i o n .

That th is is p o s s i b l e will

be d e m o n s t r a t e d be low.
C o l l o i d a l Iodine h a s b e e n d e m o n s t r a t e d to be only o n e f o u r t h a s t oxic a s T i n c t u r e
i s t e r e d o r a l l y (43).

of Iodine to r a b b i t s when a d m i n ­

I n t r a v e n o u s a d m i n i s t r a t i o n of e i t h e r p r e p ­

a r a t i o n r e s u l t e d in d e a t h of r a b b i t s with the
d o s e.

sam e m inim um

C h a n d l e r a nd M i l l e r (32) a ls o no ted th a t c o n c e n t r a t i o n s

of C o l lo i d a l Iodine s u f f i c ie n t f o r g e r m i c i d a l p u r p o s e s did not
c o r r o d e the

skin a n d o t h e r t i s s u e s to the ex ten t that alcoh o l ic

so l u t io n s did, n o r did it s ta in the skin as in t e n s e l y as did the
latter.

25
C o l lo i d a l Iodine would thus

s e e m to be m o r e d e s i r a b l e

tha n t i n c t u r e s f o r wound d r e s s i n g and f o r i n t e r n a l m e d ic a tio n .
The i n d ic a ti o n s t h a t it m a y p o s s e s s
ity m a k e it a good s u b j e c t f o r

a high d e g r e e of p e n e t r a b i l ­

st u d ie s on this

s u b j e c t and so a

l a r g e p o r t i o n of the e x p e r i m e n t a l w or k to be r e p o r t e d below i s
t h e r e f o r e c o n c e r n e d wi th the e x a m i n a t i o n of the p e n e t r a t i v e
p o w e r of th i s p r e p a r a t i o n .

MATE RIALS

AND

METHODS

The I n h i b iti o n of S p o r u la tio n of C o c c i d ia l O o c y s ts

E i m e r i a t e n e l l a was p r o p a g a t e d in six to eig ht wee k old
cockerels.

On the t e n t h day of inf e c tio n (when o o c y s t p r o d u c t i o n

is a t a m a x i m u m )
lected.

the c h ic k s w e r e

s a c r i f i c e d and the c e c a c o l ­

C e c a showing v i s ib le l e s i o n s w e r e h o m o g e n iz e d in the

W ar in g B l e n d o r a nd the o o c y s t s

sep arated from tissue fra g ­

m e n t s by s t r a i n i n g t h r o u g h f o u r l a y e r s of c l e a n c h e e s e c l o t h .
The o o c y s t s w e r e c o n c e n t r a t e d by flo ta tio n in a c o n c e n t r a t e d
sugar

so l u t io n f o ll owe d by d i f f e r e n t i a l c e n t r i f u g a t i o n in a r e ­

f rig e ra te d centrifuge.
distilled w ater

The fi n a l s u s p e n s i o n s w e r e

dilu te d with

so a s to c o n ta in a p p r o x i m a t e l y 200,000 o o c y s t s

p e r m l a n d s t o r e d in the r e f r i g e r a t o r a t 4 C f o r f u tu re

use.

F i v e —m l p o r t i o n s of iodine p r e p a r a t i o n s at the d e s i r e d
c o n c e n t r a t i o n s a n d pH v a lue w e r e b r o u g h t to 27 C in a w a t e r
b a th .

The s o l u t i o n s w e r e

in g r a d u a t e d c e n tr if u g e tu b e s

p e r e d w i th r u b b e r s t o p p e r s .

A fter te m p e ra tu re

s to p ­

e q u il i b r a ti o n ,

a o n e - m l p o r t i o n of the o o c y s t s u s p e n s i o n was added to e a c h
tube of d i s i n f e c t a n t and i m m e d i a t e l y

sha ke n by hand to i n s u r e

27
d i s t r i b u t i o n of the tube c o n te n ts .

A fte r incubation fo r the d e ­

s i r e d e x p o s u r e p e r i o d , f o u r m l of a s a t u r a t e d sodium t h io s u l fate so lu t io n was

r a p i d l y p ip e tte d into the c e n trif u g e tube to

n e u t r a l i z e the iodin e.

Co ntrol s u s p e n s io n s of the o o c y s ts w e re

t r e a t e d in the s a m e m a n n e r , with d is t il l e d w a te r re p la c i n g the
d is in f e c t a n t .

Foll owin g n e u tr a l i z a t i o n , the su s p e n s io n s we re

c e n t r i f u g e d a nd the

s u p e r n a t a n t liquid d ec an ted , the t e n - m l vol­

ume w as r e s t o r e d with d is ti ll e d w a t e r and r e c e n t r i f u g e d .

The

s u p e r n a t a n t was a g a in d i s c a r d e d to le av e a volume of one m l
of s e d i m e n t and f luid in e a c h tube.
so lu tio n of p o t a s s i u m d i c h r o m a t e

One m l of a 2.5 p e r c e nt

was added and the s e d i m e n t

w as su s p e n d e d by t r i t u r a t i o n with a wooden a p p l i c a t o r .

The

t u b es w e r e i n c u b a t e d in an in c lin e d p o sit io n f o r 48 h o u rs at
r o o m t e m p e r a t u r e to p e r m i t sp o r u la tio n of the o o c y s t s .

The

s u s p e n s i o n s w e r e t hen e x a m in e d un d e r the m i c r o s c o p e using
430X m a g n i f i c a t i o n , d u p l icate coun ts of e a c h tube w e re m ad e .
The n u m b e r s of s p o r u l a t e d o o c y s t s in a total of 100 w ere
counted..

The r e s u l t s w e r e e x p r e s s e d a s the p e r c e n ta g e of i n­

h ib itio n of s p o r u l a t i o n on the b a s i s of the s p o r u la tio n of the
untreated controls.

The

All g la s s w a r e

1' P e n e t u b e N T e c h n ic

u s e d in t h i s p o r t i o n of the

studies was

c l e a n e d by s o a k i n g f o r a t l e a s t 24 h o u r s in a c i d —d i c h r o m a t e
cleaning solution.
tap w a te r,

The a c id w as

r e m o v e d by five

rinses

in

a 24—
h o u r s o a k in d i s t i l l e d w a t e r and f o u r s u b s e ­

quent r in s in g s

in d i s t i l l e d w a t e r .

The g l a s s w a r e w as the n

d r a i n e d a n d a i r —d r i e d .
A ll c h e m i c a l s u s e d w e r e

c .p . g r a d e

unless

otherw ise

specifie d.
A ll r e a g e n t s

a nd s o lu ti o n s w e r e p r e p a r e d with d i s t i l l e d

w a t e r w h i c h h ad b e e n r e d i s t i l l e d f r o m
s o lu t io n in a g l a s s

an a lk a li n e p e r m a n g a n a t e

still.

C u l t u r e s of b a c t e r i a w e r e

checked for p urity at frequent

i n t e r v a l s by r e p e a t e d s t r e a k i n g on a tr y p to n e g lu c o s e y e a s t e x ­
tract agar
sm ears

(TGE)

an d by d i r e c t m i c r o s c o p i c

e x a m i n a t i o n of

s t a i n e d by the G r a m te c h n ic (H u c k e r m o d ifi c a tio n ).

c u ltu re s were

m a i n t a i n e d on TGE s l a n t s in s c r e w - c a p p e d v i a l s .

P y re x glass

tubing of 5 - m m o u ts id e d i a m e t e r was c ut

in 1 2 - c m l e n g t h s a n d one end g r o u n d f la t on a fine e m e r y
w h e e l.

The

A f t e r c l e a n i n g a nd d r y in g ,

the u n p o lis h e d end was

pl ug ged w ith c o t to n .

The tubes w e r e d i s t r i b u t e d in c le a n t e s t

t u b e s and s t e r i l i z e d in the d r y - a i r oven at

170 C f o r 2 h o u r s .

The c o l l o i d s y s t e m to be e x a m in e d co n ta in e d a g a r (Difco
g r a n u la t ed )

in a f in a l c o n c e n t r a t i o n of 1.5 p e r c e n t.

e s s a r y to p r e p a r e

It was n e c ­

the a g a r gel in a c o n c e n t r a t i o n su ch that, upon

dilu tio n of 16.2 m l with a d jun c t s u b s t a n c e s to 20.0 m l, the final
c o n c e n t r a t i o n would be a s

s ta t e d above.

T hus,

16.2 m l of this

a g a r g el w a s d i s t r i b u t e d p e r 50—m l E h r l e n m e y e r f la s k and a u t o c l a v e d a t 121 C f o r 20 m i n u t e s .

The gauze plug of e a c h fl a s k

w as w r a p p e d with " p a r a f i l m 11 and th e se f l a s k s could then be
s t o r e d in the r e f r i g e r a t o r at 4 C f o r p e r i o d s
in l e n g th with no d i s c e r n i b l e l o s s of m o i s t u r e .

up to one m onth
P r i o r to us e ,

the a g a r g el wa s m e l t e d and c oo led to 45 C and 2.0 m l of
m o l a r p h o s p h a t e b u f f e r s o lution added so th a t the final c o n c e n ­
t r a t i o n of the b u f f e r was te n th m o l a r at the d e s i r e d pH value .
E i t h e r 0 .8 m l of a p r o t e i n solution, d i s t i l l e d w a t e r o r a c o m b i ­
na ti o n of t h e s e to the s a m e

volum e was then added.

E a c h fl a s k

was s e e d e d wi th 1.0 m l of a s u s p e n s i o n of b a c t e r i a in d is t i l l e d
w ater.

This

s u s p e n s i o n was p r e p a r e d by washing the b a c t e r i a

f r o m the s u r f a c e of a 2 4 - h o u r TGE a g a r slope c u lt u r e with
distilled w ater.

The t u r b id it y of the b a c t e r i a l s u s p e n s i o n was

a d j u s t e d to 15 p e r c e n t t r a n s m i t t a n c e a t 490 m u in a C e nc o Sheard-Sanford P h o te lo m e te r.

The s e e d e d a g a r gel, now at a

v o lu m e of 20.0 m l , was a g i t a t e d to d i s t r i b u t e the c o n te n ts and
w as m a i n t a i n e d a t 45 C du rin g s u b s e q u e n t m a n ip u la tio n s.
To p r e p a r e

c o l u m n s of the a g a r gel within the tube s it

w as n e c e s s a r y to d e v i s e a m eth o d f o r setting the gel ra p id l y
so t h at l a r g e n u m b e r s of tub es could be p r e p a r e d without an
ag o n i zi ng w a it b e t w e e n the filling of e a c h s u c c e ed in g tube.

This

w as done by dipping a s t e r i l e tube into the m o lt e n a g a r gel and
d r aw i n g a c o l u m n of the liqu id into the tube to a height of 5 to
7 cm.

With the f o r e f i n g e r c o v e r in g the plugged end of the tube,

the tube wa s r o t a t e d o v e r the s u r f a c e of an ice cube fo r about
four seconds

so t h a t the a g a r gel r a p id ly

end of the tu be .

solid ified in the u p p e r

The tube could then be la i d in a P e t r i d i s h

h alf to c o m p l e t e g e l a t i o n while o t h e r tubes w e r e being p r e p a r e d .
It w as found c o n v e n i e n t to i n s u r e

s t a b il it y of the ice cu b es by

p l a c in g t h e m on a n o t h e r f i ll e d t r a y of ice cubes

so that the

t e n d e n c y to s l i t h e r a bout was inh ibited by the p a r t i t i o n s of the
tray.

If a m e n i s c u s d e p r e s s i o n a p p e a r e d at the lo w e r end of

a tube due to d r a i n a g e , the tube was touc he d to the s u r f a c e
the m o lt e n a g a r a t an a cu te angle and this d e p r e s s i o n filled.

of

31
T ub e s b e a r i n g a g a r g e l c o l u m n s of d if f e r in g c o n s t i t u t i o n w e r e
c o lo r coded with w a x - c r a y o n m a r k s
p l ug .

In a l m o s t a l l c a s e s , th is

a t the end having the co t ton

m e t h o d r e s u l t e d in the p r e p a r a ­

t ion of a u n i f o r m c o l u m n of a b a c t e r i a - s e e d e d a g a r ge l with a
b o t t o m n e a r l y p e r f e c t l y f l a t w hose c r o s s - s e c t i o n a l a r e a was 7.06
mm

2

± 0 . 0 03 m m
To c l e a n ,

buffer

so l u t i o n ,

2

.

dry,

2 0 -m m te s t tubes,

5.0 m l of d is in f e c t a n t ,

s a l t s o r o t h e r a d ju v a n t s u b s t a n c e s w e r e ad d e d

a nd b r o u g h t to t he d e s i r e d t e m p e r a t u r e

in a w a t e r b a th .

The

f i n a l b u f f e r c o n c e n t r a t i o n w a s h alf m o l a r and the b u f f e r s o l u ­
tions w e re

m a d e of a p p r o p r i a t e

m i x t u r e s of m o n o - and d i p o t a s ­

sium phosphates.
T he p r e p a r e d t u b e s c o n ta in in g the a g a r g e l c o l u m n s w e r e
w ip ed d r y wi th c l e a n c h e e s e c l o t h
t u b e s c o n t a i n i n g t he d i s i n f e c t i n g
s t o p p e r e d w i t h No. 2 r u b b e r
com m odate

as many as

squares

s o l u ti o n s and th e s e l a t t e r t ube s

stoppers.

7 tubes.

two t u b e s p e r t e s t tube to i n s u r e

and p l a c e d in the t e s t

There

E a c h t e s t tube c o u ld a c ­
w ere

a lw a y s a t l e a s t

a g a i n s t a tig h t s e a t b e tw e e n

the b o t t o m of the t e s t tu b e and the c i r c u m f e r e n c e
c o n t a i n in g th e a g a r gel c o lu m n .

of the tube

32
When v o l a t i l e
the r u b b e r s t o p p e r s

d i s i n f e c t a n t s w h ic h m ig h t be a b s o r b e d by
(e.g.,

h a lo g e n s )

s to p p e re d a m b e r m ixing c y lin d e r s

w ere

tested, 25-m l g la s s -

r e p l a c e d the t e s t t u b e s and

rubber stoppers.
A t t h e e n d of t he e x p o s u r e p e r i o d , the tu b e s w e r e
m o v e d f r o m the d i s i n f e c t a n t by m e a n s of a f o r c e p s
cess

and the e x ­

d i s i n f e c t a n t r e m o v e d by r i n s i n g the t u b e s in s t e r i l e

tilled w ater

or

The c o l u m n

of a g a r g e l w a s

in a s o l u t i o n of a s u i t a b l e n e u t r a l i z i n g

t e s t tu be of an a p p r o p r i a t e
n eutralizing

a g e n t to

i n the c o lu m n .

re­

dis­

agen t.

e x p r e s s e d f r o m the tube into a
sterile

n u t r i e n t b r o t h c o n ta in in g a

p e r m i t the d e v e l o p m e n t of via b le o r g a n i s m s

T h e e x t r u s i o n of the c o l u m n

was accom plished

by b lo w i n g a t the p l u g g e d en d w ith the m o u th o r by em ploy i ng
the c o t to n p lug a n d a n a p p l i c a t o r as a p i s t o n and p l u n g e r .
b r o t h tu b e s w e r e

t h e n i n c u b a t e d a t 37 C f o r

18 to 24 h o u r s

The
in

a forced—
draft incubator.

The c o n t e n t s of e a c h b r o t h tube w e r e

t h e n p o u r e d into a P e t r i

d i s h and e x a m i n e d by i l l u m i n a t i o n f r o m

b e n e a t h the p l a t e .

The c l e a r

z o n e s of in h ib itio n w e r e

w ith the a id of a s t e e l d i v i d e r and m e t r i c
tenth m i l l i m e t e r .
i nh ib i ti o n a r e

The a p p e a r a n c e

of s o m e

d e m o n s t r a t e d in P l a t e

1.

s c a le

m easured

to the n e a r e s t

typ ic a l z o n e s of

PLATE

1.

The a p p e a r a n c e of som e typical zones of inhibition
in a g a r gel c o lu m n s following d ev elop m en t.
A.

C o lu mn s ex p o se d to Colloidal Iodine for
h o u r s at pH 5.0 and 27 C.

24

B.

Co l u m n s ex p o se d to phenol fo r 24 hours
pH 5.0 and 2 7 C.

at

33
The D i s i n f e c tio n of C hicken F e c e s

Ch i ck e n f e c e s was c o l le c te d f r o m at l e a s t six c a g e s
c o n tain in g n o r m a l a d u l t b i r d s of both s e x e s on a g r a i n die t.
Twice the we ight of d i s t i l l e d w a t e r was added to the f e c e s
and the m i x t u r e h o m o g e n iz e d f o r one m inu te in the W aring
B lendor.

The r e s u l t i n g s u s p e n s i o n was

s t r a i n e d th ro u g h a

double l a y e r of c h e e s e c l o t h and c o l l e c t e d in s c r e w - c a p p e d v i a l s .
With the a i d of the m i c r o s c o p e , the s u s p e n s i o n s w e r e
c o n t ai n m a n y p a r t i c l e s of m a t t e r of v a ry in g s i z e s ,
which w e r e a s l a r g e

a s 20 m i c r a in d i a m e t e r ,

m erous bacteria freely

s u s p e n d e d in the fluid.

p r o b a b l y of p r e c i p i t a t e d u r a t e s ,
To c l e a n s t e r i l e

s e e n to

so m e of

as well a s n u ­
Some c r y s t a l s ,

were encountered.

s c r e w - c a p p e d v ia l s ,

9.5 m l of the d i s ­

in f e c ta n t p l u s p h o s p h a t e b u ff e r m i x t u r e w e r e added.

The fina l

c o n c e n t r a t i o n of the b u f f e r was half m o l a r at the d e s i r e d pH
v alu e.

The t u b e s w e r e p l a c e d in a w a t e r b a th and b ro u g h t to

tem p eratu re

e q u i l i b r i u m a t 27.5 C ± 0.5 C.

The f e c e s

s u s p e n s i o n was thoroug hly a g i ta t e d with a

p ip e tt e and 0.5 m l add ed to e a c h d i s i n f e c t a n t - b u f f e r tube and
to a d i s t i l l e d w a t e r - b u f f e r c o n tr o l tube.

The c on tents w er e

m ix e d by i n v e r s i o n of the tubes a n u m b e r of t i m e s

during the

34
c o u r s e of th e e x p o s u r e s .
sam ples
priate

A f t e r the e x p o s u r e

interval,

1.0—ml

w e r e w i t h d r a w n a nd p i p e t t e d into 9.0 m l of an a p p r o ­

n e u t r a l i z i n g a g e n t to stop the a c tio n of the d i s i n f e c t a n t .

T h is t e n f o l d d i l u t i o n w a s t h e n s h a k e n f o r five m i n u t e s on a
m e c h a n i c a l d e v i c e a t a r a t e of 200 s t r o k e s p e r m in u te th r o u g h
a one-inch excursion.

A ppropriate

in d u p li c a t e wi th TGE a g a r .
37 C, the p l a t e s w e r e
recorded.

d i lu tio n s w e r e th e n p l a t e d

ATter i n c u b a t io n f o r 48 h o u r s a t

e x a m i n e d an d the n u m b e r s of c o lo n i e s

R esults w ere

e x p r e s s e d a s the n u m b e r s

of o r g a n ­

i s m s p e r m l of th e r e a c t i o n m i x t u r e s .

S p ecial V e s s e l s

A ttem pts w ere

Three

C o m p a rtm e n t Technics

m a d e to c o n s t r u c t d if fu s io n c e l l s w h e r e

a d i s i n f e c t a n t s o l u t i o n c o u ld be

separated from

p e n s i o n by a b a r r i e r

C o llo d io n s a c s w e r e i n t u s s u s -

s o l u tio n .

a bacterial sus­

c e p t e d o v e r the e n d s of g l a s s tubing and s u s p e n d e d in a l a r g e r
v e s s e l c o n t a i n i n g th e

s u s p e n s i o n of b a c t e r i a .

c o m p a r t m e n t c o n t a i n e d th e d i s i n f e c t a n t ,
the b a r r i e r

substance

the m i d - c o m p a r t m e n t

in the f o r m of a s o lu tio n o r gel.

n a t e l y , th e p o s i t i o n of the b a c t e r i a l
fectant was

The i n n e r m o s t

r e v e r s e d in the

system .

A lter­

s u s p e n s i o n and the d i s i n ­
No r e s u l t s

w e r e o b tai n e d

35
w ith t h e s e c e l l s a s the m e m b r a n e s w e r e e x c ee d in g ly f r a g i l e
a nd h ad a g r e a t t e n d e n c y to b r e a k du ring t r i a l r u n s .
C e l l s w e r e c o n s t r u c t e d of thin r i n g s of u n g la z e d p o r c e ­
lain.

A s m a l l r i n g was c e m e n t e d to a c o v e r g l a s s with de

K h o ti n s k y c e m e n t a nd a l a r g e r rin g the n c e m e n t e d in a con­
ce n tric position.

The c e n t e r w ell was f il le d with the d i s i n f e c ­

t a n t an d the a n n u l u s wi th the b a r r i e r

substance.

A cover glass

w as s e a l e d to the top s u r f a c e s of the r i n g s and the whole a r ­
r a n g e m e n t d r o p p e d in a s u s p e n s i o n of b a c t e r i a in d i s t i l l e d
w ater.

This

s u s p e n s i o n was

s t i r r e d by m e a n s of a m a g n e ti c

s t i r r i n g d ev i c e a n d s a m p l e s w ith d r a w n at i n t e r v a l s f o r the e nu­
m e r a t i o n of the v i a b l e b a c t e r i a .

The f r a g i l i t y of the s y s t e m

and the d i f f ic u l t y i n v o l v e d in obtaining a p e r f e c t s e a l b e tw e e n
the i n n e r r i n g a n d the top c o v e r g l a s s

ob v iated continued us e

of t h i s te c h n i c a nd so the m e th o d w as a l s o a b an don ed and no
results are

r e c o r d e d f o r e x p e r i m e n t a l u se of the m ethod .

EXPERIM ENTAL

AND

RESULTS

E x p e r i m e n t s Using O o c y s t s of E_. T e n e l l a

The e f f e c t of h y d r o g e n ion c o n c e n t r a t i o n on the d i s i n f e c ­
tio n of o o c y s t s of K_. t e n e l l a by iodine w a s d e t e r m i n e d .
of Iodine S u s p e n s o i d (Me rck)
d e t e r m i n e d to be 2.0.

in 5.0 p e r c e n t c o n c e n t r a t i o n was

A 5.0 p e r c e n t L u g o l ' s Solution was a d ­

j u s t e d to t h is pH with c o n c e n t r a t e d HC1.
p re p a ra tio n were

The pH

O ther portions

a d j u s t e d to pH 7.0 with 2.5 N NaOH.

s u r e of the o o c y s t s to a l iq u o ts of th e s e p r e p a r a t i o n s
a s d e s c r i b e d a b o ve , f o r p e r i o d s of 5,

of e a c h
E xp o ­

were made,

10 and 20 m i n u t e s .

The

p e r c e n t of i n h ib i t io n of s p o r u l a t i o n of the o o c y s t s a s c o m p u t e d
from

control sporulations

is given in T ab le

1.

These

d a t a show

the m a r k e d e f f e c t of h y d ro g e n ion c o n c e n t r a t i o n on the kill of
the c o c c i d i a l o o c y s t s .

At the

s a m e pH v a l u e s , t h e r e is v e r y

l i t t l e d i f f e r e n c e in a c t i v i ty b e tw e e n the two iodine p r e p a r a t i o n s .
The c o l lo i d a l p r e p a r a t i o n h a s a sligh tly h i g h e r a c tiv ity but no­
w h e r e of the o r d e r of d i f f e r e n c e as d e t e r m i n e d by A n d e r s o n and
M a ll m a n n (14).

T h i s d i s c r e p a n c y is e a s i l y u n d e r s t o o d on the

b a s i s of the h ig h a c i d i t y of the c o llo id a l p r e p a r a t i o n as c o m p a r e d

TABLE
T HE

1

P E N E T R A T I O N O F COCCIDIAL OOCYSTS BY
C E N T IODINE P R E P A R A T I O N S AT 27.0 C

5% I odine a s

5 PER

P e r C en t In h ib itio n
of S p o r u la ti o n *

PH

5 m in

10 m in

20 m i n

S usp en so id (M erck)

2.0

76

96

100

S u sp e n so id (M erck)

7.0

43

89

99

L u g o l's Solution

2.0

69

91

100

L u g o l ’s S o l u t i o n

7.0

41

79

97

* The i o d i n e w a s n e u t r a l i z e d w ith s o d i u m t h i o s u l f a t e
i m m e d i a t e l y a f t e r t h e n o t e d e x p o s u r e p e r i o d and s p o r u l a t i o n
of the o o c y s t s a c c o m p l i s h e d in 2.5% p o t a s s i u m d i c h r o m a t e
f o r 48 h o u r s a t " r o o m " t e m p e r a t u r e .

37
to t h a t of LiUgol's So l u tio n .
are

in the

P reparations

of the l a t t e r u s u a l l y

r a n g e of pH 5 to pH 6.

T h e p r e p a r a t i o n of s u s p e n s i o n s of o o c y s t s of 12. t e n e l l a
w a s q u i te t e d i o u s a n d the r e s u l t i n g
able

sporulation p e rc en ta g e s.

c e n t in s o m e i n s t a n c e s .

s u s p e n s i o n s gave

These

quite

r a n g e d a s low a s

vari­

6 per

No f u r t h e r e x p e r i m e n t s w e r e c o n d u c t e d

w i th t h i s m e t h o d .

E xperim ents

w i t h the

Indicator o r g a n i s m s .

"P enetube"

T e c h n ic

P enetub es w ere p r e p a r e d contain­

ing a g a r and M / 1 0 p h o s p h a te b u f f e r a t pH 5.0.
penetubes contained M . pyogenes v a r. a u re u s
indicator o rg a n is m

a nd a n o t h e r

One

series

(FDA 209)

s e t c o n t a in e d K .

of

a s the

c o li ( l a b o r a ­

to ry strain)

a s th e i n d i c a t o r o r g a n i s m .

T h ese penetubes were

exposed for

24 h o u r s a t 27.0 C in tu b e s c o n ta in in g M / 2 p h o s ­

p h a te b u f f e r a t pH 5.0 a n d R o c c a l o r ph en o l in v a r i e d c o n c e n ­
trations.

A fter exposure

developed.

the t u b e s w e r e

r i n s e d and the c o l u m n s

The c o l u m n s w hic h had b e e n e x p o s e d to ph en ol w e r e

d e v e l o p e d in FD A b r o t h a n d th o s e w hich had b e e n e x p o s e d to
Roccal w ere
T am ol—
N.

d e v e l o p e d in F D A b r o t h c o n tain in g 0.1 p e r c e n t

A f t e r d e v e l o p m e n t , the

z o n e s of inhib ition w e r e

38
m e a s u r e d a n d the a v e r a g e v a lu e f o r e a c h p o in t p l o t t e d in F i g ­
ure

1.
In F i g u r e

trates

1A, i t c a n be

fu rth e r where

c o li h a s b e e n u s e d .

M. aureus
T h i s is

seen that R occal ap p aren tly pene­
is the i n d i c a t o r th a n w h e r e

m ore

a p p a r e n t tha n r e a l .

p l a n a t i o n of t h i s i s t h a t M . a u r e u s

is m o r e

sensitive

i n fe c ti n g a c t i o n of R o c c a l t h a n is Is. c o l i .
e v i d e n t t h a t on e x p o s u r e
m ore

In F i g u r e

I£.

The e x ­
to the

dis­

IB, it is

to p h e n o l, M. a u r e u s i s only s li g h t l y

s e n s i t i v e t h a n i s Is. c o l i .

These

results

are

in a c c o r d a n c e

w i th th e g e n e r a l e x p e c t a t i o n t h a t M. a u r e u s will d i s p l a y a g r e a t e r
s e n s i t i v i t y to the a c t i o n of m o s t d i s i n f e c t a n t s th an will E^. c o l i .
The

s h a p e s of t h e c u r v e s f o r e i t h e r d i s i n f e c t a n t a r e

sam e w hether M . aureus
indicates

The d i f f e r e n c e

case.

as

T hi s

i n d e p e n d e n t of the i n d i c a t o r o r g a n i s m .

in s e n s i t i v i t i e s

of the o r g a n i s m s to the d i s i n ­

r e s u l t in a d i f f e r e n t e n d p o in t b e in g
These

endpoints

M. a u r e u s

an in d ic a to r b e c a u s e

r e a c h e d in e a c h

r e f l e c t the e x t e n t of the c o n c e n t r a t i o n

g r a d i e n t of the d i s i n f e c t a n t in the
is k ille d .

indicators.

t h a t p e n e t r a t i o n i s p r o c e e d i n g in i d e n t i c a l f a s h i o n in

e a c h s e t of p e n e t u b e s ,

fectants

o r Is. c o li s e r v e

the

appears

range

to be

w here each o rganism

m ore

s u it a b le f o r use a s

of i t s low r e s i s t a n c e .

A

(A)

Roccal

(B)

Phenol

8

16
--------- ---

UJUJ

£

4

E
8

O

1.0

%

2.0

disinfectant

O

1.0

2.0

% disinfectant

Figure l-A,B.
The penetration, at pH5.0 and
27°C., of Roccal and Phenol in increasing
concentration with M. pyogenes (var. aureus) (— )o r
E.coli(
) as indicators in ag ar.

39
In a d d i t i o n a l e x p e r i m e n t s M y c o b a c t e r i u m p f l e i and B a ­
cillus
the

subtilis w ere used as indicator o r g a n is m s .

s t r i c t a e r o b i c n a t u r e of bo th o r g a n i s m s ,

B e c a u s e of

d e v e l o p m e n t was

a c c o m p l i s h e d in P e t r i d i s h e s c o n ta in in g the b r o t h r a t h e r th a n
in p l u g g e d t e s t t u b e s .
adequate

T h is t r e a t m e n t was n e c e s s a r y to p r o v i d e

a e r a t i o n a n d w a s found to be e x t r e m e l y i n c o n v e n ie n t

a s a r o u t in e p r o c e d u r e .

Under these

conditions, colum ns con­

t a i n in g 15. s u b t i l i s g ave i d e n t i c a l z o n e s of in h ib iti o n a s c o l u m n s
c o n ta i n in g M . a u r e u s f o l l o wing e x p o s u r e
p f le i did n o t g ive c o l u m n s
the

dem onstrating

to iodine s o lu ti o n s .
sharp

M.

d e m a r c a t i o n s of

z o n e s of i n h i b i t i o n , p r o b a b l y b e c a u s e of the f a i l u r e to o b ­

tain u n ifo rm
m ents w ere

s u s p e n s i o n s of th is o r g a n i s m .

No f u r t h e r e x p e r i ­

c o n d u c t e d w i th t h e s e o r g a n i s m s .

A ll s u b s e q u e n t e x p e r i m e n t s
w ith M . a u r e u s

made

a s th e i n d i c a t o r o r g a n i s m .

The e f f e c t of pH v a r i a t i o n .
c o n t a in i n g a g a r ,

r e p o r t e d b e lo w w e r e

a g a r plus

1.0 p e r c e n t h u m a n s e r u m

1.0 p e r
a lb u m in .

P enetubes were p re p a re d
c e n t g e l a t i n e and a g a r p l u s
Each

s e t a l s o c o n ta in e d

M / 1 0 p h o s p h a t e b u f f e r a t the pH value to w hich the tu b e s w e r e
to be e x p o s e d .

The

indicator o rg a n ism

was

M. a u r e u s .

40
Series

of e x p o s u r e

tu b e s

c o n ta in in g the d i s i n f e c t a n t s

the i n d i c a t e d f i n a l c o n c e n t r a t i o n s
p r e p a r e d a t pH v a l u e s c o v e r i n g

at

in M / 2 p h o s p h a t e b u f f e r w e r e
the r a n g e f r o m pH 5 to pH 9.

D u p li c a t e p e n e t u b e s w e r e p l a c e d in e a c h e x p o s u r e
c u b a t e d a t 27.0 C f o r 24 h o u r s .

A fter exposure

tube an d i n­
the t u b e s

were

r e m o v e d a n d r i n s e d a n d t h e n the a g a r c o l u m n s d e v e l o p e d i n the
appropriate

neutralizing broths.

i n h ib i ti o n w e r e
in F i g u r e s

The l e n g t h s of the z o n e s of

r e c o r d e d an d a v e r a g e d r e p r e s e n t a t i v e

data graphed

2 a n d 3.

Figure

2A i l l u s t r a t e s

the r a p i d l o s s

of i o d in e w i t h i n c r e a s i n g pH.

of p e n e t r a t i v e p o w e r

Th e d e p r e s s i n g

e f f e c t of th e p r o ­

t e i n a d d i t i v e s i s w e l l i l l u s t r a t e d in th e l o w e r l i n e s

of the g r a p h .

N o t i l l u s t r a t e d is the e x t e n t of v o l a t i l i z a t i o n of the iod ine a t the
d i f f e r e n t pH v a l u e s .

T h i s v o l a t i l i t y of iodine

of i n h i b it i o n b e i n g e v i d e n t a t the u p p e r e n d s
(see P la te
alone is
It is
io d i n e

1A).

At pH 5.0, the l e n g t h of th is

12.5 m m , d e c r e a s i n g

r a p i d l y to z e r o

results

in zo ne s

of the c o l u m n s
zone in a g a r
m m a t pH 9.0.

e v i d e n t t h e n , t h a t if it w e r e p o s s i b l e to p r e v e n t e s c a p e of
m olecules fro m

the

s o lu tio n to the g a s p h a s e ,

the e x t e n t

of p e n e t r a t i o n a t l o w e r pH v a l u e s would be a c c o r d i n g l y i n c r e a s e d .

A

(B) 1.0% Phenol

(A) .0 5 % lodine(Colloid)
20

20

01UJ

.10

-~o

cx

*o—
^

°

5

i

6

i

7
pH

—I■I

8

ra

9

0

5

6

7
pH

8

9

Figure 2 -A ,B .
The penetration of Iodine and
Phenol with respect to pH into agar(— ),
agar+ 1.0% gelatine (—
and agar + 1.0% human
serum albumen(— ).

41
F igure

2B r e p r e s e n t s

the e x t e n t of p e n e t r a t i o n of

p e r c e n t p h e n o l in to th e t h r e e

gel p h a s e s .

T he g r e a t e r

of d i f f u s i o n in a g a r a l o n e a t l o w e r pH v a l u e s
evident.

1.0
extent

(below pH 6.4) i s

T h e d i m i n i s h i n g p e n e t r a t i o n w ith i n c r e a s i n g pH in the

pro tein —
c o n t a i n i n g g e l s i s p a r t l y a r e f l e c t i o n of t h i s l o w e r dif­
fusion ra te

of p h e n o l a t

h i g h e r pH v a l u e s a n d p a r t l y to the i n ­

f l u e n c e of th e h y d r o g e n

ion c o n c e n t r a t i o n on the

the p r o t e i n w i t h p h e n o l .
f o r i o d in e

reactivity

C o m p a r i s o n of t h e s e f i g u r e s

of

w ith t h o s e

s h o w t h a t p h e n o l p e n e t r a t i o n is n o t as a d v e r s e l y a f ­

f e c t e d by i n c r e a s i n g pH

as

protein as

w ith io d in e .

inhibitory as

In F i g u r e

i s io d in e , n o r is the

3, t h e i n f l u e n c e

the q u a t e r n a r y a m m o n i u m

p r e s e n c e of

of pH on the p e n e t r a t i o n of

compounds

Hyam ine

In a l l c a s e s

1622,

C hloride

and R o c c a l is g ra p h e d .

of t h e s e

c o m p o u n d s i s g r e a t e r w ith i n c r e a s i n g pH.

Z ephiran

the p e n e t r a t i o n
The d e ­

p e n d e n c e of p e n e t r a t i o n upon pH d o e s n o t a p p e a r to be a s g r e a t
as

in t h e c a s e s of p h e n o l a nd of io d in e , n o r is the e x t e n t of

i n h ib i t i o n by p r o t e i n a s g r e a t .

T h e e f f e c t of t e m p e r a t u r e
ing a g a r

and a g a r p lu s

1.0 p e r

p r e p a r e d w i th M_. a u r e u s

as

variation.

cent human

P enetubes
serum

contain­

a l b u m in w e r e

the i n d i c a t o r o r g a n i s m .

The c o l u m n s

(A) .0 5 % Hyamine 1622

(B) . 0 5 % Zephiran Cl
10
8

U JU J

.6
E

E

-o—
,

4
2
0

pH

12

5

6

7
pH

8

9

(C) 0.1% Roccal

10

8
6

4
2
O

5

6

7
pH

8

9

Figure 3-A,B,C.
The penetration of quats with
respect to pH into a g a r ( — ), agar-*-1.0% gelatine
(— ), and agar+l.0% human serum albumen(— ).

42
w e r e b u f f e r e d a t pH 5.0 a nd pH 9.0 in the u s u a l m a n n e r and
ex p o sures w ere

m a d e to d i s i n f e c t a n t s

M /2 phosphate b u ffers.

Exposure

baths at t e m p e r a tu r e s f ro m

a t the

s a m e pH v a l u e s in

w a s f o r 24 h o u r s in w a t e r

2.5 C to 37.0 C.

n e u t r a l i z a t i o n of the c o l u m n s w e r e

D e v e l o p m e n t and

ac co m p lish ed as p rev io u sly

d e s c rib e d fo r each disinfectant.
F igure

4 represents

d i s i n f e c t a n t a t pH 5.0.

the e x t e n t of p e n e t r a t i o n of e a c h

F igure

4A i l l u s t r a t e s

the l i n e a r i n c r e a s e

in the p e n e t r a t i o n of 0.75 p e r c e n t p h e n o l w ith i n c r e a s i n g t e m ­
p erature.
shows

R o c c a l a t 0.1 p e r

cent con centratio n (Figure

4B)

a s i m i l a r l i n e a r i n c r e a s e of p e n e t r a t i o n w ith i n c r e a s i n g

tem p eratu re.
In F i g u r e
m ide
per

4C a n d D the l i n e a r

c o n t a i n i n g 0 . 02 5 p e r

r e s p o n s e s of S u c c i n c h l o r i -

cent available

c e n t I o d i n e S u s p e n s o i d (M e rc k )

chlorine

a r e plotted.

i n h ib i t i o n due to the v o l a t i l i t y of the h a lo g e n s
th e

"upper

these

latter

zo nes."
zones

The l e s s e r

A linear in crease

a nd of 0.05
The z o n e s of

are

in d icated as

in the p e n e t r a t i o n in

i s a l s o no ted.
slopes

of the l i n e s

representing

t i o n of e a c h d i s i n f e c t a n t in the p r e s e n c e
c a te th a t with i n c r e a s i n g t e m p e r a t u r e

the

the p e n e t r a ­

of the a l b u m i n in d i ­
r e a c t i o n of the

(A)

.7 5 %

Phenol

(B)

0.1% Roccal

mm

20

-O

E

E

20
30
temp. °C.

20
30
temp. °C.

40

40

(D) .05% l2(Suspensoid)

(C) Succinchlorim ide(025% av CL)
20

20

iio

upper
zone

EIO

upper
vzone
—
o------e_o_
0

10

20

temp. °C.

30

o --------

-o

40

20
30
temp °C.

40

Figure 4-A ,B ,C ,D .
The effect of temperature
on the diffusion of various disinfectants at
pH5.0 into
agar(—) and agar+l.0% human
serum albumen. (— )

43
d i s i n f e c t a n t w i t h t h e p r o t e i n i s i n c r e a s e d , o t h e r w i s e the
of th e l i n e s w o u l d b e i d e n t i c a l to t h o s e

r e p r e s e n t i n g the d i ffu­

s i o n in a g a r a l o n e , a l t h o u g h in s u c h c i r c u m s t a n c e s
of th e l i n e s

slopes

the p o s i t i o n

w o u l d s t i l l be a t a l o w e r p o s i t i o n in the g r a p h s .

W h e r e th e l i n e s
protein are

r e p r e s e n t i n g the p e n e t r a t i o n in a g a r and in

alm ost p arallel,

a s w ith S u c c i n c h l o r i m i d e ,

a hi gh

a v i d it y of t he d i s i n f e c t a n t f o r the p r o t e i n i s i n d i c a t e d .

The

t o t a l r e a c t i o n of th e d i s i n f e c t a n t with the p r o t e i n m u s t be n e a r l y
com plete

a t the l o w e r t e m p e r a t u r e

perature

do n o t h a v e a m a r k e d in f lu e n c e on the

A t p H 9.0 the n a t u r e

and th u s i n c r e a s e s

of the r e s p o n s e

in t e m ­

reaction

rate.

of p e n e t r a t i o n of

the d i s i n f e c t a n t s to t e m p e r a t u r e

v a r i a t i o n d i f f e r s f r o m the l i n e a r

responses

s e e n in F i g u r e

a t t h e l o w e r pH.

disinfectants

As

te s te d gives a lin e a r i n c r e a s e

r e s p e c t to i n c r e a s i n g t e m p e r a t u r e .
cal,
sam e

Z e p h ira n C hloride and H yam ine
data yield s tra ig h t lin es

trations

in p e n e t r a t i o n w i th

The c u r v e s f o r p h e n o l ,
1622 a r e

logarithm ic.

when the l o g a r i t h m s

a r e p l o t t e d a g a i n s t the t e m p e r a t u r e .

ing l o g a r i t h m i c p l o t s h a v e the

5, none of the

sam e

slope.

RocThese

of the p e n e ­

All t h e s e

resu lt­

No s a t i s f a c t o r y

ex­

p l a n a t i o n f o r t h i s b e h a v i o r h a s b e e n p r o p o u n d e d by the a u t h o r .
I odine S u s p e n s o i d u n d e r t h e s e

sam e

c o n d it io n s g i v e s c u r v e s

as

(A) .7 5 %

Phenol

(B) . 0 5 %

Roccal

o-

or
20
30
temp. °C.

15

(C) .0 5 %
.0 5 %

20
30
temp. °C.

40

Z ephiran Cl2
Hyamine 1622

40

(0) .05% l2(Suspensoid)

10
E

E

-o

5
-o '
0

10

20
30
temp. °C.

40

20
30
temp. °C.

40

Figure 5 - A,B,C,D. The effect of temperature on
the diffusion of various disinfectants at pH9.0
into a g a r ( — ) and ag ar+ l.0 % human serum
album en.(— ).

44
in F i g u r e

5D.

The p e n e t r a t i o n

approxim ately
plain,

15 C.

a m a x i m u m v a lu e

T h is condition is lik ew ise

i n a s m u c h a s no

pH v a l u e

reaches

"upper

a n d s o th e d e c r e a s e

c a n n o t b e due to a l o s s

at

d if f i c u l t to e x ­

z o n e s ' 1 of i n h ib i ti o n o c c u r

at this

in p e n e t r a t i o n a t high t e m p e r a t u r e s

of i o d in e t h r o u g h v o l a t i l i z a t i o n .

T h e e f f e c t of v a r i a t i o n of d i s i n f e c t a n t c o n c e n t r a t i o n .
etubes w ere p r e p a r e d
ex perim ents.
hours

i n the

m a n n e r as

D uplicate tubes w ere

to d i s i n f e c t a n t s

b u ffe r a t the

in v a ry in g

experim ents.

in the p r e c e d i n g

e x p o s e d a t 27.0 C f o r

concentrations

s e l e c t e d pH v a l u e s .

m e n t of the c o l u m n s w e r e
previous

sam e

Pen-

24

in M / 2 p h o s p h a t e

N e u t r a l i z a t i o n a nd d e v e l o p ­

accom plished as

i n d i c a t e d in the

T he p r o t o c o l of e a c h e x p e r i m e n t i s d e ­

s c r i b e d below.
T h e p e n e t r a t i o n of io d in e p r e p a r a t i o n s
containing p r o te in s

was

e t r a t i o n of L u g o l ' s

S o l u t i o n a n d Iod ine T i n c t u r e

concentrations
cent human
pH 5.0.
m anner
tions

exam ined.

into a g a r an d a g a r -

i n to a g a r p l u s

serum

album in.

1.0 p e r
These

I o d i n e S u s p e n s o i d ( M e rc k )
an d the c u r v e s

w ere

sim ilar

Shown in F i g u r e

at increasing

cent gelatine and

exposures

w ere

of F i g u r e

6.

1.0 p e r

made

w a s e x a m i n e d in the

for p e n e tra tio n at in c re a s in g

to t h o s e

6 is the p e n ­

at
sam e

concentra­

Typical c u rv e s for

A

(A)

Lugols"

(B) Tincture

16

12
JO

UJUJ

E8

8

4

0

1.0
% I

2.0

2.0
% \2

Figure 6-A,B. The penetration
of Iodine
preparations, with respect to increasing concen­
tration, into agar + 1.0% gelatine (— ) and agar+
1.0% human serum albumen (— -)

45
the C o l lo i d a l I o din e m a y b e
e n t i r e f a m i l y of c u r v e s

s e e n in F i g u r e

12A, B and C.

This

a p p e a r to be c a s e s of H i l l ' s e q ua tion and

will b e d i s c u s s e d f r o m t h i s

s ta n d p o in t in a l a t e r

p e n e t r a t i n g a b i l i t y of the t h r e e

s e c t io n .

iodine p r e p a r a t i o n s ,

The

in d e sc e n d i n g

o r d e r , i s L u g o l ' s So lu tio n, T i n c t u r e of Iodine and Colloidal Io­
dine.

T h is o r d e r i s i n a g r e e m e n t w ith the

r e s u l t s o b ta in e d by

A n d e r s o n an d M a l l m a n n (14) u sin g the p a r a f f i n - c o v e r e d a g a r
p l a te m e t h o d .
In a n o t h e r

s e t of e x p e r i m e n t s , f o u r p r e p a r a t i o n s

l o i d a l Iodine of v a r y i n g p a r t i c l e

s iz e w e r e p r e p a r e d in a c c o r d a n c e

with the d i r e c t i o n s of M i l l e r (44).
w e r e of i n c r e a s i n g p a r t i c l e

size,

r e d (I) t h r o u g h i n c r e a s i n g d a r k
(IV).

These

suspensoids w ere

centrations as p re p a ra tio n s

These

suspensoids

(I -

r a n g in g in c o l o r f r o m

IV)

a brick

s h a d e s of b ro w n (II, III) to b l a c k
d ilu te d to the

same

iodine c o n ­

of the M e r c k S u s p e n s o i d and all

a d j u s t e d to pH 5.0 w i th M / 2 p h o s p h a te b u f f e r .
t a i n in g a g a r a n d a g a r p l u s

of C o l ­

Penetubes con­

1.0 p e r c e n t g e la tin e w e r e

exposed

in t h e s e h y d r o s o l s a n d a f t e r d e v e lo p m e n t in the u s u a l m a n n e r
the z o n e s of i n h i b i t i o n r e c o r d e d .
Tab le 2.

It c a n be

These

results

are

shown in

s e e n t h a t the zones of inhibition p r o d u c e d

by a ll the p r e p a r a t i o n s

are

n e a r l y i d e n ti c a l a t id e n ti c a l

TABLE

2

THE E F F E C T O F P A R T I C L E SIZE ON THE P E N E T R A T IO N
O F C O LLOIDAL IODINE INTO AGAR P L U S 1.0 P E R CEN T
G E L A T I N E AT pH 5.0 AND 27.0 C FO R 24 HOURS
m m P e n e t r a t i o n with
Preparation
0.29% I

0.0 3% I
Cm

M e rc k Suspensoid

15.1
15.1

5.2
5.2

Suspensoid I

15.2
15.2

5.2
5.2

S u s p e n s o i d II

15.8
15.7

5.3
5.2

S u s p e n s o i d III

14.8
1 5.2

5.3
5.1

S u s p e n s o i d IV

15.6
15.6

5.1
5.2

15.3

5.2

Average

46
concentrations.

Th e p a r t i c l e

size

of the

s u s p e n s o i d s d o e s not

a p p e a r to be c r i t i c a l i n the p e n e t r a t i o n of the iodine d e r i v e d
fro m these p a r tic le s .
C h l o r i n e c o m p o u n d s w e r e i n v e s t i g a t e d in the
a s the io d ine p r e p a r a t i o n s ,
Figure

7.

The

shape

w ith the

of the c u r v e s

brought about by i n c r e a s i n g
a r e the

sam e

o b ta in e d p l o t t e d in

r e p r e s e n t i n g the p e n e t r a t i o n

concentrations

of a v a i l a b l e c h l o r i n e

a s t h o s e fou nd f o r the iodine p r e p a r a t i o n s .

c o m p a r i s o n of F i g u r e s
sodium h y p o c h lo rite
respectively,

results

sam e m anner

7A and B,

A

r e p r e s e n t i n g the d a ta fo r

(Rom an C le a n se r)

a t pH 5.0 and pH 9.0

s h o w s the m a r k e d d e p e n d e n c e of t h i s c o m p o u n d

upon h y d r o g e n ion c o n c e n t r a t i o n .

It is a l s o a p p a r e n t f r o m

c u r v e s th a t the p e n e t r a t i o n of t h e s e
m a r k e d l y l i m i t e d by the p r e s e n c e

the

c h l o r i n e c o m p o u n d s is m o s t

of p r o t e i n s

c o m p o u n d s in g e n e r a l do n o t p e n e t r a t e

and th a t c h lo r i n e

the t e s t s y s t e m s to the

sam e ex ten t a s iodine.
P h e n o l p e n e t r a t i o n is

shown in F i g u r e

8.

It c a n be s e e n

t h a t the p e n e t r a t i o n of p h e n o l is n ot a f f e c te d to the

same

by the h y d r o g e n ion c o n c e n t r a t i o n n o r by the p r e s e n c e
teins a s a r e
of the

the h a l o g e n s .

sam e nature

The c u r v e s

degree

of p r o ­

o b ta in e d with phe no l a r e

a s t h o s e o b ta in e d with the h a lo g e n s alth ough

(A)

NaOCI

pH5.0

(B) NaOCI

25

25

E 15

E 15

pH9 0

— o

o

.05
.10
% available Cl2

.10
.05
% available Cl2

(D)Succinchlorimide pH5.0

(C) Chloramine T pH5.0
30

30

E 15

o

%

.10
.05
available Cl2

.10
.05
% available Cl2

Figure 7-A ,B ,G ,D . The penetration of chlorine
compounds, with respect to increasing concentration, into agar(— ) and agarH-l.0% human
serum albumen. (— -).

(A)

pH50

(B)

pH 9 .0

20

20

-or
UUUJ

EI0

2.5
%

0.5

phenol

25

(C)

2.5
% phenol

pH5.0

%

1.0

phenol

20

Figure 8 - A,B,C.
The penetration of phenol,
with respect to increasing concentration, into
agar(— ), a g a r+-1.0% gelatine (— ), and agar+l.0%
human serum albumen. (— ).

47
the i n it ial slope is l o w e r than in the c u r v e s

r e l a t i n g the p e n e ­

t r a t i o n of the h a l o g e n s to the c o n c e n t r a t i o n t h e r e o f .
The p e n e t r a t i o n of R o c c a l at i n c r e a s i n g c o n c e n t r a ti o n s
into a g a r and a g a r p l u s
shown in F i g u r e 9.

1.0 p e r c e n t h u m an s e r u m albu m in is

The p e n e t r a t i o n of this com pound at pH

9.0 is se e n to be g r e a t e r than a t pH 5.0.

The p r e s e n c e

of the

p r o t e i n d o es n o t a p p e a r to inhibit the p e n e t r a t i o n to as g r e a t a
d e g r e e as wi th the h a l o g e n s .
M e r t h i o l a t e wa s e x a m in e d as an aq u eo u s solution and
a s a t i n c t u r e c o n t a i n i n g 35 p e r ce nt eth an o l.

The n e u t r a l i z a ­

tion an d d e v e l o p m e n t of c o lu m n s e x p o s e d to this com pound w e re
a c c o m p l i s h e d in f r e s h l y p r e p a r e d F l u i d T hio gly co llate Medium
(Difco)i

In F i g u r e

experim ents
tincture.

10A and B the r e s u l t s a r e p lo tte d f o r two

r u n c o n c u r r e n t l y with the aqueou s

solution and the

The c u r v e s o b t a ine d with the t i n c t u r e have not been

c o r r e c t e d f o r the e f f e c t of ethanol alone, w hich can be s e e n to
a c co u n t f o r a p p r o x i m a t e l y 5.0 m m of the p e n e t r a t i o n .
p e n e t r a t i o n of the t i n c t u r e

The

is not as g r e a t l y r e d u c e d by the

p r e s e n c e of g e l a t i n e a s the aqueous

solution.

m en t s of t h is type gave highly v a r i a b l e

Repeat ex p eri­

results.

a n o th e r e x p e r i m e n t with a queous M e r th io l a te

In F i g u r e

is r e c o r d e d .

10C,
It

(A)

pH 5.0

(B) pH9.0

'UJUi

E
E

2.0

1.0

%

Roccal

1.0

2.0

% Roccal

Figure 9-A,B. The penetration of Roccal, with
respect to increasing concentration, into agar
(— ) and agar + 1.0% human serum albumen(——).

(A)

Aqueous

(B)

20

20

§10

EIO

.05
merthiolate

Tincture

.02

%

25

.05
merthiolate

.02

%

(C)

Aqueous

o

0.2

% merthiolate

Figure 1 0 - A,B,C.
The penetration of mer­
thiolate at pH5.0, with respect to increasing
concentration, into agar(— ), a g a r+ 1.0% gelatine,
(— ), and agar + 1.0% human serum albumen(--).

48
can be s e e n t h a t the e x t e n t of p e n e t r a t i o n of the M e r t h i o l a t e in
th is e x p e r i m e n t i s f a r g r e a t e r th a n in the e x p e r i m e n t g r a p h e d
in F i g u r e

10A.

One w o n d e r s w h e t h e r n e u t r a l i z a t i o n of the e x ­

c e s s M e r t h i o l a t e h a s b e e n s u c c e s s f u l in a ll t r i a l s .

The v a r i a t i o n of s t a t i o n a r y p h a s e c o m p o n e n t s .

Penetubes

w e r e p r e p a r e d c o n t a i n i n g g e l a ti n e o r h u m a n s e r u m a lb u m in at
c o n c e n t r a t i o n s f r o m z e r o to 1.0 p e r c e n t.
um n s w as

The pH of th e s e c o l ­

s e t a t pH 5.0 with M /1 0 p h o s p h a te b u f f e r .

t u b es w e r e

e x p o s e d to 0.1 p e r c e n t R o c c a l,

The p e n e ­

1.0 p e r c e n t phenol

and 0.05 p e r c e n t Iodine S u s p e n s o id a t pH 5.0 and 27.0 C f o r
24 h o u r s .

N e u t r a l i z a t i o n and d e v e l o p m e n t w e r e a c c o m p l i s h e d

a s b e f o r e a n d the r e s u l t i n g
r e s u l t s a p p e a r in F i g u r e
t e s t e d it c a n be

z o n e s of inhib ition r e c o r d e d .

11.

F o r e a c h of the d i s i n f e c t a n t s

s e e n t h a t the a lb u m in is m o r e

p e n e t r a t i o n of the d i s i n f e c t a n t than g e l a ti n e .
f e a t u r e of the c u r v e s
above

These

in h ib ito ry to the

A n o th e r s a li e n t

is t h a t they le v e l off, d e m o n s t r a t i n g tha t

so m e c o n c e n t r a t i o n of the p r o t e i n the r a t e of p e n e t r a t i o n

of the d i s i n f e c t a n t s is n e a r l y in d e p e n d e n t of the p r o t e i n c o n c e n ­
tration.

T h i s o b s e r v a t i o n m a k e s lo g ic a l the use of

protein concentrations
circum stances,

in the o t h e r e x p e r i m e n t s .

m inor e r r o r s

1.0 p e r c e nt

U n d e r th e s e

in the addition of the p r o t e i n will

(A)

0.1% Roccal

(B)

1.0% Phenol

161

UiUJ

--------- -o

£8-

o - ----

4 -

%

0.5
1.0
protein

% protein

(C) .05% I.(colloid)
20

E 10

—o

1.0
0.5
% protein

Figure II - A, B,C.
The penetration of Roccal,
Phenol, and Iodine at pH5.0 and 27°C. into
an agar stationary phase containing increasing
concentrations of gelatine (— ) or human serum
albumen (— ).

49
not th en c a u s e l a r g e

errors

in the r e s u l t i n g o b s e r v a t i o n s .

Once

again , i t is n o t e d t h a t p e n e t r a t i o n of R o c c a l and phenol is l e s s
a d v e r s e l y a f f e c t e d t h a n iodine by the p r e s e n c e of p r o t e i n .
The r e s u l t s p l o t t e d in F i g u r e

12A and B d e m o n s t r a t e

the ef f e c t of a dding 0.5 p e r c e n t p o t a s s i u m iodide o r sodium
c h lo r i d e to the s t a t i o n a r y p h a s e on the p e n e t r a t i o n of iodine
into su c h g el s y s t e m s .

The p e n e t r a t i o n of C olloidal Iodine when

s o d iu m c h l o r i d e i s c o n t a i n e d in the gel p h a s e is id e n ti c a l to the
p e n e t r a t i o n when no s a l t is added.

The p r e s e n c e of p o t a s s i u m

iodide in the g el p h a s e e n h a n c e s the p e n e t r a t i o n of the co lum ns
by io dine.

T h i s e x p e r i m e n t was co n d u cted at pH 5.0 and 27.0

C f o r 24 h o u r s .
In a s u b s e q u e n t e x p e r i m e n t an u n b u ff e re d solution of
iodine in d i s t i l l e d w a t e r containing 0.028 p e r ce nt iodine was
all ow ed to p e n e t r a t e
dition of e i t h e r
to the

a g a r gel c o lu m n s and c o lu m n s with the a d ­

1.0 p e r c e nt p o t a s s i u m iodide o r

stationary phase.

A du plicate

g el at in e wa s a l s o e x p o s e d .

sodium c h l o ri d e

s e t containing

The e x p o s u r e

1.0 p e r c e n t

was m a d e at 27.0 C

f o r 24 h o u r s a nd the r e s u l t s a r e ta b u la te d in Table 3.
results

are

in a g r e e m e n t with tho se p r e v i o u s l y c ited .

These
It is

a p p a r e n t t h a t the a d d i ti o n of p o t a s s i u m iodide has o c c a s io n e d

(A).

Agar

(B) Agar+10% gelatine

30
20

NaCI
20

NaCI
10

0

.01

.02

.03

.04

% I*

% l2

(C) Agar

30

(D) Agar,

I32%KI
20

,cr

.05

pH 5.0

—o

<r

.05% I.

20

.10
% I

.10

%KI

.20

Figure I2-A,B,G,D.
The effect of salts on the
penetration of Colloidal Iodine. Explanations
in text.

TABLE

3

THE P E N E T R A T I O N O F 0.028 P E R CE N T IODINE IN
AQUEOUS SOL UT ION INTO AGAR COLUMNS WHERE
THE G E L SYST EM CONTAINS ADDITIVES AS
INDICATED.
M. AUREUS INDICATOR, 24
HOUR E X PO SU R E AT 27.0 C
m m P e n e t r a t i o n in
S a l t Added
Agar

A g a r + 1% G e la tin e

None

33.1

22.0

1% KI

52.1

34.0

1% NaCI

34.2

22.1

50
a 55 p e r c e n t i n c r e a s e
by the i o d in e .

The

in the p e n e t r a t i o n of e i t h e r gel s y s t e m

s o d i u m c h l o r i d e a d d iti o n g iv e s no i n c r e a s e

in p e n e t r a t i o n of i o d in e o v e r th a t o b t a in e d in the c o n t r o l c o l ­
u m n s , h e n c e the e f f e c t of the p o t a s s i u m iodide m u s t be
a nd n o t due to the e f f e c t of s a l t c o n c e n t r a t i o n .
a l so

s e e n to be the

c o n tain i n g
crease

s a m e f o r the a g a r

sp e c i fi c

T h is e ff e c t is

s y s t e m an d the g e l a t i n e -

s y s t e m a n d t h u s it is l o g i c a l to s u s p e c t th a t the in­

in p e n e t r a b i l i t y wh en p o t a s s i u m

to the f o r m a t i o n of t r i i o d i d e

iodide

is p r e s e n t is due

ion.

T h i s p o s s i b i l i t y w a s f u r t h e r e x p l o r e d by adding 0.132
p e r c e n t p o t a s s i u m io d id e to a s e r i e s
t a in i n g i n c r e a s i n g
The e x p o s u r e
27.0 C f o r

c o n c e n t r a t i o n s of C o llo id a l Iodine a t pH 5.0.

of a g a r c o l u m n s in t h e s e tu b e s w as c o n d u c te d a t

24 h o u r s .

The p e n e t r a t i o n of the c o lu m n s at e a c h

c o n c e n t r a t i o n i s p l o t t e d in F i g u r e
of e x p o s u r e
tassium

of e x p o s u r e t u b e s c o n ­

of r e p l i c a t e p e n e t u b e s

io d i d e .

to c o n t r o l s

co n taining no po­

It i s a p p a r e n t t h a t the p e n e t r a t i o n of the a g a r

gel is g r e a t e r w h e r e p o t a s s i u m
qu en tl y t r i i o d i d e )

12C t o g e t h e r with the r e s u l t s

iodide is p r e s e n t (and c o n s e ­

t h a n w h e r e f r e e iodine a lone is p r e s e n t .

T h i s p o i n t i s f u r t h e r d o c u m e n t e d in F i g u r e
r e s u l t s p l o t t e d in t h i s f i g u r e

12D.

The

show the p e n e t r a t i o n of 0.05 p e r

51
c e nt C o ll oi d a l Iodine a t pH 5.0 and 9.0 when i n c r e a s i n g am ou nt s
of p o t a s s i u m io dide have b e e n added to the d is in f e c t a n t.

The

l i n e a r i n c r e a s e with i n c r e a s i n g c o n c e n t r a t i o n s of the s a l t d e m ­
o n s t r a t e s the i n c r e a s i n g p e n e t r a t i o n of the a g a r gel by i n c r e a s ­
ing c o n c e n t r a t i o n s of t r i i o d id e ions.

It should be noted that

the ad d i tio n of the p o t a s s i u m iodide b r e a k s the h y d r o s o l and
th a t at pH 9.0 the h y d r o s o l is not stab le at a c o n c e n t r a ti o n of
0.05 p e r c e n t .
In o t h e r e x p e r i m e n t s ,
phase.

c a s e i n was ad de d to the s t a t i o n a r y

The i n s o l u b i l i t y of this p r o t e i n a t pH 5.0 p r e v e n t e d its

add itio n as a s olu tio n to the m e l t e d gel.

When c a s e i n was s u s ­

p en d ed in the a g a r gel a nd the m i x t u r e a u to c la v e d , a d a r k brown
gel e v e n tu a l l y wa s f o r m e d in which the zon es of inhibition could
not r e a d i l y be d i s c e r n e d .
In one e x p e r i m e n t a m m o n iu m sulfate was added to the
stationary phase.

The p e n e t r a t i o n of phenol and R o c c a l was not

a f f ected by t h i s a d d i ti o n .

The p e n e t r a t i o n of Colloidal Iodine

was a f f e c t e d to a g r e a t d e g r e e ,

an a g a r containing 0.5 p e r c e nt

a m m o n iu m s u l f a t e was p e n e t r a t e d 45.5 m m and a 1.0 p e r c e nt
c o n c e n t r a t i o n of t h is c o m pound r e s u l t e d in 54.8 m m p e n e t r a t i o n .
In a g a r alone the p e n e t r a t i o n of the co lum ns a m ou nte d to only

52
17.0

m m with the s a m e d i s i n f e c ta n t e x p o s u r e .

The a m m o n i u m

su lf ate in n e i t h e r c o n c e n t r a t i o n inh ibite d the gr o w th of the in­
h i b it o r o r g a n i s m a s d e m o n s t r a t e d by e x p o s u r e to a b u f f e r s o l u ­
tion.

These re s u lts

p l a in e d .

are

i n t e r e s t i n g but a r e not r e a d i l y ex ­

Ad dition of 1.0 p e r c e n t u r e a to the

s e e m e d l i k e w i s e to f a c i l i t a t e

station ary ph a s e

the p e n e t r a t i o n of this p h a s e by

iodine but u r e a wa s i n h i b ito r y to the i n d i c a t o r o r g a n i s m
the

exact

z o n e s of i nh ibition p r o v e d difficult to m e a s u r e .

The r a t e
p lu s

of d i f f u s io n .

Penetubes

containing a g a r and

1.0 p e r c e n t g e l a t i n e w e r e p r e p a r e d at pH 5.0.

et ub es w e r e e x p o s e d in l a r g e
f o i l - c o v e r e d No. 7 r u b b e r
iodine

and so

tubes

agar

T h e se p e n ­

s t o p p e r e d with a lu m in u m

s t o p p e r s containing a 0.028 p e r c e n t

solu t i o n a t pH 5.0.

The iodine

Iodine S u s p e n s o i d ( M e r c k ).

so lutio n was p r e p a r e d f r o m

At i n t e r v a l s up to 27 h o u r s dupli ca te

p a i r s of p e n e t u b e s w e r e r e m o v e d and t r e a t e d in the p r e s c r i b e d
m anner.

The r e s u l t i n g

zon es of inhibition following e a c h e x ­

p o s u r e p e r i o d a r e p l o t t e d in F i g u r e

13.

A f t e r an in itial r i s e ,

the p e n e t r a t i o n w a s l i n e a r with r e s p e c t to t i m e .

The a s s u m p t i o n

th at the p e n e t r a t i o n in 24 h o u r s was at a c o n s t a n t r a t e is not
far

f r o m the a c t u a l c a s e

the

r a t e of p e n e t r a t i o n .

and m a y be u s e d as an e x p r e s s i o n
T h e se

results

are considerably

of
d i ff e re n t

20

Agar
OJUU
O'

time (hr)

20

Figure 13.
The rate of diffusion of .028%
Iodine at pH50 and
27.0°C into agar and
agar +1.0% gelatine.

53
f r o m the r a t e of penetration, r e p o r t e d by A n d e r s o n and Mallma nn
(14) f o r 5 p e r c e n t Colloidal Iodine, but the differen ce in h y d r o ­
gen ion c o n c e n t r a t i o n in th e se t r i a l s does not p e r m i t a d i r e c t
com parison.

E x p e r i m e n t s on the D isinfection of
Chicken F e c e s

The v a r i a t i o n of d is in fe c ta n t c o n c e n t r a t i o n .

Iodine p r e p a ­

r a t io n s w e r e a d j u s t e d to the d e s i r e d pH value in M /2 phos phate
bu ffer.

The iodine co n te nt of e a c h p r e p a r a t i o n was d e t e r m i n e d

j u s t p r i o r to use by t h io sulfate tit r a t io n .

The ex p o su re p er i o d

of the f e c a l s u s p e n s i o n to the action of the d isin fec tan t was 5.0
m i n u t es, a t which t i m e the

1.0 ml s a m p le s w ere

re m o v e d to

9.0 m l s t e r i l e 0.05 N s o dium thiosulfate dilution blanks for
n e u t r a l i z a t i o n and s u b s e qu en t o p e r a t i o n s .

Initial counts of b a c ­

t e r i a in the f e c a l s u s p e n s io n w ere obtained f r o m co ntro l t r e a t ­
m en t s in the p h osp ha te b uf fe r.

All counts a r e the a v e ra g e of

the n u m b e r s o b t a i n e d f r o m duplicate plate counts.
of the n u m b e r s of o r g a n i s m s

The lo g a r i t h m s

surviving a f t e r 5.0 m inutes expos ure

a r e p l o tt e d at e a c h c o n c e n tr a tio n of iodine te s te d .
In F i g u r e

14A, r e s u l t s obtained with Iodine Suspensoid

(Merck) a r e plotted; in F ig u r e

14B, the r e s u l t s obtained with

(A) I, (Colloid)
Log [^Survivors)

(B) LugoPs

Solution

tn 4
o 2

0.2

0.2

0.1

% l2

%l

(C) Tincture

of

I

8
<o

6
4

o»

2
0

0.1

% I.

Figure I4-A,B,G.
The
population of chicken
pH 5 0 , 27.5° C.

0.2

reduction of bacterial
feces in 5.0 min. at

54
Lugol^s Solution ( U.S.P. XIII) a r e plo tted and in F i g u r e

14C, the

d a ta o b t a i n e d with Iodine T in c tu r e (U.S.P. XIII) a r e plotted.
dilu tio ns w e r e m a d e with d i s t i l l e d w a t e r ,
t en t of the t i n c t u r e

All

so that the alco hol c o n ­

was p r a c t i c a l l y nil and c e r t a i n l y not in the

r an g e of c o n c e n t r a t i o n of alcohol which is g e r m i c i d a l .
It c a n be s e e n f r o m the g r a p h s that the c u r v e s of b a c ­
t e r i a l r e d u c t i o n with r e s p e c t to i n c r e a s i n g
a r e p r a c t i c a l l y i d e n t i c a l,

iodine c o n c e n t r a t i o n

so that, un d e r th e se conditions, the

acti v it y of iodine i s the s a m e in a ll t h r e e p r e p a r a t i o n s te s t e d .
The c u r v e s a r e a l l s li ghtly sig m o id and it is of p o s s ib le
nif icanc e t h a t the c o n c e n t r a t io n a t which the c u r v e s

sig­

s t a r t to

b ec o m e a s y m p t o t i c to the a b s c i s s a is a t a c o n c e n t r a ti o n of 0.05
p e r cen t iodin e.
To d e t e r m i n e f u r t h e r the p r o p e r t i e s of the Colloidal
Iodine, a n u m b e r of p r e p a r a t i o n s of the h y d r o s o l w e r e made
in a c c o r d a n c e with the d i r e c t i o n s of M ill e r (44).
tion of f i n e s t p a r t i c l e
r e d in a p p e a r a n c e

size ( " f i n e 11 s u s p e n s o id )

suspensoid)

a light b r i c k

was p r e p a r e d f o r final use by centrifuging

a h y d r o s o l of s m a l l p a r t i c l e
particles.

The p r e p a r a ­

size and d is c a r d i n g the l a r g e s t

The p r e p a r a t i o n of l a r g e s t p a r t i c l e
a p p e a r i n g a l m o s t b la c k

size ( " c o a r s e 11

was p r e p a r e d by c olle cting

55
the s e d i m e n t f r o m the c e n t r i f u g a t i o n of the c o a r s e s t h y d ro s o l .
Both p r e p a r a t i o n s w e r e c o m p o s e d of p a r t i c l e s a v e r a g in g 0.7 u
in d i a m e t e r ,
"fine"

r a n g i n g in d i a m e t e r f r o m 0.2 u to 0.9 u.

suspensoid these p a r tic le s

in the s a t u r a t e d

were

In the

su sp e n d e d individu ally

s o l u t i o n of iodine, while the " c o a r s e ”

so id w a s c o m p o s e d of c l u m p s of the p a r t i c l e s .
so id s w e r e u s e d in e x p e r i m e n t s in the

suspen­

These

suspen-

s a m e m a n n e r as the

p r e v i o u s l y m e n t i o n e d iodine p r e p a r a t i o n s with the r e s u l t s a s
i l l u s t r a t e d in the c u r v e s of F i g u r e
are

15.

The r e s u l t i n g c u r v e s

s u p e r i m p o s a b l e on e a c h o t h e r and v e r y s i m i l a r to the c u r v e s

of F i g u r e

14.

T h e y d i f f e r in shape f r o m the c u r v e s in the l a t t e r

f ig u r e by n o t d e m o n s t r a t i n g i n c r e a s i n g s lo p e s as c o n c e n t r a t i o n is
i n c r e a s e d a t e x t r e m e l y low d ilu tio n s .

T his d if f e r e n c e is p r o b ­

ab ly due o nly to the l e s s e r n u m b e r of points e x a m in e d in this
r e g io n t ha n w e r e e x a m i n e d in the e a r l i e r e x p e r i m e n t s .

Again,

the p o i n t of d i m i n i s h i n g r e t u r n s is r e a c h e d a t a c o n c e n t r a t i o n
of 0.0 5 p e r c e n t iodine c o n c e n t r a t i o n .
the

so l u tio n o r

A p p a r e n tl y the f o r m of

s u s p e n s i o n of iodine is not c r i t i c a l f o r the d i s ­

i n f ec t io n of c h i c k e n f e c e s u n d e r t h e s e co n d ition s.
C h l o r i n e c o m p o u n d s w e r e e x a m in e d u n d e r the
d it io n s a s w e r e the iodine p r e p a r a t i o n s .

sa m e c on­

The c o n c e n t ra ti o n of

(B) "Coarse"

Log t#Survivors)

(A) “Fine"

§4

</>
JL,
o»

.05

.05
% \2

% l2

Figure 15-A, B.
The reduction of the bacterial
population of chicken feces in 5.0 min. at
pH5 0 , 2 5 ° C., by: A."Fine" l2 suspensoid, B."Coarse1
\2
suspensoid.

d

e a c h c o m p o u n d w a s e x p r e s s e d a s the p e r c e n t a v a i l a b l e
a s d e t e r m i n e d b y s o d i u m t h i o s u l f a t e t i t r a t i o n (45).
an d B, the d a t a f o r s o d i u m h y p o c h lo r it e
Chloram ine

T (Wyandotte)

are

The s o d i u m h y p o c h l o r i t e a p p e a r s

In F i g u r e

(R o m an C l e a n s e r )

respectively

s i m i l a r to e a c h o t h e r a nd r e s e m b l e

chlorine

graphed.

and

Both a r e

t y p i c a l 11d i e - a w a y "

to be

16A

s li g h tly m o r e

curves.

eff e c t i ve

in r e d u c i n g the b a c t e r i a l p o p u la t io n of the c h i c k e n f e c e s than
does

Chloram ine

T.

Succinchlorim ide

i s p r e s e n t e d in F i g u r e

16C.

This

(N atio n a l Dyes)

activity

c o m p o u n d d i s p l a y s an a c t i v i t y

d e c i d e d l y d i f f e r e n t f r o m t h a t of the o t h e r two c o m p o u n d s
class

w i t h r e s p e c t to c o n c e n t r a t i o n .

The

slope of the b a c t e r i a l

r e d u c t i o n a t lo w c o n c e n t r a t i o n s of s u c c i n c h l o r i m i d e
s t e e p e r t h a n t h o s e of the o t h e r
c h a n g e s to a f a r l o w e r
per

chlorine

c o m p o u n d s , bu t r a p i d l y

Succinchlorim ide

e f f e c t iv e t h a n the h y p o c h l o r i t e o r the
In F i g u r e

is m u c h

slope a t c o n c e n t r a t i o n s in e x c e s s

cent available c h lo rin e .

in i ts

appears

C hloram ine

of 0.02

to be l e s s

T.

17A a nd B the e f f e c t of i n c r e a s i n g c o n c e n t r a ­

t io n s of R o c c a l a t p H 5.0 and pH 9.0 on the d i s i n f e c t i o n of c h i c ke n
f e c e s is

shown.

The t en fo ld n e u t r a l i z i n g d il u tio n s w e r e

F D A b r o t h c o n t a i n i n g one p e r c e n t of T a m o l —
N.
w ere

These

i n c u b a t e d a t 37 C and g r o w t h o c c u r r e d in all tu b es

made

in

di l ut i on s
w ithin

(A) Sodium Hypochlorite

(B) Chloramine

T

(O

CO
cn
S2

.05

Vo
%

av.

.05
% av.

1.0
Cl2

1.0
C l2

(C) Succinchlormide
i—>

CJ>

.05
1.0
% av. Cl2

Figure I6-A,B,C.
The reduction of the bacterial
population of chicken feces in 5.0 min. at
pH 50, 27.5°C. by chlorine compounds.

57
24 h o u r s , d e m o n s t r a t i n g t h a t no b a c t e r i o s t a t i c a c t i o n r e m a i n e d
follo w in g t h i s f o r m
th i s m a n n e r a r e
curve

of n e u t r a l i z a t i o n .

very

representing

The c u r v e s o b ta in e d in

s i m i l a r a t b o th pH v a lu e s ;

d i s i n f e c t i o n at pH 9.0 is

a t low c o n c e n t r a t i o n s of the c o m p o u n d .
ness

in the f i v e - m i n u t e

0.02 p e r

i s the s a m e

made

The m a x i m u m e f f e c t i v e ­

in b o th c a s e s .
m anner,

the f i r s t d ilu t i o n s

in F D A b r o t h to stop the a c t i o n of the ph en o l by

m e a n s o f d i l u t i o n a n d c o m b i n a t i o n w ith the o r g a n i c
th e b r o t h .
g r o w th ,
ure

is a t a bout

a nd the n u m b e r of r e s i d u a l r e s i s t a n t

P h e n o l w a s e x a m i n e d in like
were

somewhat ste e p e r

e x p o s u r e p e r i o d in b o th c a s e s

cent concentration,

organism s

h o w e v e r , the

I n c u b a t i o n of t h e s e b r o t h d il u tio n s

m a t t e r of

r e s u l t e d in p r o m p t

d e m o n s t r a t i n g t h a t the n e u t r a l i z a t i o n w as e f f e c ti v e .

17C s h o w s the

s i g m o i d c h a r a c t e r of the

survival curve

ta i n e d w i t h i n c r e a s i n g c o n c e n t r a t i o n of p h en o l.
the h o r i z o n t a l b r a n c h of the c u r v e
d e s c e n d i n g b r a n c h i l l u s t r a t e the
t iv i ty of t h i s

and the

rapid loss

c o m p o u n d with d ilu tio n .

at which s u r v iv a l b e c o m e s
p e r cent phenol.

F ig­
ob­

The e x t e n t of

steepness

of the

of g e r m i c i d a l a c ­

The c o n c e n t r a t i o n of phe no l

a s y m p t o t i c to c o n c e n t r a t i o n is a t 1.0

(A) Roccal,

pH5 0

(B) Roccal, p H 9 0
CO

6

Log

[^Survivors]

8

o2
_i
O

(C) Phenol,

0.2

pH5.0

0.2

0.1

%

(D) Merthiolate, pH5.0

6

Log

t*Survivors]

8

0.1

O

0.1 Oi

0.2

Figure I7-A,B,C,D.
The reduction of the
bacterial
population
of chicken feces in
5.0 min. at 2 75°C ., by various compounds.

M erthiolate
and the r e s u l t i n g
o b ta i n e d .

in a q u e o u s

so lu tio n at pH 5.0 was e x a m i n e d

survivor curve as

shown in F i g u r e

N e u t r a l i z a t i o n and d e c i m a l d ilution w e r e

in f r e s h l y p r e p a r e d F l u i d

17D wa s

accom plished

T h i o g ly c o lla te M e d iu m (Difco), i n c u b a ­

tio n of w h i c h d e m o n s t r a t e d g r o w th and he nc e a d eq u ate n e u t r a l i ­
za ti o n of t h e b a c t e r i o s t a t i c a c t i o n of m e r t h i o l a t e .
curve d e m o n s tra te s that m erth io la te

The s u r v i v a l

is p r a c t i c a l l y ine ffec t i ve in

r e d u c i n g the b a c t e r i a l c ount of c h ic k e n f e c e s u n d e r t h e s e c o n ­
d i ti o n s.

The r a t e of d i s i n f e c t i o n of c h ic k e n f e c e s .

The five iodine

p r e p a r a t i o n s w e r e a l l a d j u s t e d to 0.05 p e r c e n t iodine c o n c e n t r a ­
tio n a t pH 5.0 in M / 2 p h o s p h a te b u f f e r .
of the b u f f e r a l o n e .

C o n tr o ls w e r e p r e p a r e d

A fter exposure periods

u t e s , n e u t r a l i z a t i o n a n d dilutio n w e r e

of 1.0 and 5.0 m i n ­

accom plished as previously

d e s c r i b e d a n d s a m p l e s p l a t e d f o r e n u m e r a t i o n of the

su rv i v i n g

organism s.

The p e r

c e n t k ill, e f f e c te d in t h e s e p e r i o d s of e x ­

p o su re, was

r e c k o n e d f r o m the co u n ts o b ta in e d by id e n tic a l

t r e a t m e n t of the f e c a l s u s p e n s i o n in the p h o s p h a te b u f fe r c on­
trols.

These

d a t a a r e p r e s e n t e d in T a b le 4.

p e r i o d of 1.0 m i n u t e , the 0.05 p e r

A f t e r an e x p o s u re

ce nt iodine in the five p r e p a ­

r a t i o n s g a v e a n a v e r a g e k ill of 99.82 p e r c e n t with a m e a n

TABLE

4

THE P E R C E N T K I L L O F THE B A C T E R IA L P O P U L A T IO N
O F CHICKEN F E C E S A F T E R EX PO SU R E F O R THE
I NDICATED PE R IO D S TO 0.0 5 P E R CENT
IODINE AT pH 5.0 AND 27 C
Per
0.05% I_ a s
2

C e n t K ill A f te r
Exposure for

1.0 m in

5.0 m in

S u sp en so id (Merck)

99.95

99.97

"F in e"

99.37

99.84

" C o a r s e 11 s u s p e n s o i d

99.98

99.99

L u g o l ' s So l u t io n

99.84

99.94

I odine

T incture

99.98

99.98

A verage

99.82

99.96

suspensoid

59
d e v ia ti o n of 0 . 16 p e r c e n t .

T h is

represents

b e t t e r th a n two p a r t s p e r t h o u s a n d .
p o s u r e , the p r e p a r a t i o n s

A fter

an a c c u r a c y

s lightly

5.0 m i n u t e s of e x ­

at th is c o n c e n t r a t i o n c a u s e d an a v e r a g e

k i l l of 99.96 p e r c e n t w ith a m e a n d e v ia tio n of only 0.4 p e r c e nt ,
r e p r e s e n t i n g a n a c c u r a c y of f o u r p a r t s p e r te n tho u sa n d .

None

of the d e v i a t i o n s f r o m e i t h e r m e a n e x c e e d the m e a n d e v i a t i o n by
a f a c t o r of f o u r ,
as r e p r e s e n tin g

so t h a t a ll v a l u e s o b ta in e d c a n be c o n s i d e r e d
s a m p l e s f r o m the

sam e

specim en.

t h a t f o r the d i s i n f e c t i o n of c h i c k e n f e c e s ,
S olu tio n a n d I odin e

T incture

are

It is e v i de n t

C ollo id a l Iodine, L u g o l ' s

i d e n t i c a l in a c ti v it y when m e a ­

s u r e d a t c o n s t a n t t e m p e r a t u r e , pH and c o n c e n t r a t i o n .
C h l o r i n e c o m p o u n d s , ph e n o l,

R o c c a l and M e r t h i o l a t e w e r e

e x a m i n e d in a s o m e w h a t d i f f e r e n t f a s h io n .

The e x p e r i m e n t a l p r o ­

c e d u r e w a s a s g i v e n f o r iod in e , b u t the i n t e r v a l s of e x p o s u r e
were

e x t e n d e d to 30 m i n u t e s .

p h o s p h a t e b u f f e r a n d d u p li c a te
5.0 an d pH 9 .0 .
c a n be

a g a in w e r e

concentrations

w ere

ru n in M / 2
s e t a t pH

The e f f e c t of the p h o s p h a te b u f f e r a t pH 5.0

s e e n to b e n i l ( F i g u r e

exposure.

C ontrols

18A) e v e n a f t e r 30 m i n u t e s of

In p h o s p h a t e b u f f e r a t pH 9.0 t h e r e

significant d e c r e a s e

in the n u m b e r s

30 m i n u t e s t h i s d e c r e a s e

is a s m a l l but

of viab le o r g a n i s m s ,

a m o u n t s to a p p r o x i m a t e l y

after

50 p e r c e n t .

bO
The c u r v e s o b t a i n e d f o r d is i n f e c t a n t s in th e se b u ff e r s have not
b een c o r r e c t e d f o r t h i s e ffect of the b u ff e r alone and thus the
b a c t e r i c i d a l e f f e c t of the a lka line b u ff e r ha s d e p r e s s e d the c u r v e s
o b tain ed a t pH 9.0 s o m e w h a t.
R e s u l t s wi th c h l o r i n e
G r a p h B i l l u s t r a t e s the r a t e

com pounds a r e

shown in F i g u r e

18.

of d is in f e c tio n of c h ic k e n f e c e s by

so d iu m h y p o c h l o r i t e c o n taining 0.03 p e r c e n t a v ailab le c h lo ri n e .
At pH 5.0 the i n i t i a l r a t e

is g r e a t e r than a t pH 9.0, although

a f t e r ten m i n u t e s e x p o s u r e both r a t e s have b ec o m e the s a m e .
With C h l o r a m i n e

T ( G r a p h C) and S u c c in c h lo r im id e

(Graph D),

b o th co nta in in g 0.05 p e r c e n t a v a ila b le c h lo r i n e , the r a t e

of d i s ­

in fectio n a t the a c i d pH is ex cee din gly g r e a t e r than at the a l ­
k ali ne pH.

Within the

30—m inute e x p o s u r e p e r i o d th e se

com­

pounds a t pH 9.0 did not effect as g r e a t a kill as they did at
pH 5.0 in c o n t r a d i s t i n c t i o n to the h y p o c h lo rite .

This d iff e re n c e

would a p p e a r to be r e l a t e d to the d is s o c i a t i o n of th e s e o rg a n i c
co m p o u nd s a t t h e s e pH va lue s to yield hy po ch lorite ion and s u b ­
seq u en t ly the f r e e c h l o r i n e .
In F i g u r e

19A, the r a t e s of d is in fe c tio n p r o d u c e d by 1.0

p e r c e n t p he no l a t pH 5.0 and 9.0 a r e

shown.

T h e r e is a high

r a t e of k ill in the f i r s t m inute and the r a t e then d e c r e a s e s

at

(A) M/2 Phosphate Buffer

(B)0.03% Sod. Hypochlorite

8
r~\
to
k_
O

V)

>

>

-o

~ry

7
o*
o
_J
6

O

10

20
min.

30

20

min.

30

(D)0.05% Succinchlorimide

(0)0.05% Chloramine T
I— V

-CX.

—o

X)

o>

min.

30

20

min.

30

Figure I8-A,B,C,D. The rate of reduction of
the bacterial population of chicken feces at
pH5.0 (— ) and pH9.0(— *), temperature 275°C.
The concentration of the chlorine compounds
is given as percent available Cl2.

61
succeedin g tim e

intervals.

Under these

c o n d iti o n s , the h y d r o g e n

ion c o n c e n t r a t i o n d o e s not a p p e a r to a ff e c t the r a t e of d i s i n f e c ­
tio n of p h e n o l .
R o c c a l a t 0 .0 2 p e r c e n t c o n c e n t r a t i o n g iv e s
s u lt s to t h o s e of p h e n o l, a s
the r a t e

is

the

a t pH 5.0.

rate

power,

re­

At pH 9.0
of tim e tha n is

19C the e f f e c t of 0.2 p e r c e n t m e r t h i o l a t e

on the d i s i n f e c t i o n of the f e c e s

r e a d i l y be

19B.

s l i g h t l y h i g h e r in the e a r l y i n t e r v a l s

In F i g u r e
( aqueous)

shown in F i g u r e

sim ilar

is

shown.

It c a n

s e e n t h a t t h is c o m p o u n d h a s v e r y li tt le d is in f e c t i n g

s c a r c e l y b e t t e r th a n a lk a l i n e p h o s p h a te b u f f e r al one.

The c h a n g e s in the b a c t e r i a l f l o r a of the f e c e s following
disinfection.

Colonies w e re

r a n d o m l y p ic k e d f r o m the p l a t e s

in the p r e c e d i n g

experim ents.

s l a n t s of s t e r i l e

TGE a g a r and c h e c k e d f o r p u r i t y by s t r e a k i n g

on s t e r i l e p l a t e s of the

sam e

T h e s e f is h i n g s w e r e p la n t e d on

com position.

The c u l t u r e s

then e x a m in e d m ic r o s c o p i c a l l y as g r a m - s t a i n e d
wet m ounts

to o b s e r v e

m otility.

a b i l i t y to p r o d u c e c a t a l a s e

sm ears

w e re
a nd as

T h ey w e r e t e s t e d f o r t h e i r

and to f e r m e n t B r i l l i a n t G r e e n L a c ­

t o s e B il e b r o t h in 48 h o u r s .

T w en ty c u l t u r e s f r o m

the c o n t r o l

(A)

(8) 0 .0 2 %

Roccal

O

20

1.0% Phenol

'> 6
if)
\_/

0

10

.2 0
min.

30

10

mm.

30

(C )0 .2 % Merthiolate
<
/>
w

>7
— — —o

--

CO

0

10

20
min.

30

Figure I9-A,B,C. The rate of reduction of
the bacterial population of chicken feces at
pH 5 0 (— ) and pH9 . 0 temperature 275°C.

62
p l a t e s w e r e e x a m i n e d and ten c u l t u r e s f r o m e a c h of the d i s i n ­
fectant tre a tm e n ts .
The s i g n i f i c a n t findings f r o m th is c u r s o r y e x a m in a t i o n
of the b a c t e r i a l f l o r a r e v e a l e d th a t in the o r i g i n a l f l o r a the
spore-form ing

r o d s c o m p r i s e d 20 p e r c e n t of the i s o l a t e s .

F o ll o w i n g t r e a t m e n t with iodine c o m p o u n d s, c h l o r i n e com p o un ds ,
p h eno l a nd R o c c a l the p r o p o r t i o n of s p o r e - f o r m i n g r o d s i n c r e a s e d
to 50 to 90 p e r c e n t .

A fter

30 m in u te s t r e a t m e n t with R o c c a l at

pH 9.0, 40 p e r c e n t of the r e s i d u a l p o pu lation w e r e

slow l a c t o s e -

f e r m e n t i n g c o l i f o r m o r g a n i s m s a s c o m p a r e d to 20 p e r c e n t of
the o r i g i n a l p o p u l a t i o n as c o l i f o r m o r g a n i s m s w hich f e r m e n t e d
lactose prom ptly.
The c o m p o s i t i o n of the o r i g i n a l b a c t e r i a l f l o r a of the
c h ic k e n f e c e s did n o t d i f f e r m a r k e d l y f r o m the c o m p o s i t i o n s
n o te d by E m m e l (46) and by J o h a n s s o n , et al_. (47).

DISCUSSION

" I t is c h a r a c t e r i s t i c of the a d ­
v a n c e of s c i e n c e th a t l e s s and
l e s s is found to be d a tu m , and
m o r e and m o r e is found to be
in feren ce."
B ertra n d R ussell

The M e a s u r e m e n t of P e n e t r a b i l i t y

In the e x p e r i m e n t a l s e c t i o n the r e s u l t s o b ta in e d with the
"penetube"

technic

been p re se n te d .

a n d the d i s i n f e c t i o n of c h ic k e n f e c e s h ave

It i s o b vio u s t h a t in the

"penetube"

tech ni c

the d if f u s i o n of a d i s i n f e c t a n t into an a g a r g el c o lu m n p e r m i t s
m e a s u r e m e n t of a l i n e a r d i s t a n c e

t r a v e l e d by th a t d i s i n f e c t a n t .

The e n d p o i n t m e a s u r e d i s no t n e c e s s a r i l y the full d i s t a n c e t h a t
h a s b e e n t r a v e l e d b y a l l m o l e c u l e s of the d i s i n f e c t a n t but t hi s
e n d p o in t d o e s

r e p r e s e n t the p o in t w h e r e

a m inim al c o n c e n tra ­

t io n of t he d i s i n f e c t a n t h a s b e e n l e t h a l to the i n d i c a t o r o r g a n ­
i s m in the p e r i o d of e x p o s u r e .
reference

T h i s po in t h a s b e e n ta k e n a s a

p o i n t f o r the c o m p a r i s o n of the p e n e t r a t i o n of d i f­

f e r e n t d i s i n f e c t a n t s o r of one d i s i n f e c t a n t u n d e r d i f f e r e n t c on­
ditions.

C om parisons

h ave b e e n m a d e

only w h e r e

i n d i c a t o r o r g a n i s m w a s u s e d in e a c h c a s e .

the

same

The p e n e t r a t i o n of

64
proteinaceous

m a t r i c e s by d i s i n f e c t a n t s h a s a l s o bee n m e a s u r e d

by m e a n s of t h i s

technic

and the d i f f e r e n t i a l e f f e c ts of the p r o ­

t e i n s on th e p e n e t r a t i o n w e r e n o te d abo ve .
A few g e n e r a li z a tio n s

are

in o r d e r .

In t h e s e p e n e t r a t i o n

s t u d i e s i t w a s fo un d, p e r h a p s f o r t u n a t e l y f o r the

s u c c e s s of the

sanitarian,

t h a t the h i g h e s t p e n e t r a t i o n o b ta in e d by

d is in fe c t i n g

compounds

w a s a t the h y d r o g e n ion c o n c e n t r a t i o n s

a t w hic h the

compounds

are m ost germ icidal.

may

to i n d i c a t e

t h a t the e x t e n t of the z o n e s of inhibition a r e

T h is c o r r e l a t i o n

seem
a func­

t io n of g e r m i c i d a l a c t i v i t y a lo n e an d n o t of p e n e t r a t i o n .
t h i s i s n o t the c a s e

c a n be s e e n f r o m

w a s a d d e d to io d i n e

s o l u t i o n s w ith r e s u l t i n g i n c r e a s e s

z o n e s of i n h i b i t i o n .

M arks

io d id e i o n s a r e l e s s

g e r m i c i d a l th a n m o l e c u l a r iodine,

the g r e a t e r

the s t u d i e s

and S t r a n s k o v (48)

T ha t

in whic h iodide
in the

sh ow ed th a t t r i ­

z o n e s of i n h ib iti o n in the g e ls c o lu m n s

so t h a t

ca n n ot be

due to a g r e a t e r g e r m i c i d a l a c t i v i t y of the t r i i o d i d e io n s .
h a s b e e n d e m o n s t r a t e d (49) t h a t ion s diffuse m o r e
t h a n do the c o m p l e m e n t a r y m o l e c u l e s .
fused fu rth e r,

It

r a p id l y

T r i i o d i d e ions the n dif­

a n d a l t h o u g h i n t r i n s i c a l l y l e s s g e r m i c i d a l tha n

io d in e , c a u s e d a g r e a t e r

zone of inh ib ition .

We m u s t conc lude

th a t t he t e c h n i c d o e s a c t u a l l y m e a s u r e
not m e r e l y r e f l e c t d i f f e r e n c e s

p e n e t r a b i l i t y and does

in g e r m i c i d a l a c tiv ity .

The d i s i n f e c t i o n of c h ic k e n f e c e s

is not a s obviou s a

t e s t of th e p e n e t r a t i v e p o w e r of a d is i n f e c t a n t .
of b a c t e r i a in o r g a n i c p a r t i c l e s

is not s t r i c t l y u n i f o r m a nd at

l e a s t h a l f of the p o p u l a t i o n of the f e c a l
the s u s p e n d i n g m e d i u m .
freely

suspension a re free

t io n in the d i s i n f e c t i o n p r o c e s s .
resem blance

to the

and M a l l m a n n (14).

in

The p e n e t r a t i o n of the b a c t e r i a , w h e t h e r

s u s p e n d e d o r e n c l o s e d in p a r t i c l e s ,

a close

The e n c l o s u r e

In th is

m u s t be a c o n s i d e r a ­

r e s p e c t the t e s t b e a r s

s p e e d of d is in f e c t i o n t e s t of A n d e r s o n

The t e s t e m p l o y e d in th is stud y has the added

c o m p l i c a t i o n s of the p r e s e n c e of o r g a n i c and p a r t i c u l a t e m a t t e r
and a n o n u n i f o r m p o p u l a tio n .
in t h is t e s t

system

able p r o p e r t i e s ,

it m u s t have a c o m b in a t io n of highly d e s i r ­

and,

so, th is t e s t is a r i g i d one, d e s ig n e d to

w eed o u t “ wea k s i s t e r s . 11
a close

resem blance

in e n v i r o n m e n t a l
reproducible
as a rig o ro u s

F o r a d i s i n f e c t a n t to be e f f i c i e n t

The c o n d iti o n s of th is t e s t a l s o b e a r

to m a n y p r a c t i c a l

situations encountered

s a n i t a t i o n and, i n a s m u c h as it y ie ld e d f a i r l y

results

in the a u t h o r ' s

t e s t of d i s i n f e c t a n t s .

han ds,

m ay find a p p l i c a t i o n

66
The a g r e e m e n t of r e s u l t s o b ta in e d with d i s i n f e c t a n t s a s
m e a s u r e d by th e d i s i n f e c t i o n of c h ic k e n f e c e s
tube"

and by the ’' p e n e ­

te c h n i c m a y be c o i n c i d e n t a l o r m a y be due to the c o m m o n

r e q u i r e m e n t of p e n e t r a t i o n in b o th t e c h n i c s .
however,

t h a t the f e c a l d i s i n f e c t i o n te c h n ic

penetrability,

a l t h o u g h the

"penetube"

1 do not b e l i e v e ,
is a s i m p l e t e s t of

te c h n ic undoubtedly is

such a te st.

The

The r a t i o n a l e
be o b v io u s .

"P enetube"

T ec h n ic

u n d e r ly i n g this te c h n ic

m a y n ot a t f i r s t

It m u s t be r e c a l l e d th a t the te c h n ic w a s d e s i g n e d

to a c c o m p l i s h th e m e a s u r e m e n t of p e n e t r a b i l i t y while e l i m i n a t i n g
o r av o id in g the o b j e c t i o n s p r o p o u n d e d in the In tro d u c tio n .
The c h a r a c t e r i s t i c s

d e s i r e d in a s y s t e m f o r m e a s u r i n g

the p e n e t r a t i v e p o w e r s of d i s i n f e c t a n t s a r e

m an ifold .

The i n­

d ic a t io n t h a t p e n e t r a t i o n has o c c u r r e d should be the v isi b l e
d e a t h of b a c t e r i a .

T his d e s id e r a t u m

has two i m p l ic a t io n s ;

f i r s t i m p l i c a t i o n b e i n g t h a t an a u x a n o g r a p h ic
c i le m e a s u r e m e n t of r e s u l t s
the i n d i c a t o r o r g a n i s m s

and the

the

te st provides fa­

s e co n d th a t the d e a t h of

i m p l i e s th a t a s u ff ic ie n t c o n c e n t r a t i o n

of the d i s i n f e c t a n t h a s b e e n a t a definite lo c u s f o r a su ffi c ie nt

67
l e n g t h of t i m e f o r d i s i n f e c t i o n to have o c c u r r e d .

The b a c t e r i a

to be k i l l e d m u s t be l o c a t e d in u n i f o r m d i s p e r s i o n in a definite
c o l l o i d a l b a r r i e r of o r g a n i c o r p r o t e i n a c e r o u s m a t t e r , p r o v i d i n g
a v o l u m e t h r o u g h w h i c h the diffusion o r p e n e t r a t i o n of the d i s ­
in f e c t a n t m u s t u n e q u i v o c a ll y o c c u r f o r the d e a th of the i n d i c a t o r
b a c t e r i a to r e s u l t .

The d i s p e r s e d o r g a n i s m s

m u s t not be p e r ­

m i t t e d to m u l t i p l y d u r i n g the e x p o s u r e to the d i s i n f e c t a n t so
t h a t a u n i f o r m c o n c e n t r a t i o n of c e l l s will be m a in ta i n e d ,

a ll

of w h ich wi l l be a t the

sa m e p h y sio lo g ical o r m orphogenetic

s ta g e of d e v e l o p m e n t .

T h i s l a s t r e q u i r e m e n t , th e n , will p r e ­

v en t the r e f l e c t i o n of d i f f e r e n t i a l g r o w th r a t e s
the I n t r o d u c t i o n , a nd w i ll n e c e s s i t a t e

a s d i s c u s s e d in

a s u b s e q u e n t t r e a t m e n t to

p e r m i t the v i s i b l e d e v e l o p m e n t of viable o r g a n i s m s .

Th i s

step of d e v e l o p m e n t c a n be c o m b in e d w ith a n e u t r a l i z a t i o n
to e l i m i n a t e t h e e f f e c t s of b a c t e r i o s t a s i s .

same
s te p

A n o th e r d e s i r e d a t ­

t r i b u t e of th e t e c h n i c m u s t be a c a p a c i t y f o r wide e x p e r i m e n t a l
v a r i a t i o n of c o m p o n e n t s and p r o c e d u r e s .

The s e p a r a t i o n of the

s t e p s of d i s i n f e c t i o n and g r o w th p e r m i t th is p o s s i b i l i t y .
In 1921, S t i l e s

a nd A d a i r (50)

r e p o r t e d m e a s u r e m e n t s of

the c o e f f i c i e n t s of d i f f u sion of e l e c t r o l y t e s into g e ls w h e r e the
gds

w ere

s u p p o r t e d in g l a s s tu b e s in a f a s h i o n s i m i l a r to the

68
’• p e n et u b e "

construction.

T heir r e s u lts

t e r m i n a t i o n s of d if fu sio n c o e f f i c i e n t s .

are

M ore

among the b e s t de ­
r e c e n tl y ,

Davis et

a l . (51) r e p o r t e d a m e t h o d f o r a n t ib io ti c t e s t i n g employing l i n e a r
d if fu sio n of the a n t i b i o t ic into i n o c u l a te d n u t r i e n t a g a r in g l a s s
capillaries.

A c c o r d i n g to th e s e a u t h o r s , l i n e a r diffusion a s s a y

a f f o r d s m a n y a d v a n t a g e s o v e r a g a r p l a te m e t h o d s .

There are

t h e r e f o r e p r e c e d e n t s f o r d e t e r m i n i n g diffu sio n o r p e n e t r a t i o n
in a l i n e a r d i r e c t i o n in g l a s s t u b e s a s has bee n done in the
"penetube"

technic.

The p r o b l e m of c a p i l l a r y c r e e p a g e

of d is in f e c t a n t betw een

the w a l ls of the g l a s s tube and the p e r i p h e r a l s u r f a c e of the a g a r
c o lu m n s was not a s e r i o u s one.

W here c a p i l l a r y c r e e p a g e was

e n c o u n t e r e d the edge of the zone of inhib ition a p p e a r e d as a
c y l i n d e r t r u n c a t e d a t an angle o r a s an i n v e r t e d cone.

This

o c c u r r e d in l e s s th a n two tub e s p e r th o u sa n d , and, i n a s m u c h
as

su c h t u b e s w e r e

rejected, cap illary creepage

cannot be c on­

s i d e r e d a s a s e r i o u s d i s a d v a n ta g e .
The i n t e r p r e t a t i o n of the
tube"

results

g a in e d using the

"pene­

t ec h n i c l e a d to the g e n e r a l i z a t i o n th a t the exten t o r ra te

of p e n e t r a t i o n of a d i s i n f e c ta n t u n d e r v ar y in g conditions of pH,
tem perature, presence

of a d ju nc t s u b s t a n c e s ,

e t c ., into gels of

69
v a r i e d c o m p o s i t i o n c a n be m e a s u r e d .

One substantive o b s e r v a ­

tio n of b o th p r a c t i c a l and t h e o r e t i c a l i n t e r e s t is that, fo r any
p a r t i c u l a r s e t of c o n d i tions in v e s tig a te d , th e r e is found some
m i n im u m c o n c e n t r a t i o n of the d is in f e c ta n t giving a l m o s t m a x i ­
m a l e x t e n t of p e n e t r a t i o n .

S tate d a n o th e r way, with in c r e a s i n g

c o n c e n t r a t i o n s of the d is in f e c ta n t a point of dim inishing r e t u r n s
s e t s in wh i c h c a n be r e a d i l y r e c o g n iz e d on in sp ec tion of the
concentration curves.
in T abl e

T h e se

‘' p l a t e a u 11 va lue s a r e p r e s e n t e d

5, along with like va lue s obtained f r o m the c u r v e s of

the d is i n f e c t i o n of c h i ck en f e c e s .

The a g r e e m e n t of the va l ue s

m a y only b e c o i n c i d e n ta l o r m a y indeed signify that the two
d i s s i m i l a r technics a re c o r r e la te d .

This c o r r e l a t i o n ,

if re a l ,

would sig nif y t h a t the d isi n fe c ti o n p r o c e s s in the e x p e r i m e n t s
with c h i c k e n f e c e s wa s e n t i r e l y dependent upon the p e n e t r a b i l i t y
of the d i s i n f e c t a n t .

This view and som e m a t h e m a t i c a l f o r m u l a ­

t io n s c o n c e r n i n g p e n e t r a t i o n and d isin fe c tion p r o c e s s e s a r e d i s ­
c u s s e d below.
It was b e l i e v e d th a t the am ount of di si n fe c ta n t diffusing
into the a g a r c o l u m n s could be r e l a t e d to the extent of p e n e ­
t r a t i o n and to the n i t r o g e n content of the c olu m ns .

Iodine can be

r e a d i ly a s s a y e d , and thus in s y s t e m s w here colum ns containing

TABLE

5

THE CONCENTRATION O F DISINFECTANTS AT WHICH
DIMINISHING E F F E C T IV E N E S S BECOMES A PP A RE N T
IN THE PE N E T RA TIO N O F GEL COLUMNS AND
IN THE DISINFECTION O F CHICKEN FEC ES

Compo un d

P l a t e a u Value in P e r Cent
C o n c e n tra tio n
------------------------------------------------------------------------From "Penetube"
F r o m Disinfe cti on
Experim ents
of Chicken F e c e s

Iodine (colloid)

0.05

0.05

Iodine ( L u g o l ’s)

0.05

0.05

Iodine ( T i n c t u r e )

0.05

0.05

Sodium Hy p o c h l o r it e

0.05

0.1

Chloram ine

0.05

0.1

Succinchlorim ide

0.02

0.01

Roccal

0.02

0.02

Phenol

1.0

1.0

0.01

0.02

M e r th i o l a t e

T

(aq.)

70
a g a r and a g a r p lu s p r o t e i n add itive s w e r e e x p o se d to ide nt ic a l
c o n c e n t r a t i o n s of iodine, the l o s s of iodine f r o m e a c h solution
was d e t e r m i n e d .

All t r i a l s gave id e n tic a l l o s s e s of iodine f r o m

the s o lu tio n s and s t i l l the extent of p e n e t r a t i o n v a r i e d in the
m a n n e r shown in the e x p e r i m e n t a l sec tion .

The n itr o g e n d e ­

t e r m i n a t i o n was the s a m e f o r gelatine and the hum an s e r u m
al b u m i n alth o ug h l e s s e r p e n e t r a t i o n o c c u r r e d in the l a t t e r s y s ­
tem s.

It would then a p p e a r th a t the r a t e at which m o le c u l e s

of iodine e n t e r the c olum n is s t r i c t l y a function of the c o n c e n t r a ­
tion of the iodine and the c r o s s - s e c t i o n a l a r e a of the c olumn in
c o n ta c t with the

s olu tion.

The ex ten t of p e n e t r a t i o n would then

be a function of the c o n c e n t r a t io n g r a d i e n t of the u n r e a c t e d
iodine in the c o l u m n s .
trinsically m ore
less

The hum an s e r u m alb u m in is then in­

r e a c t i v e with iodine than is gelatine and a g a r

r e a c t i v e than e i t h e r .

The D i s infe ctio n P r o c e s s as Affected
by P e n e t r a b i l i t y

The d a t a and o b s e r v a ti o n s r e c o r d e d in the e x p e r i m e n t a l
se c ti o n m a y be c o n s i d e r e d in the light of the disinfection p r o c ­
e s s in g e n e r a l in the hope that som e additional light m ay be
shed on t h is c o n t r o v e r s i a l su b jec t.

71
In th e e a r l y

s t a g e s of the p r o c e s s of d is in f e c tio n

o r g a n i s m (O) m a y be c o n s i d e r e d
v e r s i b l e u n io n wi th

a s f i r s t e n t e r i n g into a

re­

m o l e c u l e s of the d i s i n f e c t a n t (D) a s follow s:

_
ki
[I] O + D ='—=■
2
, ,

(OD); w h e r e

fe c ta n t c o m p le x and k

and k
-L

f o r w a r d and r e v e r s e

(OD) is an o r g a n i s m - d i s i n -

the e q u i l i b r i u m c o n s t a n t s of the
L)

reactions.

Su c c ee d in g this union, the fa te

of c o m p l e x (OD) i s d e a t h of the o r g a n i s m
reaction rate

the

(O^) a t so m e

s pe c i fi c

(h^), h e n ce:

kl
[Ill O + D = = = = =
2

k3 _
(OD) ------------^ O

at a s te a d y s t a t e , the
d

r a t e of f o r m a t i o n of (OD) m u s t eq ua l the r a t e of b re a k d o w n of
the c o m p l e x in a c c o r d a n c e with the following equation:
(1) k

N c = k [OD] + k [OD]; w h e r e N
I

I

b

J

is the n u m b e r of
1

f r e e o r u n r e a c t e d o r g a n i s m s , c the c o n c e n t r a t i o n of the d i s i n ­
f e c t a n t a n d [OD] the n u m b e r of o r g a n i s m s
E q u a ti o n (1) r e a r r a n g e s

in c o m p l e x f o r m a t i o n .

to:

N
k3 + k2
(2) ^QQ~j = ----£--------- » and the total- n u m b e r of o r g a n i s m s

is e q u a l to the

s u m of the n u m b e r of f r e e

(N^

o r g a n i s m s and those

in c o m p l e x f o r m a t i o n (n e g le ctin g the n u m b e r dead);

the e qua ti on

72
(2) b e c o m e s :

(3)

Nt
jqqj

=

k2 + k3
~

+

I; and if

the m a x im u m v eloc ity

a f u n c ti o n of t he t o t a l p o p u latio n and any i n t e r m e d i a t e

(K) is

vel oc ity

(k) a f u n c t i o n of the n u m b e r of o r g a n i s m s in c o m b in a tio n with
the d i s i n f e c t a n t , then:
N
k
+ k
K.
t
2
3
(4) ~ = j-q u j * a n d l e tti n g -----~------- = a (a no the r co nst a n t ),

t h e n on s u b s t i t u t i n g e q u a tion (4) and th is c o n s ta n t into eq uati on
(3), e q u a t i o n (5) i s o b t ained:
(5) k =

a + c

.

We m a y now c o m p a r e t h is t h e o r e t i c a l t r e a t m e n t with the
d a ta o b t a i n e d e x p e r i m e n t a l l y f r o m the f e c a l d is in f e c tio n e x p e r i ­
ences.
The d e a t h of the b a c t e r i a in the f e c a l s a m p l e s
r a p i d in the f i r s t m i n u te of e x p o s u r e
tio n t h at, in t h i s
t e r i a in the
with r e s p e c t

is e x t r e m e l y

and if we m ak e the a s s u m p ­

s m a l l tim e i n t e r v a l , the r a t e of kill of the b a c ­

sam ple

is l o g a r i t h m i c , then the death of b a c t e r i a

to t i m e will

r ia originally p re s e n t,

be a function of the
or—
dN /dt =

kN; w he re

n u m b e r s of b a c t e ­
k is the co e ffi c i e nt

of d i s in f e c t i o n , N the n u m b e r s of b a c t e r i a and t the tim e of

exposure.

I n t e g r a t i o n of th is e q u a tio n b e tw e en z e r o tim e and

one m i n u t e y i e l d s :

k = In N , 1

In N *, w h e re N and N
2
1
2

are

the n u m b e r of b a c t e r i a o r i g i n a l l y p r e s e n t and the n u m b e r of
survivors

respectively.

The value of k can then be found for

any p a r t i c u l a r c o n c e n t r a t i o n of the d i s i n f e c ta n t a t c o n s ta n t t e m ­
perature

a nd pH.

In a c c o r d a n c e with Chick (52), the k va l ue s

have b e e n d e t e r m i n e d f o r a n u m b e r of the com pounds f r o m the
e x p e r i m e n t a l d a t a r e p o r t e d above.

The value of e^ f o r the t r a n s ­

f o r m a t i o n of the l o g a r i t h m s to the b a s e

10 has b e e n o m i t t e d .

On

p lo tting t h e s e v a l u e s of k a g a i n s t the c o n c e n t ra ti o n , the c u rv e A
of F i g u r e

20 f o r the d i s i n f e c ti o n of c h ic k e n f e c e s by L u g o l ' s

Solutio n a t pH 5.0 a n d 27.5 C was o b ta ine d .

C o n s id e ri n g the

eq u a ti o n of t h i s c u r v e to be k — K c / ( a + be), a L i n e w e a v e r - B u r k
p l o t (53) e q u a t e s the
eq u a t i o n b e c o m e s :

value

of the t h r e e c o n s t a n t s

k = 4 .6 c / ( 0 ,0 3 + c).

tion i s shown in F i g u r e

20 as c u r v e B.

s t a n t b in t h i s e q u a t i on is

so tha t the

The plot of this e q u a ­
T h a t the value of c o n­

unity p r o v i d e s a ch eck on the a g r e e ­

m e n t of the e m p i r i c a l e q u a tion with the t h e o r e t i c a l l y d e r i v e d
eq u a t i o n (5).
e q u a tio n a r e
(Fig ure

The e x p e r i m e n t a l d a ta and the plot of the e m p i r i c a l
in v e r y c l o s e a g r e e m e n t .

20, c u r v e C) u n d e r the s a m e

Data ob ta ine d with phenol
c on ditions do not follow the

k
O'

0.05
0.5

1.0

c.

0.15
1.5

0.2
2.0

Figure 20. The rate of disinfection (k in log
of kill min"1) at increasing concentration ( c ) of
Lugol's Solution (curve A), and Phenol (curve C).
0 Q
Curve B is plotted from k = 0 3 +c •

74
same form .

T h i s type of deviation is explicable on the b a s i s

of the i r r e v e r s i b l e

r e a c t i o n in f o r m u l a [II] being dependen t upon

a m u l t i m o l e c u l a r r e a c t i o n be tw een phenol and the o r g a n i s m s .
Thus, at low c o n c e n t r a t i o n s of the d is in f e c ta n t the r a t e of in­
crease

of the d e a t h v e l o c ity c o n s ta n t will not be as g r e a t a s

at in term ediate concentrations.

Th is is tan tam ou nt to saying

t h at p henol h a s a high c o e ffi c ie n t of dilution.

The m a t h e m a t i c a l

t r e a t m e n t of s u c h s i t u a t i o n s is quite co m p lex , but can be done
in the

s a m e f a s h i o n a s the t r e a t m e n t of de v iate d a d s o r p t io n i s o ­

th e r m s where

' ’p a c k i n g 11 of m o l e c u l a r l a y e r s

occurs

(54).

This

t r e a t m e n t will d i s c l o s e the b e h a v io r of phenol to be a spe c i a l
c a s e following the s a m e g e n e r a l equation.
It m u s t be n o t e d that equation (5) is a r e p r e s e n t a t i o n of
the M i c h a e l i s - M e n t e n e quation (55) f o r e n z y m a tic r e a c t i o n s and, indeed , wa s d e r i v e d in analogo us fas h io n .

In this d e r iv e d e q u a ­

tion, the c o n s t a n t K r e p r e s e n t s the m a x i m u m r a te of d is in f e c ­
tion a tt a i n a b l e u n d e r the p r e v a il i n g conditions and the c o n s t a n t
a r e p r e s e n t s the c o n c e n t r a ti o n of the d is in f e c ta n t at which half
the m a x i m u m

r a t e of d i s in fe c tio n o c c u r s .

It ca n be s e e n t hat w he re d isin fe c tio n p r o c e e d s in a c ­
c o r d a n c e with t h is k i n e t ic p i c t u r e , an o r g a n i s m - d i s i n f e c t a n t

75
c o m p l e x c a p a b l e of d i s s o c i a t i o n m u s t be f o r m e d .

If the f i r s t

step in d i s i n f e c t i o n i s then p o s tu la t e d to be a c o lli si o n be tween
m o l e c u l e s of the d i s i n f e c t a n t and viable o r g a n i s m s , this c ol li s ion
n e e d not n e c e s s a r i l y r e s u l t in an i m m e d ia t e

irreversible

reac­

tio n w ith c o n s e q u e n t d e a th of the o r g a n i s m .

If the diffusion of

the d i s i n f e c t a n t s c a n p r o c e e d in any m e a n f r e e path, then the
r a t e a t w h i c h m o l e c u l e s of the d is in f e c ta n t will be d e s o r b e d
f r o m the s u r f a c e will c o r r e s p o n d to the e q u il i b r iu m c o n s t a n t
f o r the d i s s o c i a t i o n of the o r g a n i s m - d i s i n f e c t a n t co m plex .

In

th is study of the d i s i n f e c tio n of chicken f e c e s , w h e re the p e n e ­
t r a t i o n of the p a r t i c l e s

e n c lo si n g the o r g a n i s m s has been ma de

a f a c t o r in the l i m i t a t i o n of the d isi n fe c tio n p r o c e s s , the a p­
p e a r a n c e of a r e l a t i o n s h i p to this p o s t u l a t e d d isi n fe c tio n m e c h ­
a n i s m h a s b e c o m e e v i de nt.
process

This im p li e s that the disinfe c t ion

and the c h a r a c t e r i s t i c

c u r v e s ob tain e d during the c o u r s e

of d i s in f e c t i o n a r e the r e s u l t of a f i r s t o r d e r

r e a c t io n r a t e at

low c o n c e n t r a t i o n s of the d is in f e c t a n t and a z e r o o r d e r r e a c t i o n
r a t e a t high d i s i n f e c t a n t c o n c e n t r a t i o n s .

This m e c h a n i s m may

be f u r t h e r c o m p l i c a t e d by the p r e s e n c e of p o l y m o l e c u la r r e a c ­
tio ns and by a n o n u n i f o rm d i s t r i b u t i o n of r e s i s t a n c e of the o r ­
g a n i s m s to the a c t i o n of the di si n fe c ta n t.

It is obvious that, in

76
the c i t e d e x p e r i m e n t s , t he p o p u la ti o n of m i c r o b e s was a m i s c e l ­
l a n e o u s o ne a n d t h a t g r a d e d d e g r e e s of r e s i s t a n c e
an d d id o c c u r .

m u s t have

H o w e v e r , the p e r c e n t a g e of r e s i s t a n t o r g a n i s m s

p r e s e n t w a s o n l y a p p r o x i m a t e l y 0.01 p e r c e n t of the to ta l p opu ­
l a t i o n an d a p p a r e n t l y t h i s f a c t o r i s of a l e s s e r

i m p o r t a n c e t ha n

m i g h t be e x p e c t e d .
I t m u s t be a d m i t t e d t h a t the d e r i v a t i o n of e q u a tio n (5)
was not p e r f e c tly
organism s

rigorous,

was neglected.

d e r iv e d equation f r o m

i n a s m u c h a s the n u m b e r of d e a d
The a g r e e m e n t of the e m p i r i c a l l y

the e x p e r i m e n t a l f a c t s with the f o r m of

the t h e o r e t i c a l l y d e r i v e d e q u a tio n would in d ic a te th a t th is n e g l e c t
w as n o t d i s a s t r o u s .

T h is c a n be ta k e n to m e a n th a t the i n t e r ­

a c t i o n of the d e a d o r g a n i s m s w ith the r e m a i n i n g
the

system

c o m p o n e n t s of

in a n y s m a l l t i m e i n t e r v a l is nil.

A n o t h e r i n t e r p r e t a t i o n c a n be p l a c e d upon the c o i n c i d e n c e
of f o r m

of th e e q u a t i o n s e x p e r i m e n t a l l y and t h e o r e t i c a l l y d e r i v e d

f o r t he d i s i n f e c t i o n p r o c e s s .

The

r e s e m b l a n c e of the e q u a t i o n s

to th e L a n g m u i r A d s o r p t i o n I s o t h e r m

is quite good.

The n o n -

i d e n t i f i c a t i o n of c o n s t a n t a with unity m a y m e r e l y be a c o n s e ­
q u e n c e of th e u n i t a g e e m p lo y e d .
(23) t h a t y e a s t c e l l s

rem ove

The o b s e r v a t i o n of K n a y s i

iodine f r o m

so lu tio n in a c c o r d a n c e

77
w it h an a d s o r p t i o n i s o t h e r m

m i g h t be r e a d i l y ta k e n as e v i de n c e

t h a t the d i s i n f e c t i o n m e c h a n i s m p r o c e e d s

in the n o te d o r d e r a s

a r e s u l t a n t of the a d s o r p t i o n m e c h a n i s m .
this r e s e m b la n c e

is one n e c e s s a r y

C ontrariw ise,

co n d itio n f o r

a l t hough

such a m echan­

i s m , th is e x p l a n a t i o n i s n o t s u f f ic ie n t to e x p la in the w e l l -k n o w n
(but p o o r l y u n d e r s t o o d )

r e v e r s a l e f f e c t s o b ta in a b le with m e r c u r i a l s

an d q u a t e r n a r y a m m o n i u m c o m p o u n d s .

The m e c h a n i s m p r o c e e d ­

ing t h r o u g h a n o r g a n i s m —d i s i n f e c t a n t c o m p l e x s u f f ic e s b e t t e r to
e x p l a in a l l the kn own f a c t s

c o n c e r n i n g d i s i n f e c t i o n and a ff o r d s

a r e a d y a n s w e r to the r o l e of p e n e t r a t i o n by the d i s i n f e c t a n t
p r e c e d e n t to the e n s u i n g l e t h a l r e a c t i o n s .
T h e k i n e t i c s of c h e m i c a l a d s o r p t i o n m a y give us a d d i ­
t io n a l i n s i g h t in to the
apparent fro m
to

r o le

of p e n e t r a t i o n in d i s i n f e c ti o n .

It is

i n s p e c t i o n of the c u r v e s p r e s e n t e d in F i g u r e s

10, t h a t t h e r e

is a re se m b la n c e

to a d s o r p t i o n i s o t h e r m s .

6
In

a c c o r d a n c e wi th the c u s t o m a r y d e r i v a t i o n of the L a n g m u i r A d­
sorption Iso th e rm

(54), an a lo g y l e a d s to the d e r i v a t i o n of an

e q u a ti o n of the s a m e f o r m f o r the
i n f e c t a n t in the
tio n (6); k

"penetube"

te c h n ic .

r a t e of p e n e t r a t i o n of a d i s ­
T h is t r e a t m e n t y ie l d s e q u a ­

= K c / ( b + c), w h e r e k^ is the le n g th of the

i n h i b it i o n o b t a i n e d in 24 h o u r s following e x p o s u r e to the

zone of

78
d i s i n f e c t a n t a t c o n c e n t r a t i o n c a t a c o n s ta n t t e m p e r a t u r e and
pH.

K t h e n r e p r e s e n t s the m a x i m u m r a t e of p e n e t r a t i o n ob­

ta i n a b l e u n d e r the c o n d itio n s of the t e s t and b r e p r e s e n t s the
c o n c e n t r a t i o n a t w h i c h half the m a x i m a l r a t e is obtained .

As

in the p r e v i o u s c a s e f o r d is i n f e c t io n of ch ic k e n f e c e s , the e m ­
p i r i c a l d a t a c a n be f i t t e d to t h is e qu ation upon e v a lu a tio n of the
c o n s t a n t s K a nd b.

In F i g u r e

21, the e m p i r i c a l data fo r the

r a t e of p e n e t r a t i o n ( e x p r e s s e d as m m p e r 24 hr.) of Iodine
T i n c tu r e a t v a r y i n g c o n c e n t r a t i o n s

into a g a r containing

1.0 p e r

c e n t g e l a t i n e a nd 0.5 p e r c e n t p o t a s s i u m iodide a r e p lo tt e d
( cu r v e A).

F r o m t h e s e d a ta , the e q ua tion

is o b t ai n e d ,

a p l o t of w hich a p p e a r s

B.

in the

= 22 .7c/( 0 .00 5 + c)
s a m e f ig u r e a s c u rve

It is t e m p t i n g , t he n, ag a in to a t t e m p t to explain dis inf e c t i on

m echanics

a s b e i n g e n t i r e l y de p en de n t upon the r a t e of p e n e t r a ­

ti o n of t he d i s i n f e c t a n t in a c c o r d a n c e with equation (6).

On the

s a m e g r o u n d s a s b e f o r e , th is is n e i t h e r n e c e s s a r y n o r a r e t h e r e
su f f i c ie n t b a s e s f o r

so doing.

It m a y only be a co incide nc e tha t

the i n t e r c o n n e c t e d p r o c e s s e s follow the sa m e

rate p ro c e s s .

A

d i r e c t c a u s a t i o n c o n n e ctio n b e tw e e n two se q u e n tia l ev en t s is not
su f f ic i e n t in s t e a d y s t a te m e c h a n i c s fo r a single
s t r u e d a s the

s ole

step to be c o n­

r a t e - d e t e r m i n i n g f a c t o r in the o v e r - a l l

— o

20

.02

.03
c

.04

.05

Figure 21.
The rate of penetration (kp in
mm.) at increasing
concentration (c) of iodine
in agar +1.0% gelatine and 0.5% Kl. Curve Aexperimental data, Curve B: calculated from
« 22.7c
p=.005+ c

79
process.

I t a p p e a r s to th is a u t h o r th a t the k i n e ti c s of de a t h

of m i c r o o r g a n i s m s

d u r ing c h e m i c a l d is in fe c tio n m u s t be d i c ­

ta te d by the a d s o r p t i o n of the d is in f e c ta n t f r o m the e n v ir o n ­
m en t , the s u b s e q u e n t f o r m a t i o n of a d is s o c ia b l e com plex, p e n e ­
t r a t i o n of the c y t o p l a s m and a c o n se q u e n t i r r e v e r s i b l e
t h e r e i n wi th u l t i m a t e d e a th of the o r g a n i s m .

Th.'

reaction

s u r v iv a l c u r v e s

m u s t be a f u n cti on of t h e s e p a r a m e t r i c functions and can l e a d
to a r e p r e s e n t a t i o n of f i r s t and z e r o o r d e r r e a c t i o n r a t e
chanics.

The p o s s i b i l i t y of h i g h e r o r d e r

me­

r e a c ti o n r a t e s is not

ex cl u d ed , n o r any c o m b in a tio n with the l o w e r o r d e r r e a c ti o n
rates.

The n o n u n i f o r m d i s t r i b u t i o n of r e s i s t a n c e in the popu­

lati o n e x p o s e d to the d i s in f e c ta n t is c e r t a i n l y a f a c t o r inf lue nc ­
ing the e v e n t s p o r t r a y e d .

T his d is tr i b u t i o n c a n be like ne d to

the d i s t r i b u t i o n of a c t i v a tio n e n e r g i e s among the m o le c u le s r e ­
ac ti n g in any c h e m i c a l r e a c t i o n and so the m e c h a n is t ic a p p r o a c h
e n v is i o n e d h e r e i s c a p a b le of r a t i o n a l i z a t i o n with the biologic a l
considerations.

The P e n e t r a t i v e P o w e r of Colloidal Iodine

As h a s b e e n n oted in the e x p e r i m e n t a l section, the p e n e ­
t r a t i v e p o w e r of Co l lo id al Iodine has bee n shown not to exc e e d

80
t h a t of o t h e r iodine p r e p a r a t i o n s with which it h a s been c o m ­
p a r e d u n d e r the s a m e c o n d i ti o n s .

T h e se findings a r e

in d i s a ­

g r e e m e n t wi th t h o s e of M a llm a n n and C h a n d le r (7) on the d i s i n ­
f e c t io n of a v i a n f e c e s and of A n d e r s o n and M allm an n (14) on the
in h ib it io n of s p o r u l a t i o n of c o c c i d i a l o o c y s t s .
m e n ts w e r e

T h e se d i s a g r e e ­

shown to be a fu n c tion of the pH of the v a r i o u s

p r e p a r a t i o n s a s t e s t e d a g a i n s t the o o c y s ts and f e c a l s u s p e n ­
si o n s.

When t e s t e d a t equal h y d r o g e n ion c o n c e n t r a t i o n s , C o l ­

lo i d a l Iodine a n d L u g o l ' s Solution w e r e

shown to be equall y a c ­

tive in a l l t e s t s y s t e m s .
Co l l o i d a l Iodine do es not have any i n t r i n s i c a l l y hi g h e r
p e n e t r a t i v e p o w e r t h a n o t h e r iodine p r e p a r a t i o n s .

The p r e p a r a ­

ti on a s m a r k e t e d is e x t r e m e l y acid, and b e c a u s e of this a c i dit y
ex h i bi t s h ig h p e n e t r a b i l i t y and g e r m i c i d a l ac tiv ity .

Iodine, in

the a u t h o r ’ s e x p e r i e n c e , has b e e n the d is in f e c ta n t p o s s e s s i n g
the b e s t p e n e t r a t i v e p o w e r of the v a r i o u s d is in f e c ta n ts t e s t e d .
C o l lo i d a l Iodine is m o r e
L u g o l 1s Solution o r Iodine
a re acidula ted.

e f f ic ie n t in this

r e s p e c t than a r e

T i n c tu r e u n l e s s the se p r e p a r a t i o n s

The low tox icity and low i r r i t a n t n a tu r e of

C o ll oi d a l Iodine in c o m b in a t io n with the high g e r m i c i d a l and

81
p e n e t r a t i v e p o w e r s of iodine m ak e

C ollo ida l Iodine a d e s i r a b l e

a d j u n c t to a ny h o u s e h o l d f i r s t a id k it.
F o r environm ental
in an y f o r m

s a n i ta t io n ,

is its high c o s t a s

the only o b je c tio n to iodine

c o m p a r e d to c h l o r i n e .

w o u ld s e e m to b e , on o t h e r g r o u n d s , m o r e
sa n ita tio n than c h lo r in e .

effective.

desirable for general

Iodine p r e p a r a t i o n s a r e

th a n c h l o r i n e p r e p a r a t i o n s

Iodine

m ore

stable

a t the a c i d pH v a lu e s w h e r e bot h a r e

Chlorine compounds, because

of this la b il it y , a r e

g e n e r a l l y m a r k e t e d a s a l k a li n e p r e p a r a t i o n s , a co n d itio n i n c o m ­
p a t i b l e w i th h i g h g e r m i c i d a l a c t i v i t y .
to t h i s a u t h o r t h a t if c r u d e
at reaso n ab le

It would s e e m

iodine p r e p a r a t i o n s

reasonable

could be m a r k e t e d

c o s t io d ine would be the d i s i n f e c t a n t of c h oi c e f o r

m a n y j o b s now p r e e m p t e d by c h l o r i n e .
i n v e s t i g a t e d a p r e p a r a t i o n of c r u d e
w i th g oo d r e s u l t s .
in the p r e s e n c e

G e r s h e n f e l d and F o x (56)

iodine d i s s o l v e d in a l k a l i

P e r h a p s t h i s p r e p a r a t i o n co uld be a c i d u l a t e d

of s o m e p r o t e c t i v e

substance

to yield a c r u d e

io d in e h y d r o s o l of l o w c o s t f o r use a s an e n v i r o n m e n t a l d i s ­
infectant o r

sanitizer.

SUMMARY

A l i n e a r d i f f u s i o n m e t h o d em p loy ing a g a r gel s t a t i o n a r y
p h a s e s h a s b e e n d e v e l o p e d f o r the m e a s u r e m e n t of the p e n e t r a ­
b i l i t y of c h e m i c a l d i s i n f e c t a n t s .
arates

This

"penetube"

technic

sep­

the d i s i n f e c t i o n p r o c e s s f r o m the g r o w th of the i n d i c a t o r

organism s

a nd p e r m i t s the d i s t i n c t i o n of b a c t e r i c i d a l and b a c ­

teriostatic

effects.

disinfectants w ere

O b s e r v a t i o n s on the p e n e t r a t i o n of v a r i o u s
m a d e u n d e r d i f f e r e n t c o n d iti o n s by m e a n s of

th is n e w t e c h n i c .
The

r e l a t i o n of the p e n e t r a t i v e p o w e r s of d is in f e c t a n t s

a s d e t e r m i n e d by the
chicken f e c e s

1' p e n e t u b e 11 te c h n ic to the d i s i n f e c t i o n of

was in v estig ated .

A h y p o t h e s i s c o n c e r n i n g the

r o l e of p e n e t r a t i o n in the d i s i n f e c t i o n p r o c e s s

w as f o r m u l a t e d .

T h is h y p o t h e s i s c o n c e r n s the f o r m a t i o n of a d i s s o c i a b l e o r g a n i s m disinfectant com plex as a n e c e s s a r y
the d i s i n f e c t i o n p r o c e s s
reaction m echanism s.

a s a c o m b in a t io n of z e r o and f i r s t o r d e r
The p o s s i b l e

i n f e c ta n t m o l e c u l e s by the o r g a n i s m s
resistance

ste p in the a s s u m p t i o n of

r o l e s of a d s o r p t i o n of d i s ­
and of the d i s t r i b u t i o n of

to d i s i n f e c t i o n am o n g the c o m p o n e n t p o p ulati on a r e

a l s o d i s c u s s e d in r e l a t i o n to the h y p o t h e s i s .

83
The high p e n e t r a b i l i t y p r e v io u s l y c la im e d f o r Colloidal
Iodine wa s shown to be a function of its acid condition r a t h e r
than an i n h e r e n t p r o p e r t y of Colloidal Iodine as such.

BIBLIOGRAPHY

Chick, H., a nd M a r t in , C. J .
The P r i n c i p l e s Involved in
the S t a n d a r d iz a t io n of D is in f e c ta n ts and the Influence
of O r g a n i c M a t t e r upon G e r m i c i d a l Value.
Jour.
Hyg. 8; 654, 1908.
G arrod, L. P .
T e s tin g of D is in fe c ta n ts in the P r e s e n c e
of O r g a n i c M a t t e r .
J o u r . Hyg. 35: 219, 1935.
Salle , A. J . a n d Catlin, B. W.
P r o f i l e E v a lua tion s of
G erm icides.
J o u r . Am. P h a r m . A s s o c . , Sci. Ed.
36: 129, 1947.
Wyss, O.
C h e m ic a l D is in f e c ta n ts .
2: 413, 1948.

Ann. Rev. M icrobi ol .

B r u i n s , H. R.
C oe fficients of Diffusion in L iq u id s.
C r i t . T a b l e s 5: 63, 1929.

Int.

Vincent, M. H.
Sur l a D e sin fe ctio n des M a t i e r e s F e c a l e s
N o r m a l e s e t P a th o lo g iq u e s ; Etude de la Val e ur C om p a r e e d e s D iv e r s D e si n f e c ta n ts C h im iq u e s A c t u e l s .
Ann. l ’I n s t. P a s t . 9: 1, 1895.
M a l l m a n n , W. L ., and C h a n d le r, W. L.
On the D i s i n fe c ­
tio n of A v ian F e c a l M a t e r i a l .
J o u r . Am. Vet. Med.
A s s o c . 36; 190, 1933.
J o h n s , C. K.
A Method f o r A s s e s s i n g the Sanitizing E ff i ­
c i e n c y of Q u a t e r n a r y A m m o n iu m and Hypoc hlorit e
P roducts.
Am . J o u r . Pub. H ea lth 37: 1322, 1947.
Se e lig , M. G., and Gould, G. W.
O s m o s i s as an I m p o r ­
t a n t F a c t o r in the A ction of A n ti s e p ti c s .
Surg.
Gynec. O bs t. 12: 262, 1911.

85
10.

N yiri,

W., a n d J a n n i t t i , M.
About the F a t e of F r e e Iodine
upo n A p p l i c a t i o n to the U n b ro k e n A n i m a l Skin.
An
E x p e r i m e n t a l Study.
J o u r . P h a r m . Exp. T h e r . 4j4:
85, 1932.

11.

K a r n s , G. M.
Th e B e h a v i o r of Iodine Solutions at Li qui d
So lid I n t e r f a c e s .
I. The Wetting P o w e r of Iodine
f r o m V a r i o u s A n t i s e p t i c S o lu tio n s.
J o u r . Am . P h a r m .
A s s o c . 2J_: 779, 1932.

12.

K arns,

13.

B is k i n d , M. S.
P e n e t r a t i o n t h r o u g h T i s s u e of Iodine in
D i f f e r e n t S o lv e n t s .
P r o c . Soc. E x p . Biol, and Med.
30: 35, 1932.

14.

A n d e r s o n , L . P . , and M a ll m a n n , W. L .
The P e n e t r a t i v e
P o w e r s of D i s i n f e c t a n t s .
Mich. A g r . Exp. Sta .,
T e c h . B u i . 183, 1943.

15.

E t c h e l l s , J . L . , a nd F a b i a n , F . W.
T e c h n ic f o r Skin I r ­
ritation T ests:
T e c h n ic f o r D e t e r m i n i n g I r r i t a t i n g
E f f e c t of C h e m i c a l C o m p o u n d s .
J o u r . Ind. Hyg. 17:
298, 1935.

16.

B e i j e r i n c k , M. W.
V e r s l . en Meded. Kon. Akad. v. W e t e ns h.,
Afd. N a t u u r k . , A m s t e r d a m 3de R e e k s , 6; 123, 1889.
c i t e d i n : van I t e r s o n , G. J r . , den D o o r e n de Jong,
L . E . , a n d K l u y v e r , A. J .
M a r t i n u s W illem B e i j e r i n c k .
M a r t i n u s Nijhoff, The Hague, 1940.

17.

K e n d a l l , A. I., a n d E d w a r d s , M. R.
A M ethod f o r D e t e r ­
m i n i n g th e G e r m i c i d a l Value and P e n e t r a t i n g P o w e r
of L i q u i d D i s i n f e c t a n t s .
J o u r . Inf. Dis. 8; 250, 191 1.

18.

C a r n o t , P . , a nd D um ont, J .
T ec h n iqu e d 'E t u d e de l a P e n e ­
t r a t i o n d e s A n tis e p ti q u e s en M ilieux S o lid e s.
C om pt.
Ren d. Soc. B io l., 81: 1199, 1918.

G. M., C r e t c h e r , L . H., and B e a l , G. D.
II. The
I m p o r t a n c e T h e r e o f in the P r e p a r a t i o n of New Iodine
A ntiseptics.
J o u r . A m . P h a r m . A s s o c . 21: 783, 1932.

86
19.

R u e h l e , G. L . A., and B r e w e r , C. L .
U nited S ta te s F ood
a n d D r u g A d m i n i s t r a t i o n M ethod s of T e s tin g A nt i ­
s e p tic s and D isin fe c ta n ts.
USDA C i r c . 198, 1931.

20.

C h a n d l e r , W. L .
On the V e r m i c i d a l Value of Iodine.
A g r . Ex p . Sta. Q u a r . Bui. 7: 1, 1924.

21.

K n a y s i , G.
D i s i n f e c t io n I.
The D e v e lo p m e n t of Knowledge
of D i s i n f e c t io n .
J o u r . Inf. Dis. 4 7 : 293, 1930.

Mich.

22. K n a y s i , G . , a n d G o rd o n , M.
II.
The M a n n e r of D e a t h of
C e r t a i n B a c t e r i a an d Y e a s t s when S u b je c te d to Mild
C h e m i c a l and P h y s i c a l A g e n ts .
J o u r . Inf. D i s. 47:
303, 1930.
23.

K n a y s i , G.
III.
J o u r . Inf.

The Taking up of Iodine by Y e a s t C e l l s .
D is. 4 7 : 318, 1930.

24.

K n a y s i , G.
IV.
Do B a c t e r i a Die L o g a r i t h m i c a l l y ?
Inf. D i s . 47: 322, 1930.

25.

K n a y s i , G.
V.
Som e P r o p e r t i e s of F r e q u e n c y C u r v e s and
T h e i r U s e in S tud ies of D is in f e c tio n .
J o u r . Inf.
D i s . 42: 328, 1930.

Jour.

26.

Ch ic k , H.
An I n v e s t i g a t i o n of the L a w s of D isin fe c t i on .
J o u r . Hyg. j8: 92, 1908.

27.

R a h n , O.
I n j u r y and D e a th of B a c t e r i a by C h e m i c a l A g e n t s .
B i o d y n a m i c a , N o r m a n d y , Mo.
1945.

28.

F ish,

29.

M a l l m a n n , W. L .
P r a c t i c a l A s p e c t s of D is in fe c tio n .
a nd S a n i t. C h e m , 2A (3): 141, 1948.

30.

A m a n n , J . v.
6: 235,

F.
The E f fe c t of P h y s i c a l and C h e m i c a l Age nts on
the O o c y s t s of E i m e r i a t e n e l l a .
Sc ienc e 75: 292,
1931.

U ltram ikroskopie
1910.

d e r Jo d lo s u n g e n .

Soap

K olloid-Z.

87
31.

B o r d i e r , H., a n d Roy, G.
S ur l 'l o d e
R e n d . 163: 567, 1916.

32.

C h a n d l e r , W. L . ,
and M i l l e r , E. J .
P h y s . C h e m . 31: 1091, 1927.

33.

34.

B e n n e t , F . C.
Co lloid al Iodine.
51, 1931.

Colloidal.

Compt.

C o lloid a l Iodine.

Am. Jo u r. P h a rm .

Jour.

10 3:

C h a n d l e r , W. L.., and F e r g u s o n , C. M.
V e r m i c i d a l Iodine
P reparatio n s.
M ich. Sta. Bd. A g r . , Ann. R e pt. for
y e a r ending Jun e 30, 1927, 311.

35.

A l d e r m a n , E . M.
(Chandler).

V e r m i c i d a l Value of C olloidal Iodine
T h e s i s (M.S.) Mich. Sta. Col., 1928.

36.

C h a n d l e r , W. L .
A d m i n i s t r a t i o n of Iodine V e r m ic i d e to
Sheep.
Mich. Sta. Bd. A g r ., Ann. Rept. f o r y e a r
e nding J u n e 30, 1929, 159.

37.

W e i s n e r , E. S.
On the Value of C o llo id a l Iodine in the
C o n t r o l of C o c c i d i o s i s in P o u l t r y and R a b b i t s .
The­
s i s (M.S.) M ich. Sta. Col., 1928.

38.

C h a n d l e r , W. L .
C hickens.
1932.

39.

W i ls o n , F . H.
Stu d ies on the
I n f e s t a t i o n s of H o r s e s .
C o l . , 1939.

40.

S t a f s e t h , H. J .
On the C o n tr o l of T a p e w o r m In f e s t a t i o n in
C h i c k e n s with N o te s on the P a th o lo g y of the I n t e s t i n e s
of the H o s t s .
Mich. A g r . Exp. Sta., T e c h . Bui. 148,
1935.

41.

A n d e r s o n , L.. P .
P e n e t r a t i v e P o w e r s of D i s in f e c ta n t s .
T h e s i s (Ph.D.) Mich. Sta. Col., 1939.

On the C o n tr o l of C e c a l C o c c i d i o s i s in
Mich. A g r . Exp. Sta., T e c h . Bui. 127,

T r e a t m e n t of H a b r o n e m a
T h e s i s (M.S.) Mich. Sta.

88
42.

G e r s h e n f e l d , L.., and Witlin, B.
Iodine a s a n A n t i s e p t i c .
An n . N. Y. Acad. Sci. 53, A r t . 1; 172, 1950.

43.

N yiri,

44.

M iller, E. J.

W., a n d D ubois, L.
T o x i c i t y of C o llo id a l Iodine.
J o u r . Ana. P h a r m . A s s o c . 20; 546, 1931.
P e r s o n a l C om m unication,

1951.

45.

W i l l a r d , H. H., and F u r m a n , N. H.
E l e m e n t a r y Q ua nt i t a t i ve
A nalysis.
3rd Ed.
D. van N o s t r a n d Co., Inc ., New
Y o r k , 1940.

46.

E m m e l , M. W.
B a c t e r i a l F l o r a in F e c e s of the N o r m a l
Fow l.
J o u r . Inf. Dis. 4 6 ; 293, 19 30.

47.

J o h a n s s o n , K. R . , S a r l e s , W. B., and S h a p ir o , S. K.
The
I n t e s t i n a l M i c r o f l o r a of H en s a s In fl u e n c e d by V a r i o u s
C a r b o h y d r a t e s in a B i o t i n - D e f i c i e n t R a tio n.
Jour.
B a c t . 5 6 ; 619, 1948.

48.

M a r k s , H. C . , a nd S t r a n d s k o v , F . B.
H a lo g e n s and T h e i r
Mod e of A ction.
Ann. N. Y. A ca d. Sci. 53, A r t . 1:
163, 1950.

49.

W i l l i a m s , J . W., and Cady, L . C.
M o l e c u l a r D iffus ion in
So l u t i o n .
Che m . Rev. 14; 171, 1934.

50.

Stiles,

W., a n d A d a i r , G. M.
T he P e n e t r a t i o n of E l e c t r o ­
l y t e s in to G e l s .
III. T he In flue nc e of the C o n c e n t r a ­
t i o n of t h e Gel on the C o e f f ic i e n t of Diffusion of
Sodium C hloride.
B i o c h e m . J o u r . 15; 620, 1921.

51.

D avis,

W. W., P a r k e , T. V., D aily, W. A ., Ru shton, L . M.,
a n d M c G u i r e , J . M.
So m e New P r o c e d u r e s a nd In­
s t r u m e n t s U seful fo r M i c r o b i o l o g i c a l Antibioti c
T e s t i n g by Diffusion M e th o d s .
V. A T e s t P r o c e d u r e
E m p l o y i n g L i n e a r D iffusion into I n o c u la t e d A g a r F ille d G lass C ap illaries.
Jour. Am. P h a rm . A ssoc.,
S c i . E d . 39: 331, 1950.

52.

C h i c k , H.
The T h e o r y of D is i n f e c t i o n .
t e r i o l o g y _f* 179, 1930.

A S y s t e m of B a c ­

89
53.

L i n e w e a v e r , H., a n d B u r k , D.
The D e t e r m i n a t i o n of E n ­
zyme D issociation C onstants.
J o u r . A m . C he m . Soc.
56. 658, 1934.

54.

B r u n a u e r , S.
Th e A d s o r p t i o n of G a s e s and V a p o r s .
I. P h y s ­
ical A dsorption.
P rin c e to n U niversity P r e s s , Princeton,
N. J . , 1943.

55.

M i c h a e l i s , L.., a n d M e n te n , M. L,.
Die K ine tik d e r I n v e r t i n wirkung.
B i o c h e m . Z. 4 9 : 333, 1913.

56.

G e r s h e n f e l d , L,., and F o x , D.
The B a c t e r i c i d a l E f f i c i e n c y
of C r u d e I odine in (0.1 N) A lk a li.
Am. Jour. P h a rm .
120: 279, 1948.