A COMPARATIVE STUDY OP THE 
EFFICIENCY OF CERTAIN GERMICIDES IN THE 
PRESERVATION OF BIOLOGICS
By
WILBUR G* MALCOLM'S\
L M A f t V .  IW rT -R S lT Y  0 F  M A R Y L A lto
T h e s is  su b m itte d  t o  t h e  F a c u l ty  o f  th e  G rad u a te  S choo l o f  
th e  U n iv e r s i ty  o f  M ary land  i n  p a r t i a l  f u l f i lm e n t  
o f  th e  re q u ire m e n ts  f o r  th e  d e g re e  o f  
D o c to r o f  P h ilo so p h y *
\
1990
UMI Number: DP70134
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A COMPARATIVE STUDY OF THE 
EFFICIENCY OF CERTAIN GERMICIDES IN THE 
PRESERVATION OF BIOLOGICS*
The m a t t e r  o f  p r e s e r v a t io n  i s  o f  th e  g r e a t e s t  im p o rta n c e  
i n  th e  m a n u fa c tu re  o f  b io lo g ie s  t h a t  a r e  to  be em ployed in  th e  
t r e a tm e n t  o f  human d is e a s e s *  The u se  o f  p h e n o l and t r i k r e s o l  i n  
th e  c a p a c i ty  o f  b io lo g ic  p r e s e r v a t i v e s  i s  a lm o st u n i v e r s a l .  T hese 
c h e m ic a ls , how ever, a r e  n o t e n t i r e l y  s a t i s f a c t o r y .  W ith th e  
c o m p a ra t iv e ly  r e c e n t  developm en t o f  a  number o f  g e rm ic id a l  s u b s ta n c e s ,  
i t  seemed a p p ro p r ia te  to  make a  s tu d y  o f  some o f  them  to  d e te rm in e  
w h e th e r  o r  n o t th e y  m igh t be em ployed t o  adv an tag e*
T here  a r e  f i v e  d i s t i n c t  f e a t u r e s  o f  m a jo r  im p o rta n c e  w hich 
m ust be known in  o r d e r  to  judge th e  p o t e n t i a l i t i e s  o f  a  g e rm ic id e  
a s  a  b io lo g ic  p r e s e r v a t iv e *  T h e re fo re ,  th e  p r e s e r v a t iv e s  w ere 
s tu d ie d  w ith  a  v iew  to  d e te rm in in g  th e  fo l lo w in g :
(1 ) The g e rm ic id a l  p o te n c y  upon s p o re s  and v e g e ta t iv e  
c e l l s  o f  b a c t e r i a  i n  d i f f e r e n t  m ed ia .
(2 ) The e f f e c t  upon th e  im m unizing pow er o f  th e  v a r io u s  
p roduc  t s •
(3 ) T o x ic i ty  o f  th e  g e rm ic id e , w ith  s p e c i a l  r e f e re n c e
t o  th e  m arg in  o f  s a f e t y  in c lu d in g  in tr a v e n o u s ,  in tr a m u s c u la r  
and i n t r a s p l n a l  t o x i c i t y  t e s t s *
(4 ) E f f e c t  upon th e  ap p ea ra n c e  and th e  p r e c i p i t a t i o n  
o f  p r o t e i n  su b s ta n c e s*
(5 ) The h i s t o l o g i c  c h a rg e s  p ro d u ced  in  th e  an im al t i s s u e s *
M ethod o f  P ro c e d u re *
The p rob lem  was d iv id e d  i n to  two d i s t i n c t  p a r t s  w ith  
d i f f e r e n t  m ethods o f  approach* The f i r s t  p o r t io n  was a  s tu d y  
o f  th e  g e rm ic id a l  power o f  th e  v a r io u s  ch em ica l s u b s ta n c e s  t o ­
g e th e r  w ith  t h e  p h y s ic a l  e f f e c t  upon th e  b a s ic  m edia em ployed. 
O nly  s u b s ta n c e s  found  s a t i s f a c t o r y  i n  th e  f i r s t  p h a se  o f  th e  p ro ­
blem  w ere g iv e n  f u r t h e r  study* The second  d i v i s io n  o f  th e  work 
w as a  s tu d y  o f  th e  t o x i c i t y  o f  th e  p r e s e r v a t iv e ;  i t s  e f f e c t  
upon th e  p o te n c y  o f  th e  b i o lo g i e s ;  and th e  h i s t o l o g i c  changes 
p ro d u ce d .
The fo l lo w in g  g e rm ic id e s  w ere ch o sen :
1* P h e n o l ( c r y s t a l s )
2 . H e x y re s o rc in a l  (S .T *3 7 )(1 -1 0 0 0 )
3 . C o l lo id a l  c h lo r in e  e r e s o l  -  25$ s t r e n g th
4* C o l lo id a l  c h lo r in e  thym ol -  25$ s t r e n g th
5* C o l lo id a l  c h lo r in e  c r e s o l  -  50$ s t r e n g th
6 . C o l lo id a l  c h lo r in e  thym ol -  50$ s t r e n g th
7 .  T r ik r e s o l
8* Y a tre n  (pow der)
9# M ercurophen (pow der) -  2$ aqueous s o lu t io n  
1 0 . M etephen (1 -5 0 0 ) 2$ aqueous s o lu t io n
5GERMICIDAL STUDIES*
D e te rm in a tio n  o f  g e rm ic id a l  pow er in  s a l t  
s o l u t i o n ,  b o u i l l o n ,  end serum* to g e th e r  w ith  c o lo r a ­
t i o n  and p r e c i p i t a t i o n  o f  p r o t e i n  s u b s ta n c e s .
A s t e r i l e  p h y s io lo g ic a l  s a l t  s o l u t io n  was used* P l a in  
b o u i l l o n  was em ployed made from  b e e f  in f u s io n  to  w hich 1% p e p to n e  
and .5 ^  sodium  c h lo r id e  h ad  been  added* The t i t r a t i o n  was a d ju s te d  
t o  a  pH. v a lu e  o f  6 .8 .  The medium was checked  f o r  s t e r i l i t y  a f t e r  
h a v in g  b een  a u to c la v e d  f o r  one h a l f  h o u r  a t  15 pounds p r e s s u r e .
H orse  serum  was em ployed , th e  b lo o d  b e in g  ta k e n  a s e p t i e a l l y ,  
d e f i b r i n a t e d  and th e  serum  c a r e f u l l y  checked  f o r  s t e r i l i t y .  I t  
was th e n  p la c e d  in  a  r e f r i g e r a t o r  a t  4 0°F . u n t i l  used*
M ethod o f  Making D i lu t io n s .
Two p ro c e d u re s  w ere em ployed i n  m aking d i l u t i o n s .  I n  th e  
f i r s t  m ethod , X% s ta n d a rd  s o lu t io n s  o f  th e  c h e m ic a ls  w ere m ade, 
u s in g  d i s t i l l e d  w a te r  a s  th e  d i l u e n t .  From th e  o r i g i n a l  1 f0 s o lu ­
t i o n s ,  th e  h ig h e r  d i l u t i o n s  w ere  made by th e  a d d i t i o n  o f  th e  b a s ic  
m ed ia  em ployed in  each  p a r t i c u l a r  study* The t o t a l  amount o f  th e  
ch em ica l and m ed ia  was made up to  5 c .c*  An e x a u p le  i s  a s  fo l lo w s :
O r ig in a l  s t r e n g th  o f  g e rm ic id e  1%.
Amt. o f  c h em ic a l Amt. o f  m edia  P e r  c e n t  d i l u t i o n  T o ta l  c . c .
2 .5  c . c .  2 .5  c . c .  0 .5  5
3 .0  c . c .  2 .0  c . c .  -0*6 5
3 .5  c . c .  1 .5  c . c .  0 .7  5
4 .0  c . c .  1 .0  c . c .  0.8- 5
4 .5  c . c .  0 .5  c . c .  0 .a  5
4The d i l u t i o n s  w ere made i n  s t e r i l e  t e s t  tu b e s  by  means o f  
s t e r i l e  c a l i b r a t e d  p i p e t t e s , m ea su rin g  i n  te n th s *
T h is  m ethod was d is c a rd e d  a f t e r  a  number o f  d e te rm in a t io n s  
had  been  made* T h e re  was n o t  s u f f i c i e n t  o rg a n ic  m a t te r  p r e s e n t  
i n  th e  m ed ia  to  f u r n i s h  n u t r i t i v e  m a te r i a l  f o r  th e  b a c t e r i a  t f i i c h  
had  s u rv iv e d  th e  b a c t e r i o s t a t i c  a c t io n  o f  th e  c h em ic a l su b s ta n c e *
An e x c e s s iv e ly  h ig h  e f f i c i e n c y  was o b ta in e d  f o r  a l l  th e  g e rm ic id e s  
g iv in g  a  f a l s e  i n t e r p r e t a t i o n  o f  th e  r e s u l t s .  The second  m ethod 
o f  d i l u t i o n  in s u re d  a  s u f f i c i e n t  amount o f  b a s ic  m ed ia  f o r  th e  
b a c t e r i a  t o  grow i n  and ap p ro x im ated  more c lo s e ly  th e  c o n d i t io n  
found i n  t h e  b io lo g ie s *
The d i l u t i o n s  w ere made so t h a t  th e  t o t a l  volum e o f  ch em ic a l 
and m edia  was 5 c * c . The g e im ic id e  was added d i r e c t l y  to  th e  m ed ia  
w ith o u t d i l u t i o n  w ith  d i s t i l l e d  w a te r .  I t  may th e r e f o r e  be se e n  
t h a t  th e  m anner i n  w hich th e  d i l u t i o n s  w ere made I n f lu e n c e s  to  a  g r e a t  
e x te n t  th e  b a c t e r i o s t a t i c  a c t io n  o f  th e  g erm ic id e*
O rganism s Em ployed.
F our s p e c ie s  o f  b a c t e r i a  w ere en p lo y ed  in  e ac h  d i lu t i o n  
o f  th e  g e rm ic id e ;  nam ely , B a c i l lu s  c o l i f S ta p h y lo c o c c u s  pyogenes 
a lb u s ,  B a c i l lu s  m y co id es , and B a c i l l u s  s u b t i lu s *  I t  h as  been  
d e m o n s tra te d  t h a t  many g e rm ic id e s  a r e  more e f f e c t i v e  a g a in s t  
some s p e c ie s  o f  b a c t e r i a  th a n  o t h e r s .  O th e r r e a s o n s  f o r  th e  
c h o ic e  o f  th e s e  fo u r  s p e c ie s  w ere  th e  f re q u e n c y  w ith  w hich  th e y  
a re  found  i n  c o n ta m in a te d  b io lo g ie s *  The f i r s t  two s p e c ie s  a r e  
v e g e t a t i v e  c e l l s  o n ly  and th e  l a t t e r  tw o a r e  sp o re  fo rm in g  b a c t e r i a .
6I n o c u la t i o n s  and C o n t r o ls *
One t e n t h  o f  a  c . c .  o f  each  o f  th e  c u l t u r e s  w as added to  
10 c . c .  o f  p l a i n  b r o t h  and  th e  w hole in c u b a te d  f o r  24 h o u rs . A t 
th e  e x p i r a t i o n  o f  t h i s  tim e  th e  c u l t u r e s  w ere f i l t e r e d  t o  remove 
th e  clum ps and to  p ro d u ce  a s  u n ifo rm  a  d i s t r i b u t i o n  o f th e  b a c t e r i a  
a s  p o s s ib l e .  One t e n th  o f  a c . c .  o f  e a c h  o f  th e s e  c u l t u r e s  was 
added  t o  th e  s p e c i f i e d  d i l u t i o n s  o f  th e  g e im ic id e  r e q u i r in g  f o u r  
tu b e s  f o r  e a c h  d i l u t i o n ;  i . e . ,  one d i l u t i o n  f o r  B. c o l i ,  one 
d i l u t i o n  f o r  S ta p h y lo c o c c u s  pyogeneee  a lb u s ,  one d i l u t i o n  f o r  
B. m yco ides and one f o r  B. s u b t i l u s .  A f te r  in o c u la t in g  th e  
v a r io u s  d i l u t i o n s ,  th e y  w ere a llo w e d  to  s ta n d  a t  room te m p e ra tu re  
f o r  24 h o u rs .
D e te rm in in g  th e  G e rm ic id a l A c tio n  o f  th e  C h em ica ls .
The tu b e s  w ere  n e x t exam ined f a r  v i s i b l e  grow th and th e  
r e s u l t s  w ere  r e c o rd e d . Then e a c h  d i l u t i o n  was p l a t e d  s e p a r a t e ly  
on p l a i n  a g a r  m ed ia . One c . c .  o f  e a c h  o f  th e  d i l u t i o n s  was seed ed  
o v er th e  s u r f a c e  o f  p l a t e s  t h a t  had  p r e v io u s ly  been  p o u re d , a llo w e d  
to  h a rd e n ,  and in c u b a te d  f o r  s t e r i l i t y .  The in o c u la te d  p l a t e s  
w ere th e n  p la c e d  in  a  37°C. in c u b a to r  f o r  24 h o u rs  and examined 
f o r  b a c t e r i a l  g ro w th . The p re s e n c e  o f  g ro w th  on th e  p l a t e s  
i s  i n d ic a te d  in  th e  t a b u l a t i o n s  by a p lu s  s ig n  and th e  ab sen ce  o f  
g ro w th  by  a  m inus s ig n .
P h y s ic a l  E f f e c t  o f  th e  G e rm ic id es  upon th e  M edia .
( a ) C a r e f u l  a t t e n t i o n  was p a id  to  th e  c o lo r in g  a c t io n  o f  each  
g e rm ic id e  upon th e  m edia  em ployed, (b ) Change i n  th e  t u r b i d i t y
6o f  th e  s a l t  s o l u t i o n ,  ( c )  P r e c i p i t a t i o n  o f  th e  a lbum inous 
m a te r i a l  o f  th e  b o u l l io n  and serum  a n d , (d ) M i s c i b i l i t y  o f  
th e  g e rm ic id e s  w ith  th e  m ed ia . T hese  f a c t o r s  w ere  c o n s id e re d  
i n  e a c h  o f  th e  d i l u t i o n s  m ade.
S tu d y  o f  G e rm ic id a l A c tio n .
P h e n o l (CgHgQH) • -
P h e n o l , due to  i t s  a n t i s e p t i c  p r o p e r t i e s ,  h a s  been  
em ployed a s  a  p r e s e r v a t i v e  in  b io lo g ic  p ro d u c ts*  C h e m ica lly  
i t  i s  C-H-OH, w ith  th e  s t r u c t u r e
R e s u l ts
C h a rt # 1 .
P r e s e r v a t iv e * B. c o l i S ta p h . B. my- B .sub-- M edia P h y s ic a l
a lb u s c o id e s t i l u s E f f e c t s
1 2 3 4
P h e n o l 1-200 0.5 - - - — S a l t  S o l .  None
t» 1-250 0.4 - - - - tt " tt
a 1-333 0.3 - - - - *• » tt
« 1-500 0.2 - - z X it » it
n 1-1000 0.1 - - X z It w n
w 1-200 0.5 •• - - m B o u illo n P r e c i p i t a t e
* 1-250 0.4 - z 4 X tt n
it 1-333 0.3 x z z z tt it
«» 1-500 0.2 X z X X tt Very l i g h t  p p t
» 1-1000 0.1 z X X X tt tt it n
it 1-200 0.5 - - - - Serum P r e c i p i t a t e
* 1-250 0.4 - z - - n n
» 1-333 0.3 - z X X it ft
it 1-500 0.2 - - X - it L ig h t p p t .
it 1-1000 0.1 z z X X it it tt
7P h e n o l k i l l e d  a l l  f o u r  s p e c ie s  o f  b a c t e r i a  in  s a l t  s o lu t io n  
1 - 3 0  p a r t s ;  b o u i l lo n  1 -200  p a r t s ;  serum  1 -200  p a r ts *
I t  c a u se d  no d i s c o l o r a t i o n  o f  th e  s a l t  s o l u t io n ,  b o u i l l o n ,  o r  
servmu T h e re  was a  p r e c i p i t a t i o n  o f  th e  p r o t e i n  i n  th e  bouillon 
and se rum  a t  th e  d i l u t i o n s  in  w hich  i t  was g e rm ic id a l*  T h is  p r e c i p i ­
t a t e  form ed a lm o st im m e d ia te ly  and in c re a s e d  w ith  a g i t a t io n *  I n  
a  c o m p a ra tiv e ly  s h o r t  tim e i t  s e t t l e d  t o  th e  b o tto m  o f  th e  tu b e  
le a v in g  tb s  m ed ia  p r a c t i c a l l y  c le a r*  I t  was a l s o  found  to  be m is -  
c lb l e  w ith  th e  m edian a t  th e  d i l u t i o n s  employed*
T r ik r e s o l
T h is  i s  a c o a l  t a r  d e r i v a t i v e ,  e x te n s iv e ly  u se d  a s  a  b io lo g ic  
p r e s e r v a t i v e ,  b e c a u se  o f  i t s  a n t i s e p t i c  p r o p e r t ie s *  C h em iea lly  i t  
i s
P r e s e r v a t iv e
H e su its*
C h a r t  # 2 .
B* c o l i  S taph* B* my- B. s u b -  M edia P h y s ic a l
a lb u s c o id e s t i l u s E f f e c t s
s o l .
T r ik r e s o l 1-200 0*5 - - - — S a lV W ater c l e a r no p p t
tt 1-250 0*4 - - - tt ▼ tt it « it
tt 1-333 0.3 - - - - it *» tt n w t*
tt 1-500 0*2 - - - - •t it n tt » it
it 1-1000 0*1 - X X X tt *» tt n tt n
«t 1-200 0*5 w - - - TEPuillon L ig h t p p t .
n 1-250 0*4 - - - - It t» tt
« 1-333 0*3 - - - - tt tt tt
tt 1-500 0*2 - - X X It
#
Very l i g h t  ppt*
it 1-1000 0*1 - X X X None
it 1-200 0*5 - - - - Serum W ater c l e a r no p p t
tt 1-250 0*4 - - - - w tt tt » tt
it 1-333 0.3 - - X - it tt n it tt
tt 1-500 0*2 - X - - tt tt tt it tt
tt 1-1000 0*1 X X X X tt tt tt tt «
8T r ik r e s o l  l e a v e s  th e  s a l t  s o l u t io n  q u i t e  c l e a r .  I t  d id  n o t  
c au se  any d i s c o lo r a t i o n  o f  th e  serum  o r  b o u i l l o n .  T h ere  was a  
p r e c i p i t a t i o n  o f  th e  p r o t e i n  m a te r i a l  in  th e  g e rm ic id a l  d i l u t i o n s  
i n  b o th  th e  b o u i l lo n  and serum . I n  s a l t  s o lu t io n  b a c t e r i a  w ere 
k i l l e d  i n  1 -500  p a r t s ;  b o u i l lo n  1 -333  p a r t s ;  and  serum  1 -250  p a r t s .
C o l lo id a l  c h lo ro - th y m o l -  25%
T h is  i s  a n  am ber-yellow  s o l u t io n ,  w a te r  s o lu b le  w ith  a  
weak thym ol o d o r  and  c o n ta in in g  c h lo ro - th y m o l up to  25$ . Thymol 
i s  a  m e th y l- is o p ro p h y l-p h e n o l  o f th e  fo rm u la e  (1 ) -CgH7 ( 4 ) 0H(3) •
I n  t h i s  p r e p a r a t i o n  by a  s u b s t i t u t i o n  o f th e  h yd rogen  atom  th e  
h a lo g e n  c h l o r i n e ^ s  in tro d u c e d  i n t o  th e  b e n z o l n u c le u s .  G ra p h ic a l ly
i t  i s
I t  was e x p e c te d  t h a t  by  th e  in t r o d u c t io n  o f a  h a lo g e n  i n
th e  b e n z o l n u c le u s  i t  would cau se  a  f u r t h e r  in c r e a s e  i n
(14)
d i s i n f e c t i o n  v a lu e  by a  d e c re a s e  i n  i t s  t o x i c i t y  «
9R e s u l t s .
C h a r t  # 5 .
P r e s e r v a t iv e D i l . B .c o l i  
1
S ta p h . B. my- 
a lb u s  c o i des 
2 3
B. su b -  
t i l u s  
4
M edia P h y s ic a l  e f f e c t s
C o l lo i d a l -
thym ol 25# 1-200 — — — — S a l t  S°1  C loudy , 
w p e a r l
b lu is h - w h i te
1 -250 — — — — w C lo u d y , 
c o lo r
p e a r l - g r e y
1-333 — — — — " " C loudy, 
c o lo r
p e a r l - g r e y
1-500 X X X X n " C loudy , 
c o lo r
p e a r l - g r e y
1-1000 X X X X n " C loudy , 
c o lo r
p e a r l - g r e y
1-200 - - - B o u il lo n  M ilk y -w h ite  c o lo r ,
1-250
1-333
1-500^
1-1000
1-200
1-250
1-333
1-500
1-1000
z
X
X
X
X
X
— c
X
X
X
X
X
X
X
X
X
X
X
f i n e  pp t*  Which 
s t a y s  in  su s p e n s io n . 
n Sane a s  above
" G rey  c o lo r ,  p p t .  i n
su s p e n s io n  
w G rey c o lo r ,  p p t .  i n
su s p e n s io n  
n G rey c o lo r ,  l i g h t  ppt
Serum V ery heavy  c o ag u lan
some h e ld  i n  su sp e n ­
s io n ,  g r e a t e r  s h a re  
s e t t l i n g  r a p i d l y  
n S traw  c o lo r e d ,  heavy
p p t .  t h a t  s e t t l e s  
r a p id ly  
n S e ru #  no rm al c o lo r ,
v e ry  s l i g h t  p p t .  
t h a t  s e t t l e s  
w N e g l ig ib le  p p t .  c o lo r
norm al
" N e g l ig ib le  p p t .  c o lo r
norm al
10
T h is  s u b s ta n c e  was m is c ib le  w ith  th e  m edia*
I t  gave  th e  s a l t  s o l u t i o n  a  c lo u d y  b lu is h - w h i te  a p p e a ra n c e ,
and was e f f e c t i v e  i n  1 -333  p a r t s *  The g e rm ic id e  k i l l e d  i n  1 -800
p a r t s  i n  b o u i l l o n  and se ru n u  I n  b o th  o f  th e  l a t t e r  m ed ia  t h e r e  was
a  v e ry  h eav y  p r e c i p i t a t i o n  to g e th e r  w ith  a  m arked t u r b id i ty #
C o l lo id a l  c h lo ro —c r e s o l  — 25%
T h is  a ls o  i s  an am b er-y e llo w  s o l u t io n ,  w a te r  s o lu b le ,  w ith
a  weak o d o r o f  c r e s o l  and c o n ta in s  c h lo r o - c r e s o l  up to  25^# C re s o l
c h e m ic a lly  i s  CHgC^H^OH# I n  t h i s  p r e p a r a t io n  a s  w ith  c o l l o i d a l
c h lo ro - th y m o l, t h e  h yd rogen  atom  i s  s u b s t i t u t e d  by  th e  h a lo g e n  c h lo r in e #
R e su lts#
C h a rt # 4 .
D il#  B . c o l i  S taph* B* my- B *sub- M edia P h y s ic a l
a lb u s  c o id e s  t i l u s  e f f e c t s
1 2 3 4
Sol*
-  -  -  -  S a l t Z  V ery p a le  p e a r l -
g re y  c o lo r
P r e s e r v a t iv e
C o l lo id a l  
C re s o l  25% 1-200
1-250
1 -333
1-500
1-1000
1-200
1-250
1-333
1-500
1-1000
+
x
n V ery l i g h t  b lu is ]  
t i n t
n M V ery l i g h t  p e a rl*
g re y  c o lo r  
n »* V ery l i g h t  p e a r l -
g re y  c o lo r  
w M Normal c o lo r
B o u il lo n  B lu is h -w h ite
c o lo r  w ith  fcrme 
t i o n  o f  f lo c c u -  
l a n t  pp t*  w hich 
s e t t l e s  r a p id ly  
n L ig h t pB a r l  co la
w ith  p p t .  r e ­
m ain in g  in  s u s ­
p e n s io n  
n No change in
c o lo r ,  f i n e  p p t, 
h e ld  in  su sp en ­
s io n
w No change in
c o lo r*  No pp t#  
w No change in
c o lo r .  No p p t .
11
C h a r t  #4 ( c o n t 'd )
P r e s e r v a t i v e  D il*  B * c o li  S taph*  B* my- B* su b -  M edia P h y s ic a l
a lb u s  c o id e a  t i l u s  e f f e c t s
C o l lo id a l  1 2  3 4
C re s o l  25$ 1 -200  -  -  -  -  Serum H eavy pp t*  o f
serum  w hich i s  
h e ld  in  s u s ­
p e n s io n
1-250  -  -  -  -  n L ig h t  s tra w
c o lo r ,  pp t*  
w ith  r a p i d  
s e t t l i n g
1 —333  — x  — — n Serum norm al
c o lo r ,  pp t*  in  
b o tto m  o f  tu b e
1 -500
1-1000
V ery  s l i g h t  p p t  
no rm al c o lo r
Normal c o lo r ,  
n e g a t iv e  pp t*
C o l lo id a l  c r e o l  (25$) i n  th e  s a l t  s o l u t io n  was e f f e c t i v e  
i n  1 -500  p a r t s  g iv in g  a  v e ry  p e le  p e a r l - g r e y  c o lo r  to  th e  s o lu ­
t io n *  I n  th e  b o u i l l o n  and  serum  th e r e  was a  heavy  p r e c i p i t a t i o n  
o f  th e  p r o te in o u s  m a t e r i a l ,  w ith  a  r a p id  s e t t l i n g  o f  th e  p r e c i p i ­
t a t e .  The g e rm ic id e  was e f f e c t i v e  i n  b o u l l io n  in  1 -333  p a r t s  
and serum  in  1 -250  p a r t s .
C o l lo id a l  c r e s o l  -  50$
C o l lo id a l  thym ol -  50$
T h is  s t r e n g th  o f  th e  above s u b s ta n c e s  was recommended by 
th e  m a n u fa c tu re r  a f t e r  th e  f a i l u r e  o f  th e  25$ s o lu t io n  t o  p ro v e
12
s a t i s f a c t o r y  i n  t h i s  w ork dus t o  th e  h eav y  p r e c i p i t a t i o n  o f  th e  
a lbum inous m a te r ia l*  D i lu t io n s  w ere made i n  s a l t  s o l u t i o n ,  b o u i l l o n ,  
and serum  from  1% to  0.1% , i n  b o th  th e  c o l l o i d a l  c r e s o l  and c o l l o i d a l  
thym ol* A lth o u g h  t h e r e  w as an  i n c r e a s e  i n  th e  g e rm ic id a l  a c t i o n  o v e r  
th e  25% c o l l o i d a l  c h lo r o - c r e s o l  and th y m o l, th e r e  was a l s o  in c r e a s e d  
p r e c i p i t a t i o n *
E th e r  a s  a  c a r r y in g  a g e n t  o f  p r e s e r v a t i v e s  i n  b io lo g ie s  i s  q u i t e  
e f f e c t i v e  f o r  p h e n o l and so  was t e s t e d  i n  th e  above c o l l o i d s  i n  b o th  
th e  25% and th e  50% s t r e n g th s  w ith  th e  fo l lo w in g  r e s u l t s :
C h a rt #5*
D i lu t io n  i n  % P r e c i p i t a t i o n  o f
B o u il lo n  Serum
C h lo r in e  thym ol (25%) 0*9 z
x
X
X
X
X
X
X
X
X
X
0*8
0*7
0*6
0*5
0*4
0*3
0*2
0.1
s l i g h t
s l i g h t
s l i g h t
C h lo r in e  c r e s o l  (25%) 0 .9 x
X
X
X
X
X
X
X
X
X
X
0*8
0 .7
0.6
0 .5
0 .4
0 .3
0.2
0.1
s l i g h t
s l i g h t s l i g h t
- 13-
The r e s u l t s  i n  t h e  50$ c o n c e n tr a t io n  w ere  th e  same a s  th o s e  
i n  th e  2 5 $  e x c e p t  i n  t h e  c a s e  o f th e  c h lo r in e  thym ol* H ere  th e  
b o u i l lo n  was p r e c i p i t a t e d  q u i te  h e a v i ly  i n  0*4$ and s l i g h t  i n  
0*2$  w ith  a  p r e c i p i t a t i o n  o f  th e  seSrum i n  0 .1 $ .
The c h lo r in e  c r e s o l  p r e c i p i t a t e d  s l i g h t l y  i n  0*3$ b o u i l lo n  
and 0 .3 $  serum . From t h e  r e s u l t s  o b ta in e d  in  th e  p r e c i p i t a t i o n  
o f  t h e  p ro te in o u s  m a t e r i a l s ,  th e  c o l l o i d a l  c r e s o l  and c o l l o i d a l  
thym ol i n  b o th  th e  25 and 50$ s t r e n g th s ;  e th e r  would n o t  p re v e n t  
p r e c i p i t a t i o n  to  such  an e x te n t  a s  to  make t h e i r  employm ent 
p r a c t i c a b le *
Y a tre n
T h is  i s  a  German p r e p a r a t i o n .  I t  i s  a  brown powder 
w hich  h a s  a  maximum s o l u b i l i t y  i n  w ater o f  4 $ . I t  was n o t  g e rm ic id a l  
i n  1 -5 0  p a r t s .  The m edia  v/ere c o lo re d  f r a n  a  deep brown to  a  
l i g h t  ember dep en d in g  upon t h e  d i l u t i o n .  T here  was no p r e c i p i t a t i o n  
o f  t h e  p r o te in  m a t e r i a l s  in  a  4$ s o l u t io n .  The c h em ic a l s t r u c t u r e  
o f  t h i s  p ro d u c t  w as n o t l e a r n e d .
14
P r e s e r v a t i v e
Y a tre n
R e su lts *  
C h a rt #6 .
D i l .  B* c o l l Staph* B. my- B* sub
a lb u s c o id e s t i l u s
1 2 3 4
1-50 x X X X
1-52 X X X X
1-55 X X X X
1-58 X X X X
1 -6 2 X X X X
1-66 X X X X
1 -7 1 X X X X
1 -76 X X X X
1 -83 X X X X
1-90 X X X X
1-100 X X X X
1-111 X X X X
1-125 X X X X
1-143 X X X X
1-170 X X X X
1-200 X X X X
1-250 X X X X
1-333 X X X X
1-500 X X X X
1-1000 X X X X
P h y s ic a l  e f f e c t s
S a l t ,s o l*  f G ives th e  m edia  
b o u i l lo n *  a c o lo r  r a a g in g  
and serum  from  a  deep
brown to  a  l i g h  
am ber, th e  i n ­
t e n s i t y  d e c r e a s ­
in g  w ith  th e  
d i lu t io n *
15
H e x y l r e s o r e in a l  (S*T*37) (1 -1 0 0 0 )
A w a te r  c l e a r  s u b s ta n c e  p re p a re d  in  a  1-1000 s o lu t io n *  I t  
was q u i t e  m is c ib le  w ith  th e  m edia and h a s  th e  fo rm u la e  C^HgfOHjgtCgH^g) 
T h is  w as n o t  fe e rm ic id a l i n  1 -5 0  p a r t s  w ith  any  o f  th e  b a s ic  m edia*
I t  c a u se d  n o  d i s c o l o r a t i o n  and no p r e c i p i t a t i o n  o f  th e  b o u i l lo n  and 
serum*
R e su lts *
C h a rt # 7 .
O )
P r e s e r v a t iv e
H e x y l r e s o r e in a l
D il*  B. c o l i S ta p h . 3* my- B*
a  lb  us c o id e s t i l l
1 2 3 4
1-50 x X Z z
1-52 x X X z
1 -55 z X z z
1 -58 z z z z
1 -62 z z z z
1-66 z z z z
1-71 X z z z
1-76 z z X X
1 -83 z X X X
1 -90 z X X X
1-100 z z X X
1-111 X z X z
1-125 z z X z
1-143 X z X X
1-170 X X X X
1-200 z X X X
1-250 z X X X
1-333 X X X X
1-500 X X X X
1-1000 z X X X
>- M edia P h y s ic a l  e f f e c t s
S a l t , 3<»1* * 
b o u i l l o n , 
and serum
No change in  
any  o f  th e  
t h r e e  m edia  
i n  c o lo r .  No 
p r e c i p i t a t i o n .
As o u t l in e d  in  th e  s a l t  s o l u t io n s ,  th e r e  was g ro w th  in  a l l  
d i l u t i o n s  o f th e  b o u l l io n  and serum* No change was n o te d  in  th e  
p h y s i c a l  a p p ea ra n c e  o f  t h e  b o u i l lo n  o r  se rum , w ith  no p r e c i p i t a t i o n *
16
M etaphen d i lu t e d  i n  a  3% aqueous NaOH s o l u t io n .
I t  i s  an o rg a n ic  m e r c u r ia l .  C h e m ica lly  i t  i s
( 2 )
k n o m  a s  4—n i t r o - 3 ,  5 b isa c e to x y m ^ iv ju r i- 2 - c r e s o l  • I t s
s t r u c t u r a l  fo rm u la e  i s :  H_CCOOHgo
^ryuriL **w*Gr8sol
0 ; : ^ ,
A/0.
The s o l u t io n  i s  amber c o lo re d  i n  a  3% c o n c e n t r a t io n .  I t  m ixed 
r e a d i l y  w ith  th e  m edia em ployed. I t  was n e c e s s a ry  t o  make an  
i n i t i a l  d i l u t i o n  o f  1-1000 u s in g  d i s t i l l e d  w a te r  b e fo re  m aking 
th e  d e s i r e d  d i l u t i o n s  w i th  th e  m ed ia .
P r e s e r v a t iv e  D i l .
M etaphen Zf> 1 -12500 
1-10000 
1-8333  
1-7142  
1-6250  
1-12500 
1-10000 
1-8333  
1-7142  
1-6250
R e s u l t s .
C h a rt #8
B .c o l i  S ta p h . B.my- B. su b -  M edia 
a lb  us c o id e s  t i l u s  
3 4
P h y s ic a l  e f f e c t s
1
x
2
x S a l t
s o l .
X
X
V ery l i g h t  a n h e r ;  
no p p t .  
n V ery l i g h t  am ber;
no p p t .  
w V ery l i g h t  a n b e r ;
no p p t .  
w V ery l i g h t  am ber;
no p p t .  
w V ery l i g h t  a n b e r ;
no p p t .
B o u illo n  L ig h t am ber; no ppi
it n
»
17
C h a rt #8 ( e o n t 'd )
P r e s e r v a t i  ve  D i l .
M etaphen 2$6 1-12500
1-10000 
1-8333  
1-7142  
1-6250 
1-5555  
1-5000
B• c o l i  S ta p h . B .m y- B. su b -  M edia P h y s ic a l  e f f e c t s  
a lb u s  c o id e s  t i l u s  
2 31
z
X
X
X
X
X
4
X Serum L ig h t am ber;
no p p t .
* L ig h t  amba*;
no p p t .
n L ig h t am ber;
no pp t#
** L ig h t am ber;
no pp t#
w L ig h t am ber;
no p p t .
" L ig h t am bar;
no p p t#
* L ig h t am ber;
no pp t#
M etaphen gave th e  m ed ia  a  v e ry  l i g h t  amber c o lo re d  a p p e a ra n c e , 
b u t  one t h a t  was n o t e a s i l y  d i s c e r n i b l e .  W ith a  c o n c e n tr a t io n  a s  
h ig h  a s  1 -200  p a r t s  t h e r e  was no p r e c i p i t a t i o n  o f  t h e  p r o t e i n  sub­
s ta n c e s  i n  e i t h e r  th e  b o u i l l o n  o r  serum . I t  was g e rm ic id a l  i n  d i l u ­
t i o n s  o f  1-10000 p a r t s  o f  s a l t  s o l u t io n ;  1 -8333  p a r t s  b o u i l lo n ;  and 
1-5000  p a r t s  serum#
As a  f u r t h e r  s tu d y  o f  th e  g e rm ic id a l  a c t io n  o f  t h i s  s u b s ta n c e  a 
number o f  b o t t l e s  w ere  p r e p a r e d ,  seme c o n ta in in g  serum , and some con­
t a i n i n g  b o u i l lo n .  T hese w ere p re s e rv e d  w i th  1-8000 p a r t s  b o u i l lo n  and 
1-5000  p a r t s  serum . Each was in o c u la te d  w ith  a b o u t o n e -h a l f  a gram o f  
d u s t  p a r t i c l e s .  T hese c u l t u r e s  showed no g row th  when p la c e d  i n  d i r e c t  
s u n l ig h t  f o r  s e v e r a l  w eek s , a f t e r  t h i s  tim e  th e r e  ap p ea red  in  th e
16
serum  a  grow th o f  a i r  ty p e  b a c t e r i a .  A t th e  end o f  6 weeks th e  
b o u i l l o n  showed th e  p re s e n c e  o f  m olds and a i r  ty p e s  o f  b a c t e r i a .
One hund red  c . c .  o f  a  1-8000 d i l u t i o n  o f t e t a n u s  to x in  was p la c e d  
i n  e ac h  o f  f i v e  b o t t l e s .  A lso f i v e  b o t t l e s  c o n ta in in g  a  s i m i l a r  
am ount o f  a  1 -8000  d i l u t i o n  o f  d i p h th e r i a  to x in  w ere in o c u la te d  
w ith  d u s t  and in c u b a te d  f o r  two weeks a t  37°C . A t th e  end o f  t h i s  
tim e  th e y  w ere  ex an in ed  and a l l  showed no g ro w th . From t h e s e ,  f i v e  
sam p les  w ere p la c e d  in  t h e  s u n l ig h t  and f i v e  i n  th e  d a rk . At th e  
end  o f  a  m onth a l l  f i v e  sam ples k e p t from th e  sun  r a y s  w ere s t e r i l e  
w h ile  th r e e  o f th e  f i v e  s a n p le s  exposed  to  th e  a c t io n  o f  s u n l ig h t  
showed g row th . W hile no d e f i n i t e  c o n c lu s io n s  a r e  draw n a s  to  th e  
e f f e c t  o f s u n l i g h t  in  c a u s in g  a  l o s s  o f  th e  g e rm ic id a l  p r o p e r t i e s  
o f  m etap h en , i t  does seem to  have some a p p a re n t d e s t r u c t i v e  a c t io n .
M ercurophen  2# aqueous s o lu t io n .
T h is  i s  an o r g a n ic  m e r c u r ia l .  C h em ica lly  i t  i s  sodium - 
h y d ro x y m e rc u r i -n i tro -p h e n o la te .  The 2% aqueous s o lu t io n  i s  q u i te  
o r o d le s s .  I t  h a s  a b r ic k  re d  c o lo r  b u t  i n  h ig h e r  d i l u t i o n s  i s  amber 
c o lo r e d .  In  p r e p a r a t io n  f o r  th e  g e rm ic id a l  t e s t s ,  due to  i t s  
a b i l i t y  to  k i l l  b a c t e r i a  i n  h ig h  d i l u t i o n s ,  a  p r e l im in a ry  d i l u t i o n  
o f  1 -1000  p a r t s  in  d i s t i l l e d  w a te r  was made b e fo re  c o m p le tin g  th e  
f i n a l  d i l u t i o n s  by  th e  a d d i t i o n  o f  th e  b a s ic  m ed ia.(?^  S t r u c t u r a l l y
i t  i s
R e s u l t s * 
C h a rt $ 9 .
P r e s e r v a t iv e  D i l .
M ercurophen 1—50000 
1-25000 
1-16666 
1-12500  
1-10000 
1-8533
1-25000
1-16666
1-12500
1-10000
1 -8 3 3 3
1-10000
1-8333
1-7142
1 -6 2 5 0
1 -5555
B. c o l i  S ta p h . B. my- B. su b -  M edia P h y s ic a l  e f f e c t s  
a lb  us c o id e s  t i l u s  
1 2  3 4 S o l .
x  — x  — S a l t /  No p r e c i p i t a t i o n
x -  -  -  n , r *in any o f  th e  d i l u
-  -  -  -  n w t i o n s ;  c o lo r  o f
-  -  -  -  tt " th e  s u s p e n s io n
-  -  -  -  n ft changes from
-  -  -  -  w m d a rk  to  a l i g h t
am ber, d e c re a s in g  
w ith  th e  d i l u t i o n .
x  -  x  -  B o u i l lo n  Amber c o lo re d ;
-  -  x -  " same p h y s ic a l
-  -  -  -  M e f f e c t s  a s
-  -  -  — 19 d e s c r ib e d  ab o v e .
Serum No p r e c i p i t a t i o n .
* Same c o lo r  a s
n o b se rv ed  in  th e
* ab o v e .
20
T h is  s u b s ta n c e  was m is c ib le  w ith  th e  m ed ia , and was g e n n ie id a l  
i n  s a l t  s o lu t io n  i n  d i l u t i o n s  o f  1 -16500  p a r t s ;  b o u i l lo n  1-12500 
p a r t s ;  and serum  1-8500  p a r t s .  I t  im p a r ts  a  c o lo r  o f  an  amber hue 
n o t  u n l ik e  t h a t  o f  norm al serum  and b o u i l lo n ,  b u t  a  t r i f l e  more 
i n t e n s e .  T here  was no p r e c i p i t a t i o n  o f  p r o t e i n s  i n  serum  i n  d i l u t i o n s  
a s  c o n c e n tr a te d  a s  1 -200  p a r t s .
A s e r i e s  o f  p r e p a r a t i o n s  w ere made a s  i n  m etaphen  u n d e r i d e n t i c a l  
c o n d i t io n s ,  w ith  exposure  to  sun  l i g h t  to  a s c e r t a i n  i f  i t  c au sed  
a  l o s s  o f  g e n n ic id a l  pow er. B o u illo n  was p re s e rv e d  w ith  1-12000 
p a r t s ;  m ercu rophen  and serum  1-8000 p a r t s .  A f te r  a f o u r  m onths 
e x p o su re  a t  room t e n p e r a tu r e  th e  serum  and b o u l l io n  w ere found  to  be  
s t e r i l e .  A f te r  i n o c u la t i n g  f i v e  sam ples o f  te ta n u s  t o x in ,  and f i v e  
sam p les o f  d i p h th e r i a  a n t i t o x i n  each c o n ta in in g  1 -10000  p a r t s  m ercu ro ­
phen  and in c u b a t in g  a t  37°C . f o r  th r e e  w eeks, a l l  were found  to  be 
s t e r i l e .  T hese  w ere th e n  d iv id e d  in to  two l o t s ,  one exposed to  th e  
a i r  and sun  l i g h t ,  and th e  o th e r  exposed  to  th e  a i r  b u t  p r o t e c te d  
fv <vn su n  l i g h t .  A t th e  end o f  a  m onth b o th  l o t s  were s t e r i l e .  I t  
was assum ed from  t h i s  e x p e rim e n t t h a t  l i g h t  d oes n o t  a f f e c t  th e  
g e rm ic id a l  power o f  m ercu ro p h en .
D is c u s s io n .
The g e rm ic id a l  pow er r e f e r s  to  th e  d e s t r u c t io n  o f  a l l  th e  
s p e c ie s  o f  b a c t e r i a  em ployed i n  th e  e x p e rim e n ts .
Y a tre n  g iv e s  th e  m ed ia  a  c o lo r  r a n g in g  from  a  deep brown to  a  
l i g h t  am ber, th e  i n t e n s i t y  d e c re a s in g  w ith  th e  in c re a s e d  d i l u t i o n .
21
Their© i s  no p r e c i p i t a t i o n  o f  m ed ia , b u t  th e  g e rm ic id a l  a c t io n  i s  
n o t  e f f i c i e n t  a t  1—50 p a r t s ,  so  th e  volum e o f  th e  ch em ic a l needed  
f o r  th e  l u l l i n g  o f  th e  b a c t e r i a  would be to o  g re a t*
H e x y l r e s o r e in a l  (1 -1 0 0 0 ) was n o t  e f f e c t i v e  i n  1 -5 0  p a r t s  
and a l th o u g h  i t  i s  n o n - p r e c i p i t a t i n g ,  th e  q u a n t i t y  o f  th e  g e m ic id e  
em ployed i n d ic a t e d  t h a t  th e  k i l l i n g  pow er was n o t  g r e a t  enough 
to  u se  i n  th e  c a p a c i ty  o f  a  b io lo g ic  p r e s e r v a t iv e *  H e x y l r e s o r e in a l  
i s  c o lo r l e s s  and c a u se d  no change i n  th e  p h y s ic a l  c o n d i t io n  o f  any 
o f  th e  p r o d u c ts .
C o l lo id a l  c h lo ro - th y m o l, 25%, was g e n n ic id a l  in  s a l t  s o lu t io n  
a t  1—333 p a r t s ,  b o u i l lo n  1-200  p a r t s ,  and serum  1 -200  p a r ts *  I t  
had  such  a  m arked p r e c i p i t a t i n g  a c t io n  upon th e  p r o t e i n  s u b s ta n c e s  
t h a t  i t  sh o u ld  n o t be  employed*
C o l lo id a l  c h l o r o - c r e s o l , 25%, d e s tro y e d  b a c t e r i a  i n  1 -500  
p a r t s  s a l t  s o l u t io n ,  1 -333  p a r t s  b o u i l lo n  and 1 -250  p a r t s  serum* 
Heavy p r e c i p i t a t i o n  o f  th e  b o u i l lo n  and serum s made i t s  u se  p ro ­
h i b i t i v e .  A s tu d y  o f  th e  50% c h lo ro - th y m o l and 50% c h lo r o - c r e s o l  
p ro v ed  them to  be p o w e rfu l g e rm ic id a l  s u b s ta n c e s ,  m is c ib le  w ith  
th e  m edia  b u t  p r e c i p i t a t e d  so  h e a v i ly  t h a t  th e y  c o u ld  n o t  be u sed  
in  th e  c a p a c i ty  o f  b io lo g ic  p r e s e r v a t i v e s .
P h e n o l i s  g e rm ic id a l  i n  a  0 *J% s a l t  s o l u t io n ,  0*5% b o u l l io n  
and 0*5% serum . T here  was no d i s c o lo r a t i o n  o f  th e  s a l t  s o lu t io n *  
The b o u i l lo n  and serum  showed a  p r e c i p i t a t i o n  in  a  c o n c e n tr a t io n  
o f  1 -200  p a r t s .
22
T r ik r e s o l  had  a  g e r m ic id a l  p o te n c y  in  s a l t  s o l u t io n  o f  1 -500  
p a r t s ,  b o u i l lo n  1—333 p a r t s ,  and serum  1—250 p a r t s *  T h ere  was no 
d i s c o lo r a t i o n  o f  th e  m edia  w i th  seme p r e c i p i t a t i o n  o f  th e  p r o t e i n  
su b s ta n c e s*
M etaphen was g e n n ic id a l  in  s a l t  s o l u t io n  in  1-10000  p a r t s ,  b o u i l lo n  
1 -8 3 3 3 , and  serum  1-5000* T h is  s u b s ta n c e  gave  s a l t  s o l u t io n ,  b o u i l lo n  
and  serum  a  l i g h t  am ber c o lo r*  T here  was no p r e c i p i t a t i o n  o f  p r o t e i n  
s u b s ta n c e s  i n  d i l u t i o n s  o f  1 -200  p a r t s .  D ir e c t  e x p o su re  t o  sun  l i g h t  
seemed to  l e s s e n  th e  g e rm ic id a l  pow er o f  th e  s u b s ta n c e .
M ercurophen  k i l l e d  b a c t e r i a  i n  s a l t  s o lu t io n  i n  1-16500 p a r t s ,  
b o u i l l o n  1-12500 p a r t s ,  and  serum  1^8500 p a r t s .  I t  was n o n - p r o te in  
p r e c i p i t o u s  i n  d i l u t i o n s  o f  1 -200  p a r t s .  T h is  su b s ta n c e  gave th e  
m edia  a  l i g h t  am b er-red  c o lo r  b u t  was n o t o b je c t io n a b le .
The g e n n ic id a l  pow er o f  each  su b s ta n c e  d e c re a se d  w ith  an in c r e a s in g  
amount o f  o rg a n ic  m a t e r i a l  p r e s e n t  in  th e  m edia  em ployed. T here  was 
a l s o  e v id e n c e d  a  s e l e c t i v e  a c t io n  o f  th e  ch em ic a l s u b s ta n c e s ,  th e  
o rg a n ic  m e r c u r ia ls  b e in g  p a r t i c u l a r l y  e f f e c t i v e  a g a in s t  S ta p h y lo c o cc u s  
pyogenes a lb u s .  T here  w ere c e r t a i n  d i s c r e p a n c ie s  m a n ife s te d  i n  t h i s  
s e r i e s  o f  e x p erim e n ts*  How ever, th e  t e s t s  were re p e a te d  a  num ber o f  tim e s  
and th e  g e rm ic id a l  and p h y s ic a l  e f f e c t s  w ere found  to  be a s  s t a t e d .
Of th e  c h e m ic a ls  s tu d ie d  th o s e  b e s t  s u i t e d  f o r  b io lo g ic  p r e s e r v a ­
t i v e s  from  th e  s ta n d p o in t  o f  th e  g e rm ic id a l  e f f i c i e n c y ,  th e  p h y s ic a l  
e f f e c t ,  and th e  a p p ea ra n c e  o f  th e  p ro d u c t w ere ; nam ely , m ercu ro p h en , 
m etaphen , t r i k r e s o l ,  and p h e n o l. T hese f o u r  w ere  f u r t h e r  s tu d ie d *
23
TOXICITY TESTS*
T h is  p h a se  o f  t h e  iro rk  was u n d e rta k e n  c h i e f l y  t o  d e te rm in e
th e  t o x i c i t y  o f  m etaphen  and m ercu rophen  b u t in c lu d e d  in  p a r t ,
th e  t o x i c i t y  o f  p h e n o l and t r i k r e s o l *  The to f l t ic i ty  o f  th e s e
( 4 ) ( 5 )
s u b s ta n c e s  * h a s  b e e n  d e te rm in e d  by  Leake , H a le , and a  number 
o f  o t h e r  w o rk e rs  w ith  com parab le  r e s u l t s ,
Leake i n  1917 u s in g  m ice a s  e x p e r im e n ta l  a n im a ls  u n d e rto o k  
san e  e x p e r im e n ts  t o  d e te rm in e  th e  t o x i c i t y  o f  c e r t a i n  d i s i n f e c t a n t s  
u sed  in  b io lo g ic  p ro d u c ts *  H is  r e s u l t s  in d ic a te d  t h a t  assum ing  
th e  M*L*D* t o  be th e  maount n e c e s s a ry  to  k i l l  80 p e r  c e n t  o f  
th e  m ioe  i n  a  g iv en  d o s e ,  p h e n o l h a s  an  M*L*D* o f  *00037 gms. 
p e r  ©n. w e ig h t o f  m ouse. T r ik r e s o l  h a s  th e  same M. L.D . a s  found 
f o r  pheno l*  The t o x i c i t y  o f  t r i k r e s o l  and  p h e n o l was n o t 
l e s s e n e d  when d i l u t e d  w ith  n o m a l  h o rs e  serum* T hese  r e s u l t s  
a g re e  w i th  t h e  f i n d in g s  o f  H ale*
M ethods*
The e x p e r im e n ta l  a n im a ls  em ployed in  t h i s  p a r t  o f  th e  p rob lem  
w ere r a b b i t s  and a lb in o  r a t s .  Two r o u te s  o f  i n j e c t i o n  w ere 
fo l lo w e d , one in tr a v e n o u s  and th e  o th e r  in tra m u sc u la r*
The p h e n o l was p re p a re d  by wei $ i t  and d i l u t e d  to  th e  r e q u i r e d  
volume w ith  d o u b le  d i s t i l l e d  H^O* T r ik r e s o l  was in  th e  l l q i i d  
s t a t e  and d i lu t e d  b y  volum e as w ere a l s o  m ercurophen and  m e tq jh en . 
The q u a n t i ty  o f  each  g e rm ic id e  t o  b e  i n je c t e d  i n t o  each  an im al
24
w as fo u n d  by m u l t ip ly in g  th e  w e ig h t o f  th e  r a t  o r  r a b b i t  by 
t h e  d o se  i n  g ram s o f  th e  g e rm ic id e  and  d iv id in g  th e  t o t a l  by  
th e  gram a o f  g e rm ic id e  p e r  c .c *  o f  s o lu t io n *  I n  t h e  r a t ,  
in t r a v e n o u s  i n j e c t i o n s  w ere made i n  th e  saphenous ve in*  
I n t r a m u s c u la r  i n j e c t i o n s  w ere  made i n  th e  f l e s h y  p o r t i o n  o f  th e  
h in d  le g *  W ith  r a b b i t s  in t r a m u s c u la r  i n j e c t i o n s  w ere made in  
t h e  h in d  le g  and in tr a v e n o u s  i n j e c t i o n s  were made in  th e  m a rg in a l 
e a r  v e in s#  The r a t s  u se d  w ere b e tw een  100 and  150 gms* i n  
w e ig h t, th e  w e ig h t b e in g  ta k e n  b e fo r e  a  heavy  fee d in g *  R a b b its  
w ere not l e s s  th a n  !§■ k ilo g ra m s  in  w e ig h t and w ere  a p p a r e n t ly  
i n  e x c e l l e n t  p h y s ic a l  c o n d itio n *  The am ounts I n j e c te d  w ere  
m easu red  i n  L uer s y r in g e s ,  a  2 c .c *  g ra d u a te d  in  1 /1 0  c«c# and 
a  1 c . c .  g ra d u a te d  in  1 /1 0 0  c .c #  The q u a n t i t y  to  be  i n j e c t e d  
g o v ern ed  th e  ch o ic e  o f  th e  s y r in g e  b e in g  used*
A nim als s u rv iv in g  14 d ay s a f t e r  th e  tim e  o f  i n j e c t i o n  w ere  
d is c h a rg e d  end c o n s id e re d  a s  h a v in g  s u rv iv e d  th e  d o sa g e .
D i lu t io n s  o f  G erm ic ides*
I n  o r d e r  to  t e s t  th e  e f f e c t  o f  d i l u t i o n  upon th e  t o x i c i t y  
o f  th e  g e rm ic id a l  s u b s ta n c e s ,  m ercu rophen  and m etaphen  w ere 
a d m in is te r e d  in t r a v e n o u s ly  i n  r a t s  in  0#1  and 0#5% s o l u t i o n s ,  
and in t r a m u s c u la r ly  in  1 and 2% s o lu t io n s *  A l l  i n j e c t i o n s  
i n  r a b b i t s  b o th  in tr a v e n o u s ly  and in t r a m u s c u la r ly  w ere made in  
2% s o lu t io n s #
Z5
TOXICITY TEST PROTOCOLS
The fo l lo w in g  d e t a i l e d  p r o to c o l s  r e p r e s e n t  th e  r e s u l t s  
o f t h e  t o x i c i t y  t e s t s  o f  m etaphen  and  m ercurophen* The r o u te  o f 
i n j e c t i o n ;  t e s t  an im al em ployed; w e ig h t o f  a n im a l;  mgs* d o sa g e ; 
p e r c e n t  s u r v i v a l ;  and  t h e  Maximum T o le r a te d  L ose (M .T.L*) f o r  
t h e  d i l u t i o n s  o f  e ac h  m e r c u r ia l  s tu d ie d  a r e  g iv e n  i n  t a b u l a r  
form*
R at
101
102
103
104
105
106
107
108
109
110 
111 
112
113
114
115
116
117
118
119
120
26
Metaphen -  intramuscular
1# so lu tion
T est Animal -  Albino Rat
No. W eight
gms.
150
130
122
115
155
120
97
118
125
147
132
158
135
155
152
146
157
112
135
154
Mg. dosage
1 6 .6
16*6
1 6 .6
1 6 .6
1 6 .6
20.0
20.0
20.0
20.0
20.0
2 4 .0
2 4 .0
2 4 .0
2 4 .0
2 4 .0  
2 8 .0  
2 8 .0  
2 8 .0  
2 8 .0  
2 8 .0
R e su lt
S u rv iv e d
w
tt
it
tt
it
«i
it
tt
It
£ e th a l
S u rv iv e d
L e th a l
n
•t
it
n
»♦
Time
D isch arg ed
tt
it
t*
w
it
it
tt
it
it
n
w
9 d ay s 
D isch a rg ed  
6 days 
1  day  
5 d ay s 
3 days 
3 days 
3 days
Mgs. d osage  P e r c e n t  s u r v iv a l
1 6 .6  100#
20.0 100 #
2 4 .0  60#
2 8 .0  0
M.T.D.— 24
27
M etaphen -  in tr a m u s c u la r
No* o f  R at
4
4
4
4
M .T.D *- 20
2$  s o lu t io n  
T e s t  a n im a l -  A lb in o  r a t
Mgs* Dosage P e rc e n t
14*0 IQOfo
1 6 .6  100#
2 0 .0  75°/o
24*0 0
s u r v iv a l
28
Metaphen -  intravenous
0*1# so lu tio n
Test animal -  Albino rat
B at No. W eight
g n s .
Mg. dosage R e s u l ts Time
140 123 4 S u rv iv e d Discheu
141 108 4 i« w
142 105 4 n tt
143 127 4 tt tt
144 108 4 « «
145 107 6 it n
146 119 6 L e th a l 8  d ay s
147 120 6 S u rv iv e d Di scha]
148 111 6 tt tt
149 110 6 L e th a l 8  days
150 120 8 »t 5 d ays
151 122 e n 6 d ays
152 110 8 tt 6 days
153 132 8 tt 7 days
154 100 8 tt 6 days
M gs*dosage P e r c e n t  s u r v iv a l
4 100#
6 60#
8 0
M.T.D.=6
No.<
220
221
222
223
224
225
226
227
228
229
230
29
Metaphen -  intravenous
0*5% so lu tio n
T est animal -  Albino rat
W eight gras• Mgs. dosage R e s u l t Tim e.
139 2*6 S u rv iv e d D isc h a rg e d
124 2.6 h tt
115 2*6 «
130 2.6 n tt
160 4.0 L e th a l 1  day
138 4*0 tt 1  d a y
132 4.0 S u rv iv e d D isc h a rg e d
120 4.0 n tt
130 6.0 L e th a l 1 d a y
160 6.0 tt 1  day
130 6.0 tt 1 d a y
125 6.0 n 1 d ay
Mgs* dosage  P e rc e n t  s u r v iv a l
2.6 100%
4.0 50$
6.0 0
M .T.D. = 2 .6
Ho<
Hal
55
56
57
58
59
60
61
62
65
64
65
66
67
80
81
82
83
84
85
86
87
88
89
90
30
U etaphen~  in t r a m u s c u la r  
2#  s o lu t io n *
T e s t  an im al -  r a b b i t t *
re ig h t gins* IJgs* dosage R e s u l t Time
2300 6 S u rv iv e d
1960 6 ««
2075 6 *t
2745 6 «
2975 8 •«
2530 8 it
1975 8 i»
2840 8 it
2445 10 L e th a l 9 days
2730 10 S u rv iv e d
2840 10 it
2570 10 tt
2070 12 n
1625 12 L e th a l 7 days
2000 12 S u rv iv e d
1925 12 L e th a l 6 days
1620 14 « 6 days
1810 14 it 7 days
2200 14 S u rv iv e d
2150 14 L e th a l 5 days
2020 16 S u rv iv e d
2480 16 L e th a l 4  days
1650 16 L e th a l 4  days
2020 16 L e th a l 2 days
Mgs* 2)osage p e rc e n t  S u rv iv a l
6 100^
8 100^
x* 75%
12 50%
14 25$
16 26%
M.T.D.= 10.
No,
ra l
18
19
20
21
22
25
24
25
26
27
28
29
50
31
52
51
Metaphen -  Intravenously
2 % so lu tio n
Test animal -  rabbit
W eight gms* Mgs* dosage  R e s u l t  Time
1210
2570
2850
2170
2160
2150
2250
1600
2585
2050
2515
1750
1860
1375
1875
1700
1400
3
3
3
3
4 
4 
4 
4
4
5 
5 
5
5
6 
6 
6 
6
S u rv iv e d
L e th a l
S u rv iv e d
L e th a l
w
n
tt
tt
w
6 days
8 days 
8 days 
days 
days 
days 
days 
days
Mgs* dosage  P e rc e n t  s u r v iv a l
3 100 %
4 80 %
5  26 %
6 0
U.T.D.a 4
32
Mercurophan -  intramus cular
1.0% so lu tion
T est animal -  Albino ra t
NO. O f r a t  W eight 
©ns.
Mgs* d osage R e s u lt Tims i n
170 122 1 6 .6 S u rv iv e d
171 148 16*6 »
172 124 1 6 .6 «
173 130 1 6 .6 n
174 115 1 6 .6 tt
175 145 2 0 .0 tt
176 108 2 0 .0 tt
177 90 2 0 .0 L e th a l 12  days
178 136 2 0 .0 S u rv iv e d
179 140 2 0 .0
180 178 2 4 .0 it
181 132 2 4 .0 it
182 118 2 4 .0 L e th a l 3 days
183 130 2 4 .0 » 8  d ay s
184 151 2 4 .0 S u rv iv e d
185 145 2 8 .0 L e th a l 3 days
186 116 2 8 .0 tt 3 days
187 123 2 8 .0 it 3 days
188 126 2 8 .0 tt 2  d ays
189 142 2 8 .0 n 3 days
Mgs* dosage  P e rc e n t  s u r v iv a l
1 6 .6  100%
20*0
2 4 .0  60%
28*0 0
M.T.D.- 24.
33
M ercurophan  -  in tr a m u s c u la r  
2$  s o l u t io n  
T e s t  a n im a l -  A lb in o  r a t
No* o f  r a t  Mgs* dosage  P e rc e n t  s u r v iv a l
4 14*0 100$
4 16*6 80$
4 2 0 .0 20$
4 24*0 0
No.
54
Mercurophen -  intravenous
0.1% so lu tion
T est animal -  Albino rat
o t  r a t  W eight M gs. do sag e  R e s u lt  ^'ime
gms
155 8  S u rv iv e d  D isc h a rg e d  14 days
N M
»♦ »
137 8
195 8
102  8  n w
140 8  » n
109 10 * »
120 lO  L e th a l  1 d a y
147 10  "  1 day
195 10 " 1  day
125 10 " 1 day
107 12 " 1  d a y
115 12  " 1  day
147 12  •  1  day
146 12  " 1  day
150 12  " 3  d ays
M gs. d o sag e  P e r c e n t  s u r v i v a l
8 100%
10 20%
12 0
M.T.D. = 8
35
M ercurophen -  in t r a v e n o u s ly  
0*5^ s o lu t io n  
T e s t  a n im a l -  A lb in o  r a t
Jo* o f r a t  W eight 
gas
Mgs* d o sag e R e s u l t
155 110 4 S u rv iv e d
156 115 4 it
157 135 4 it
156 137 4 ft
159 120 4 L e th a l
160 142 6 »t
161 157 6 tt
162 110 6 tt
168 147 6 it
164 147 6 tt
165 148 8 «
166 152 8 it
167 117 8 tt
4.68 127 8 n
169 115 8 n
Time
Di sc h a rg e d
tf
n
tt
6 d ays 
1 d ay  
1  d a y  
1  dey 
3 days 
1  day  
1  d ay  
1  day 
1  day 
1  d a y  
1  day
Mgs* dosage  P e rc e n t  s u r v iv a l
4 80%
6 0
6 0
M*T.D.=4
36
M ercurophen  -  In tra m u s c u la r  
2 % s o l u t io n  
T e s t  an im al -  r a b b i t
No. o f  
r a b b i t
43
44
45
46
47
48
49
50
51
52
53
54
67
68
69
70
71
72
73
74
75
76
77
78
W eight gms,
1775
2350
2340
2060
1860
2370
2370
2370
2410
2200
1530
2240
2480
1710
1660
2165
1830
1970
2275
2000
2140
1820
1680
1930
Mgs. dosage
6
6
6
6
8
8
8
8
8
10
10
10
10
10
12
12
12
12
14
14
14
14
16
16
R e s u l t
S u rv iv e d
t«
tt
tt
n
L e th a l
S u rv iv e d
■t
tt
tt
n
tt
L e th a l
S u rv iv e d
L e th a l
S u rv iv e d
S u rv iv e d
S u rv iv e d
L e th a l
S u rv iv e d
L e th a l
tt
tt
Mgs. dosage  P e rc e n t  s u r v iv a l
6 100  %
a 80 %
10 80 %
12 76 %
14 50 %
16 0  %
Time
7 days
8 days 
7 days
4  days
3 days
5 days 
2  days 
5 days
M .T .L . r  12
Ho,
ra l
9
10
11
12
15
14
15
16
17
55
56
57
58
59
40
41
42
57
M ercurophen  -  In tra v e n o u s  
2  % s o l u t io n .
T e s t  a n im a l -  r a b b i t*
t ig h t  gms* Mgs* dosage R e s u l t Time
1010 3 S u rv iv e d
2600 3 m
1775 3 «
1580 3
2500 4 n
2040 4 tt
2175 4 l e t h a l 3 days
2225 4 S u rv iv e d
1325 4 t«
1575 5 L e th a l 3 days
1550 5 S u rv iv e d
3970 5 L e th a l 5 days
1770 5 tt 3 days
2015 6 tt 3 days
2040 6 n 4  days
1465 6 tt 5 days
1780 6 tt 5 days
Mgs* dosage  P e rc e n t  s u r v iv a l
3 100 %
4  80 %
5  26 %
8  0
M.T.D. -  4
36
R e s u l ts  o f  P h e n o l a id  T r i k r e a o l  E x p e r im e n ts* 
t e s t s
The t o x i c  i t  t h e s e  two s u b s ta n c e s  was c a r r i e d  o n ly
f a r  enough to  compare t h e i r  r e a c t i o n s  w i th  t h o s e  p ro d u ced  by 
me ta p  hen and m ercurophen*
The H’^ T-D* im  r a t s  was 250 m gs. p e r  k i l o  in t r a v e n o u s ly ,
and  350 mgs* in t r a m u s c u la r ly  f o r  b o th  p h en o l and t r i k r e s o l *  F u r­
t h e r  t e s t s  f o r  th e  M .T.D. w ere d is c o n t in u e d  b ecau se  o f  th e  
ex trem e tr e m o rs  m a n ife s te d  a t  a s  low d o sag es  a s  30 mgs* i n t r a -  
v e n o u s ly  and 70 m gs. in tr a m u s c u la r ly *  Many t im e s  th e r e  w as an  
o n s e t  o f  t h e s e  symptoms b e fo re  th e  i n j e c t i o n  was com pleted* 
T o x ic i ty  o f  M etaphen and M ercu rophen .
I n  d e te rm in in g  th e  M*T*D* o f  t h e s e  su b s ta n c e s  and to  
p ro p e r ly  e v a lu a te  them  a num ber o f  f a c t o r s  were co n sid e red *
Of f i r s t  im p o rta n c e  was th e  s t r a i n  o f r a t s  u sed  in  
th e  t e s t s .  The a lb in o  r a t s  em ployed i n  th e  s t a n d a r d i z a t io n  
o f  a rs p h e n a n in e  a t  th e  U n ite d  S t a t e s  H y g ie n ic  L a b o ra to ry  w ere 
used  e x c lu s iv e ly  i n  o b ta in in g  th e  r e s u l t s  w hich a r e  g iv e n  i n  
th e  p r o to c o l s .  P a r a l l e l  e x p e r im e n ts  c a r r i e d  o u t on a n o th e r  
s t r a i n  of r a t s  showed a  low er t o le r a n c e  f o r  th e  g e rm ic id e s  th a n  
i s  r e p o r t e d ,  som etim es a s  much a s  40 p e r  c e n t  on i d e n t i c a l  
dosages*  A lso  d u r in g  p e r io d s  o f  in te n s e  h e a t  an im al to le r a n c e  
f o r  th e  m e r c u r ia l s  was v e ry  much le s se n e d *
39
M etaphen*
The M* T . D. dose  i s  a s  fo l lo w s ;
Method o f  i n j e c t i o n Animal % o f  s o lu t io n M.T.D*
I n t  r a v e n o u s ly R at 0*1 6 xngs*
tt it 0 ,5 2*6  mgs*
«t R a b b it 2 . 0 4 mgs*
I n t r a m u s c u la r ly R at 1*0 24 mgs*
«t tt 2*0 20  mgs*
« R a b b it 2*0 12  mgs*
M ercurophen*
The M. T . D* d o se  i s  a s fo l lo w s :
Method o f  i n j e c t i o n Animal % o f  s o l u t io n M.T.D*
I n t r a v e n o u s ly R at 0*1 8  mgs*
It 0*5 4 mgs*
R a b b it 2*0 4 mgs*
I n t r a m u s c u la r ly R at 1 . 0 24*0 mgs
it it 2*0 16*6 mgs
it R a b b it 2*0 1 2 .0
40
I t  i s  q u i t e  e v id e n t  t h a t  th e  M .T.D . i s  n o t  a  f ix e d  va lue*
As shown by th e  e f f e c t  o f  d i l u t i o n  and th e  t o l e r a n c e  o f  i n d i v i ­
d u a l  a n im a ls ,  t h e r e  i s  a  b ro a d  ra n g e  o f  s u s c e p t i b i l i t y .  I n  
some p r e l im i n a ry  e x p e r im e n ts  an in d iv id u a l  an im al w ould o c c a s io n a l ly  
t o l e r a t e  a  dosage  a s  g r e a t  a s  40  m gs. p e r  k ilo g ra m . The h ig h e r  
c o n c e n tr a t io n s  may p ro d u ce  th ro m b o s is  and c a u se  sudden  dea th *  I t  
w i l l  b e  n o te d  i n  th e  in tr a v e n o u s  e x p e rim e n ts  in  r a t s  t h a t  when 
s o lu t io n s  w ere  em ployed d e a th  o c c u r re d  g e n e r a l l y  w i th in  34  h o u rs ,  
g iv in g  a  dosage  t o l e r a t i o n  o f  50$ l e s s  th a n  t h a t  o f  th e  0 . 1$  
s o l u t io n .  So fro m  t h i s  d a t a  i t  may b e  i n f e r r e d  t h a t  d i l u t i o n  
in f lu e n c e s  th e  s u s c e p t i b i l i t y  o f  th e  a n im a ls . T h is  i s  p a r t i c u l a r l y  
t r u e  w ith  in t r a v e n o u s  i n j e c t i o n s .
The M .T.D. d o se  in  r a b b i t s  was found  to  b e  4  m gs. p e r  
k ilo g ra m  in t r a v e n o u s ly  and 12 m gs. p e r  k ilo g ra m  in t r a m u s c u la r ly .
M ercurophen  showed 100$ g r e a t e r  t o le r a n c e  in tr a v e n o u s ly  
f o r  th e  0 .1 $  s o lu t io n  th a n  f o r  th e  0 .5 $  s o l u t io n .  R a b b its  had  
a  lo w er to le r a n c e  th a n  was e x h ib i t e d  by r a t s  bo th  in t r a v e n o u s ly  
and in t r a m u s c u la r ly .
I f  th e  t o x i c i t y  o f  m et ap hen  and m ercurophen  w ere p la c e d  upon 
a  c o m p a ra tiv e  b a s i s ,  t h e r e  i a  l i t t l e  d i f f e r e n c e  betw een them . M ercuro— 
phen  ap p ea re d  t o  be  somewhat l e s s  to x ic  th a n  m etaphen when u se d  
in t r a v e n o u s ly  in  r a t s  b u t  b o th  had th e  same M.T.D* i n  r a b b i t s
41
i n t  ram us c u l  a r l y  and in t r a v e n o u s ly .  B oth s u b s ta n c e s  when
i n j e c t e d  in t r a m u s c u la r ly  in  d o se s  o f  12  m gs. p e r  k ilo g ra m  o r
more p ro d u ce d  some i r r i t a t i o n ,  w h ich  was m a n ife s te d  f o r  s e v e r a l
( $ )m in u te s . I t  was s t a t e d  by  K olm er t h a t  th e  s o l u b i l i t y  i n  th e  
t i s s u e ,  p e rc e n ta g e  o f  m e rc u ry , and r a t e  o f  d i s s o c i a t i o n  o f
m ercu ry  in f lu e n c e  t o x i c i t y  and t h e r a p e u t i c  a c t i v i t y .
(7 )
M cN ider p o in te d  o u t  t h a t  a n im a ls  p o iso n e d  by b i c h lo r id e  
o f  m erc u ry , d e p en d in g  upon th e  r e a c t i o n ,  may be d iv id e d  in to  
s e v e r a l  g ro u p s . I n  th e  f i r s t  g roup  were a n im a ls  w hich d e v e lo p ed  
an  in te n s e  g a s t r o e n t e r i t i s  and d ie d  w i th in  48 h o u rs  from  th e  
e a r l y  deve lopm en t o f  th e  shock  s t a t e ,  w ith  i t s  a s s o c ia te d  
d i s tu r b e d  c i r c u l a t i o n ;  p r e v e n t in g  th e  m ercu ry  from  re a c h in g  
and  s e r i o u s l y  damaging th e  k id n e y s .  The seco n d  g roup  o f  
a n im a ls  re c o v e re d  fro m  th e  g a s t r o e n t e r i t i s  and a s s o c ia t e d  sh o c k , 
and d ie d  w i th in  seven  d a y s . The k id n e y s  o f  th e s e  a n im a ls  showed 
no n a rk e d  i n j u r y  to  th e  g lo m e ru li  b u t  a  d i f f u s e  W ie g e r t 's  l i k e  
n e c r o s i s  o f  th e  e p i th e l iu m  o f  th e  tu b u le s  w ith o u t s w e l l in g  o f  
th e  c e l l s .  A f i n a l  group shewed a  d e la y e d  a c u te  r e n a l  i n ju r y .
These a n im a ls  had  a  s e v e re  ty p e  o f  a c id  i n to x i c a t io n  and In te n s e  
s w e l l in g  o f  r e n a l  e p i th e l iu m , fo llo w e d  by n e c r o s i s .  M cNider s t a t e s  
t h a t  I t  a p p e a rs  t h a t  t h i s  ty p e  o f  r e n a l  i n j u r y  I s  n o t due to  th e  
m ercu ry  b u t  i s  i n  some way d ep en d en t upon th e  d is tu r b a n c e s  o f  th e  
a c id  b a se  e q u i l ib r iu m  o f  th e  b lo o d .
42
INTRASPINAL TESTS.
To T e s t  th e  A c tio n  o f  M etaphen , M ercu ro ­
p h a n . P h e n o l, and  T r i k r e s o l ,  when i n j e c ­
t e d  I n t r a s p i n a l l y .
( 3 )I n  1913 , Kramer o b se rv e d  a  number o f  c a s e s  o f  sudden d e a th  
a f t e r  th e  t r e a tm e n t  o f  c e r e b r o - s p in a l  f e v e r  w ith  a s p e c i f i c  a n tis e ru m  
and advanced  th e  h y p o th e s is  t h a t  t h e  p r e s e r v a t i v e  t r i k r e s o l  was th e  
cau se  o f  th e  symptoms* T h a t th e  to x ic  a c t io n  o f  th e  serum  when i n ­
t ro d u c e d  i n to  t h e  su b a ra c h n o id  sp a c e  o f  dogs and c a t s  was cau sed  by
th e  p r e s e r v a t i v e  t r i k r e s o l  was l a t e r  p ro ^ v en  in  an  e x p e r im e n ta l  m anner
(9 ) (IQ)by H a l e ' .  I n  1914 , V o e g t l in  co n firm ed  th e  r e s u l t s  o f  Kramer and
H ale  and fo u n d  t h a t  t r i k r e s o l  and  p h e n o l w e re  e q u a l ly  t o x ic  when i n ­
j e c te d  i n  l i k e  q u a n t i t i e s ,  a n d  s t a t e d ,  a s  a  g e n e r a l  r u l e ,  "The 
e f f e c t  on b lo o d  p r e s s u r e  i n c r e a s e s  i n  p r o p o r t io n  to  th e  r a t e  o f  i n ­
j e c t i o n  and th e  c o n c e n tr a t io n  o f  th e  p h e n o l p r e s e r v a t i v e . "
The f a c t  t h a t  p h e n o l and t r i k r e s o l  p o s s e s s  su ch  to x ic  p r o p e r t i e s  
when i n j e c t e d  i n t r a s p i n a l l y  made i t  n e c e s s a ry  to  know th e  e f f e c t s  
p ro d u ced  by th e  m s r c u r i a l s  m etaphen  and m ercurophen  upon th e  c e n t r a l  
n e rv o u s system #
P ro c e d u re .
The e x p e r im e n ts  were c a r r i e d  o u t on dogs* The a n a e s th e t i c  
em ployed was e t h e r  and th e  d e g re e  o f  a n a e s th e s ia  o b ta in e d  was q u i te  
c o n s ta n t  by  p e rfo rm in g  trach eo to m y  and in tro d u c in g  th e  v ap o rs  from
43
a  b o t t l e  d i r e c t l y  i n t o  t h e  t r a c h e a *  The b lo o d  p r e s s u r e  was ta k e n  
from  th e  c a r o t i d  a r te r y *  an d  t r a c i n g s  w ere  o b ta in e d  by  u s in g  a  m ercu ry  
m anom eter. R e s p i r a to r y  r a t e s  w ere  r e c o rd e d  g r a p h i c a l l y  by  i n s e r t i n g  
a  c a n u la  i n t o  t h e  p l e u r a l  c a v i ty  j u s t  above th e  d iaph ragm  and r e c o r d in g s  made 
on a  tam b o u r.
M ethods o f  I n j e c t i o n .
Two m ethods o f  i n j e c t i o n  w ere  em ployed; namely* p r e s s u r e  and 
g r a v i t y .
S e v e ra l  v e r t e b r a e  i n  th e  lum bar r e g io n  w ere exposed  and i n j e c t i o n s  
made by  in t r o d u c in g  a  n e e d le  d i r e c t l y  I n to  th e  s p i n a l  c a n a l .  S p in a l  
f l u i d  was p e rm it te d  to  e s c a p e . The n e e d le  w as th e n  c o n n e c te d  w ith  a  
L uer ty p e  o f  s y r in g e  o r  g ra d u a te d  b u r e t t e  d e p en d in g  upon th e  m ethod 
u se d .
A n tlm en ingocoecus serum  was p re s e rv e d  w ith  m ercurophen  and m etaphen 
i n  1-2500 p a r t s  and p h e n o l 1 -200  p a r t s .  T h is  p e r m i t te d  a  m arg in  o f  
more th a n  d o u b le  th e  q u a n t i ty  n eed ed  f o r  p r e s e r v a t io n  p u rp o se s  f o r  
m etaphen and m ercu ro p h en . S e ra  p re s e rv e d  w ith  each  o f  th e  above sub ­
s ta n c e s  w ere  i n j e c t e d  a l t e r n a t e l y  u s in g  b o th  th e  p r e s s u r e  and g r a v i t y  
m ethods o f  a d m in i s t r a t io n .
P r o t o c o l s .
Dog # 1 . 1 2 .3  fc tlo g g w a ., a n a e s th e t ic  -  e th e r  
11 ;50  t o  12 M. 5 c . c .  serum  p r e s e rv e d  w i th  1-2500  p a r t s  o f  m ercu ro p h en ,
«r
a c k n in is te re d  by g r a v i ty  m ethod . No change in  r e s p i r a t i o n  o r  b lo o d  p r e s s u r e .
44
1 2 :0 6  t o  1 2 :0 9  p*m* 5 c .c *  o f  m ercu rophen  serum  1*2500 p a r t s ,  
a d m in is te re d  by  g r a v i t y  m ethod . S l ig h t  r i s e  i n  b lo o d  p r e s s u r e ,  
r e s p i r a t i o n  norm al*
1 2 :1 4  to  1 2 :1 5  p*m* 2  c .c *  1 -2500  p a r t s  m ercu rophen  a d m in is te r e d  by 
g r a v i t y  method* R e s p i r a t io n  and b lo o d  p r e s s u r e  c o n s ta n t .
1 2 :2 5  t o  1 2 :3 0  p*m* 3 c . c .  p h e n o l serum  a d m in is te r e d  by g r a v i ty  
method* S l i g h t  r i s e  i n  b lo o d  p r e s s u r e  fo llo w e d  by  a  g r a d u a l  d e c r e a s e ;  
r e s p i r a t i o n  norm al*
12 :40  t o  1 8 :4 5  p.m* 5 c .c *  o f  1*200 p a r t s  p h e n o l serum , a d m in is te r e d  
by  p r e s s u r e  m ethod . Drop i n  b lo o d  p r e s s u r e ,  r e s p i r a t i o n  became 
sh a llo w  and checked  b e fo re  th e  p r e s s u r e  was r e l e a s e d  a f t e r  w hich  
tim e  r e s p i r a t i o n  and b lo o d  p r e s s u r e  a g a in  became n o rm al.
1 2 :5 5  t o  1 2 :5 8  p*m* 1*7 c . c .  1*100 p h e n o l i n  no rm al s a l t  s o l u t io n ,  
a d m in is te re d  by g r a v i t y  m ethod . An im m ed ia te  drop i n  h lo o d  p r e s s u r e ;  
r e s p i r a t i o n  norm al*
1 :0 0  t o  1 :0 1  p*m* 2  c .c *  1*100  p h e n o l i n  no rm al s a l t  s o l u t i o n ,  
a d m in is te re d  by  p r e s s u r e  m ethod . An im m ed ia te  d ro p  i n  th e  b lo o d  
p r e s s u r e ,  u n t i l  i t  was r e l e a s e d  when i t  became n o rm al; r e s p i r a t i o n  
normal*
1 :0 6  to  1 :1 0  p*m* 4  c . c .  o f  norm al s a l t  s o l u t io n ,  a d m in is te re d  by 
g r a v i ty  m ethod , b lo o d  p r e s s u r e  and  r e s p i r a t i o n  norm al*
1 :1 8  t o  1 :2 6  p.m* 5 .3  c . c .  o f  m etaphen  serum  1-2500  p a r t s ,  a d m in is te r e d  
by g r a v i t y  m ethod . No change i n  b lo o d  p r e s s u r e  o r  r e s p i r a t i o n .
46
1 :3 3  to  1 :3 4  p.m* 3 c .c *  1 -2500  p a r t s  m etaphen serum , a d m in is te r e d  by 
p r e s s u r e  method*
1 :3 6  p*m* p r e s s u r e  r e le a s e d *  B lood  p r e s s u r e  and r e s p i r a t i o n  norm al* 
1 :39  t o  1 :4 4  p.m* 7 c .c *  o f  1 -100  p h e n o l i n  n o rm al s a l t  s o lu t io n  
a d m in is te r e d  by p r e s s u r e  method*
1 :4 6  p*m* p r e s s u r e  r e l e a s e d ;  im m edia te  d rop  i n  th e  b lo o d  p r e s s u r e ,  
r e s p i r a t i o n  became sh a llo w  b u t  a f t e r  th e  p r e s s u r e  had been  r e l e a s e d  
th e r e  was no re c o v e ry  and th e  a n im a l succumbed*
p °g  # » . 8*2 k i lo g ra m s , a n a e s t h e t i c  -  e th e r  
1 0 :3 2  a*m* w ith d rew  1*5 c . c .  s p i n a l  f l u i d  -  c l e a r  
1 0 :4 1  t o  1 0 :5 0  a*nu 10 c .c *  o f  serum  p re s e rv e d  w ith  m etaphen  
1-2500 p a r t s  by  g r a v i t y  m ethod*
10 :59  a*m» p r e s s u r e  r e l e a s e d  and serum  p e rm it te d  t o  flo w  out*
Blood p r e s s u r e  and r e s p i r a t i o n  n o rm a l.
11 :10  to  11 :12  a»m» 4 .1  c . c .  o f  p h e n o ly z ed  serum  a d m in is te re d  
by g r a v i t y  m ethod . A d rop  i n  b lo o d  p r e s s u r e  w ith  r e s p i r a t i o n  
sh a llo w  and u n e v en . Im m e d ia te ly  b e fo re  r e l e a s in g  p r e s s u r e  c o a rs e  
tre m o rs  w ere m a n ife s te d *  A f te r  th e  p r e s s u r e  was r e l e a s e d  th e r e  
was an im m ediate  r i s e  i n  b lo o d  p r e s s u r e  to  no rm al and r e s p i r a t i o n  
became c o n s ta n t*
11 :27  to  11 :30  a.m* 4 c . c .  o f  1-2500  p a r t s  m ercurophen  serum  
a d m in is te re d  by g r a v i t y  method*
11:30 to  1 1 :3 3  a .m . 1*5 c . c .  o f  a d d i t i o n a l  serum .
46
1 1 :3 4  a .m . p r e s s u r e  r e l e a s e d .  B lood p r e s s u r e  n o rm a l, r e s p i r a t i o n  
n o rm al. No change i n  r e s p i r a t i o n  and b lo o d  p r e s s u r e  a f t e r  th e  
r e l e a s e  o f  th e  p re s e rv e d  serum s*
1 1 :4 5  to  1 1 :5 2  a*m* 4 c .c *  m ercu rophen  i n  1 -2500 p a r t s  serum  
a d m in is te re d  by  g r a v i t y  method* B lood  p r e s s u r e  and r e s p i r a t i o n  
norm al*
12 :0 1  t o  1 2 :0 6  p*m* 4*2 c .c *  o f  p h e n o l serum  a d m in is te r e d  
by g r a v i t y  method* A s l i g h t  r i s e  i n  b lo o d  p r e s s u r e  f a l lo w e d  by 
a  g r a d u a l  low ering*  When p r e s s u r e  w as r e l e a s e d  b lo o d  p r e s s u r e  
became n o rm al.
12 :1 0  to  1 2 :1 1  p*m* 2*5 c .c *  o f  p h e n o l serum ; r a p id  d e c re a s e  
i n  b lo o d  p r e s s u r e ,  r e s p i r a t i o n  norm al*
12 :19  to  1 2 :2 3  p*m* 9 c . c .  o f  1 -2500 p a r t s  m etaphen  serum  a d m in is te r e d  
under p r e s s u r e  i n j e c t i o n .  B lood  p r e s s u r e  and r e s p i r a t i o n  n o rm al.
12 :27  to  12 :29  p*m* 7*3 c .c #  o f  1-2500  p a r t s  m ercu ro p h en . R e s p i r a t io n  
and b lood  p r e s s u r e  n o rm a l.
12 :37  to  1 2 :3 9  p*m* 2*9 c . c .  o f  p h e n o l serum  a d m in is te r e d  by 
g r a v i ty  m ethod . T h ere  was a  g ra d u a l  d e c re a s e  in  b lo o d  p r e s s u r e  b u t  
i t  r e tu r n e d  to  norm al when p r e s s u r e  was r e l e a s e d .
12 :45  to  1 2 :4 7  p*m* 7 c .c *  -fe% p h e n o l serum a d m in is te r e d  u n d e r p r e s s u r e .  
T here was a  m arked d e c r e a s e  in  b lo o d  p r e s s u r e ;  r e s p i r a t i o n  became 
sh a llo w  and f i n a l l y  s to p p e d ; p r e s s u r e  was r e l e a s e d  a f t e r  w hich th e  
r e s p i r a t i o n  and b lo o d  p r e s s u r e  became norm al*
47
1 2 :5 4  to  1 2 :5 8  p*m* 4 #  p h e n o l i n  n o im a l s a l t  s o l u t io n  a d m in is te r e d  
by p r e s s u r e  m ethod . T h e re  was a  m arked d e c re a s e  i n  b lo o d  p r e s s u r e ,  
tre m o rs  w ere  shown, r e s p i r a t i o n  became sh a llo w  and f i n a l l y  s to p p e d *
The E f f e c t  o f  P h e n o ly ze d  Serum .
The r e s u l t s  o b ta in e d  from  a  1 -200  p a r t s  p h e n o ly z ed  serum  
w ere i n  g e n e r a l  an a lo g o u s to  th o s e  o f  V o e g t l i n ^ ^  I n  a l l  c a s e s  
th e r e  w as an im m ediate  and m arked d rop  in  th e  b lo o d  p r e s s u r e  
w ith  a  s lo w in g  up and f i n a l l y ,  c h e c k in g  o f  th e  r e s p i r a t i o n  i f  
th e  p r e s s u r e  was n o t  r e le a s e d *  Serum i n j e c t e d  u n d e r  p r e s s u r e  
gave a  more p r e c i p i t o u s  d rop  th a n  when i n j e c t e d  by th e  g r a v i t y  
m ethod. The amount o f  p h en o ly zed  serum  n e c e s s a ry  t o  cau se  
th e s e  e f f e c t s  was q u i te  s m a l l ,  2 c .c * ,  w i th in  s e v e r a l  m in u te s  
tim e . The r e s p i r a t o r y  and b lo o d  p r e s s u re  changes a re  shown 
i n  th e  t r a c in g s  1 and 2*
The ex trem e symptoms m a n ife s te d  i n  th e  v aso m o to r and r e s p i r a t o r y  
c e n te r s  by th e  u se  o f p h e n o l and t r i k r e s o l ,  e l im in a te  th e s e  
su b s ta n c e s  a s  p r e s e r v a t i v e s  i n  b io lo g ie s  w hich  a r e  to  be  
a d m in is te re d  v i a  th e  s p i n a l  c m d l-
M etaphen and M ercurophen*
A re v ie w  o f  th e  p r o to c o l s  and th e  r e s p i r a t o r y  and b lo o d  
t r a c in g s  f o r  th e  m e r c u r ia ls  w i l l  show an ab sen ce  o f  th e  s e v e re  
symptoms t h a t  w ere  m a n ife s te d  in  th e  p h en o l e x p e r im e n ts . I n j e c t i o n s
48
o f  l a r g e  am ounts o f  se rum  p re s e rv e d  w ith  m etaphen  and  m e rc u ro -  
p h sn  had  no n o t ic e a b le  e f f e c t  upon th e  b lo o d  p r e s s u r e  o r  r e s p i r a t o r y  
movements a s  shown in  t r a c in g s  3 and 4 .  The p r e s s u r e  m ethod
o f  i n j e c t i o n  p ro d u ced  a  s l i g h t  i n c r e a s e  in  t h e  b lo o d  p r e s s u r e  w hich 
was n o t  dangerous* I t  may, t h e r e f o r e ,  b e  assum ed t h a t  n e i t h e r  
m etaphen n o r  m ercurophen  may b e  e x p e c te d  to  have any  d e t r im e n ta l  
e f f e c t s  upon th e  v a so m o to r o r  r e s p i r a t o r y  c e n te r s  in  th e  
dosages u se d .
T r a c in g  Number 4 .  4 c c .  o f  1 -2 5 0 0  p a r t s  mercurophen and serum, a d m in i s t e r e d  by 
g r a v i t y  method i n  7 m in u t e s .  Upper t r a c i n g  r e s p i r a t o r y ;  lower t r a c i n g  
b lo o d  p r e s s u r e .
■ m n m w M M W P i
T r a c in g  7?umber 2 
in  2 m in u te s .
p re s j ur e svmnt omi
a d m in i s t e r e d  by p reau u . 
and th e  lo w er  t r a c i n g
‘e method
b l ood
49
POTENCY TESTS#
M ethod o f  P ro ced u re#
U n p re se rv e d  s e r a  w ere  c o l l e c t e d  from  v a r io u s  b io lo g ic  h o u se s
f o r  t h i s  p h a se  o f  th e  study# T e ta n u s  to x in  and  a n t i t o x i n ,  d ip h th e r i a
to x in  and a n t i t o x i n ,  w e re  u se d  to  d e te rm in e  th e  e f f e c t  o f  th e  g e rm ic id a l
s u b s ta n c e s  on t h e i r  im m unizing  powers* To th e  above to x in s  and a n t i t o x in s
w ere added th e  q u a n t i t i e s  o f  each  p r e s e r v a t i v e  w hich had  been  found  to
be g e rm ic id a l#  The s u b s ta n c e s  u se d  and q u a n t i t i e s  added w ere :
P h e n o l, i n  a n t i t o x i n s  t o x in s  ■§%•
T r Here s o l ,  #4$ i n  to x in s  and a n t i to x in s #
H e tap h e n , a n t i t o x i n s  1 -5000  p a r t s ,  to x in s  1-10000 p a r t s #
M ercu rophen , a n t i t o x i n s  1 -8000  p a r t s ,  t o x in s  1-12000  p a r t s #
These w ere th e n  p la c e d  i n  a  r e f r i g e r a t o r  a t  40°F# to g e th e r  w ith  th e
c o n tr o ls  o f  u n p re s e rv e d  serum# A p r e l im in a r y  p o te n c y  t e s t  was made
on th e  d ip h th e r i a  and te ta n u s  to x in s  and a n t i t o x in s  to  d e te rm in e  th e
e x a c t  u n ita g e #  They w ere  th e n  p la c e d  i n  an in c u b a to r  a t  37°C# to
h a s te n  any a c t i o n  o f  th e  p r e s e r v a t iv e s #  A t i n t e r v a l s  p o te n c y  t e s t s
w ere made on p r e s e rv e d  and u n p re se rv e d  s e r a  t o  n o te  any l e s s e n in g  o f
th e  u n ita g e #
P o te n c y  T e s ts  ( p r o p e r )
The m ethods em ployed i n  t e s t i n g  th e  p o te n c y  o f  th e  b io lo g ie s
*
s tu d ie d  i s  e s s e n t i a l l y  th e  same a s  th& t u sed  a t  th e  U n ite d  S t a t e s  
H y g ie n ic  L a b o ra to ry  in  t e s t i n g  th e  p o te n c y  o f  s ta n d a rd  com m ercial 
d ip h th e r i a  and te ta n u s  a n t i to x in s *  T hese t e s t s  may be found  i n  a
50
pam ph le t o f  th e  U n ite d  S t a t e s  H y g ien ic  L a b o ra to ry  e n t i t l e d  " T e s ts  o f  
S ta n d a rd  and Com m ercial D ip h th e r ia  and T e ta n u s  An t i  to x in "  .
T e s t in g  th e  A n ti to x in s *
S ta n d a rd  d i p h th e r i a  t o x in .  -
A s ta n d a rd  to x in  was em ployed w hich had  been  p r o p e r ly  aged 
and r ip e n e d  and th e  M.L.D* d e te rm in ed *  E ach s ta n d a r d  to x in  was t e s t e d  
a g a in s t  a  s ta n d a rd  a n t i t o x i n  and e ac h  L p lu s  dose  o f  t o x in  was u sed  
on fo u r  a n im a ls . The d e s i r a b l e  q u a n t i t y  o f  d i l u t e d  to x in  sh o u ld  
be i n  th e  n e ig h b o rh o o d  o f  2 c .c *  and th e  d i l u t i o n s  made a c c o rd in g ly .
D e te rm in a tio n  o f  D i lu t io n s  o f  th e  A n tito x in s*
T h is  was done so t h a t  1 c .c *  o f  a f i n a l  d i l u t i o n  was r e p r e s e n te d  
by one u n i t  o f  a n t i to x in *  Not l e s s  th a n  1 c*c* o f  th e  sam ple was 
used  in  th e  p r e l im in a r y  d i l u t i o n .  The r e q u ir e d  amount o f  s a l t  s o lu t io n  
was p i p e t t e d  i n to  a  f l a s k ,  th e  a n t i t o x i n  sam ple was drawn in  s l i g h t  e x c e ss  
i n to  a  1 c .c *  w a sh -o u t p ip e t te *  The m en iscus was th e n  b ro u g h t 
down t o  th e  p ro p e r  c a l i b r a t i o n  m ark by w ip in g  th e  t i p  o f  th e  p i p e t t e  
w ith  s t e r i l e  g au ze . A f t e r  d e l iv e r y  e ac h  p i p e t t e  was washed o u t a num ber 
o f  tim es*  I f  se co n d a ry  d i l u t i o n s  w ere r e o u i r e d  s a l t  s o lu t io n  was 
p la c e d  in  im m unity f l a s k s  i n  an amount d e te rm in ed  by th e  d i l u t i o n  
d e s i r e d ,  and th e  p r im a ry  d i l u t i o n  d e l iv e r e d  from  w ash -o u t c a p a c i ty
p i p e t t e s  o f  1 o r  2 c .c*  c a p a c i t i e s .
F i l l i n g  th e  S y r in g e s *
Two s y r in g e s  w ere  s e t  up f o r  e ac h  ssm ple* Four a d d i t io n a l  s y r in g e s  
w ere s e t  up f o r  c o n t r o l s ,  each  c o n ta in in g  th e  L p lu s  dose o f  s ta n d a rd  
to x in  and one u n i t  o f  s ta n d a rd  hom ologous a n t i to x in *
51
B osenau s y r in g e s  w ith o u t th e  s id e  a m  were a t t a c h e d  to  
s t e r i l e  v a s e l in e d  n e e d le s  by  m eans o f  s h o r t  heavy  ru b b e r  tu b in g *
The s ta n d a r d  to x in  was f i r s t  p i p e t t e d  i n t o  th e  s y r in g e  by m eans 
o f  g ra d u a te d  d e l iv e r y  p i p e t t e s ,  th e n  fo llo w e d  th e  a d d i t io n  o f  
1 c .c #  o f  th e  a n t i t o x i n  sam ple m easu red  by 1 c . c .  d e l i v e r y  p i p e t t e s .
As each  c . c .  o f  th e  d i l u t e d  a n t i t o x i n  was added to  th e  L p lu s  d ose  
o f  t o x in ,  th e  s y r in g e  was b r i s k l y  r o t a t e d  to  in s u r e  p ro p e r  and 
th o ro u g h  m ix in g .
I n j e c t i o n .
The i n j e c t i o n s  w ere made i n t o  g u in e a  p ig s  one h o u r a f t e r  th e  
a n t i t o x i n  had  been  p la c e d  in  th e  s y r in g e s ,  re m a in in g  d u r in g  th e  i n te r im  
a t  room te m p e ra tu re *  The g u in ea  p ig s  em ployed conform ed a s  n e a r ly  a s  
p o s s ib le  to  a  250 gram  w e ig h t. W eights o f  l e s s  th a n  240 o r  more th a n  
260 gram s made th e  a n im a ls  u n s u i t a b l e .  Any v a r ia n c e  i n  w e ig h ts  in s id e  
th e s e  l i m i t s  w ere c o r r e c te d  f o r  by d i s t r i b u t i n g  th e  a n im a ls  i n  su c h  
a  m anner t h a t  each  g roup  was r e p r e s e n te d  by heavy and l i g h t  a n im a ls .
B e fo re  i n j e c t i o n s  w ere  made th e  abdom inal w a ll  o f  th e  g u in e a  p ig  was 
c lo s e ly  c l ip p e d  b u t n o t d i s i n f e c t e d .  I n j e c t i o n s  w ere made su b c u ta n e o u s ly  
i n  th e  abdom inal r e g io n ,  th e  n e e d le  b e in g  in s e r t e d  i t s  e n t i r e  l e n g th ,  
p a s s in g  t r o u g h  th e  l i n e a  a lb a  to  p re v e n t le a k a g e  upon w ith d ra w a l. 
F o llo w in g  th e  i n j e c t i o n  o f  t h e  t o x in - a n t i t o x i n  m ix tu re  a  volume o f  
p h y s io lo g ic a l  s a l t  s o lu t io n  s u f f i c i e n t  to  b r in g  th e  t o t a l  volume to
5 Z
ab o u t 4 c*c* was s w ir le d  th ro u g h  th e  s y r in g e  end in je c te d *
I n t e r p r e t a t i o n  o f  R e su lts *
I f  th e  a n t i t o x i n  had  n o t l o s t  any o f  i t s  p o te n c y , th e  a n im a ls  
i n j e c t e d  w i th  th e  L p lu s  dose  o f  to x in  d ie d  in  a p p ro x im a te ly  f o u r  
days* (T h is  c o u ld  n o t be s t r i c t l y  ad h e red  to  and acme leaw ay  f o r  
th e  i n t e r p r e t a t i o n  o f  r e s u l t s  had  to  be g iv en  due to  th e  b io lo g ic  
f a c t o r  o f  in d iv id u a l  r e s i s t a n c e  o f  th e  a n im a ls )*  F o u r c o n t r o l  
anima l s  employed i n  th e  t e s t i n g  o f  th e  s ta n d a rd  a n t i t o x i n  and to x in  
d ie d  upon th e  f o u r th  day a f t e r  in je c t io n *
T e s t in g  o f  T e tan u s  A n t i to x i n *
The t r e a tm e n t  o f  th e  s ta n d a rd  t e t a n u s  to x in  and a n t i t o x in  w as 
s im i la r  to  th e  m ethods em ployed i n  d ip h th e r i a  s ta n d a rd  to x in  and 
a n t i t o x in  w ith  th e s e  d i f f e r e n c e s *  The m ix in g  o f  t e ta n u s  to x in  and 
a n t i t o x in  was done by  th e  l i g h t  o f  a carb o n  f i la m e n t  in c a d e s c e n t  
lam p, and s e c o n d ly , th e  d i l u t i o n s  we r e  c a r r i e d  one d e c im a l f u r t h e r  
th an  w ith  d ip h th e r ia  a n t i t o x i n  and to x in *
I n t e r p r e t a t i o n  o f  R e s u l ts .
Due to  th e  f a c t  t h a t  a u to p s ie s  y ie ld  n o t  d e f i n i t e  in fo rm a tio n  
a s  to  th e  c a u se  o f d e a th  from  te ta n u s  i t  was n e c e s s a ry  to  make ob­
s e r v a t io n s  o f  th e  a n im a ls  f o r  t e t a n i c  symptoms. These w ere o b se rv ed  
on th e  se c o n d , t h i r d ,  and  f o u r th  days by m aking a  th o ro u g h  in s p e c t io n  
o f  th b  an im als*  An e x p re s s io n  o f  th e  d e g re e  o f  t e t a n i c  symptoms was 
re c o rd e d  a s  fo l lo w s :  H y p e r te n ic i ty ,  s l i g h t  symptoms, symptoms, m arked
53
sym ptom s and  s e v e re  symptoms* A llow ance f o r  th e  in d iv id u a l  
v a r ia n c e  o f  th e  an im al to  re sp o n se  was made and e v a lu a te d  os 
i n  th e  d i p h th e r i a  a n t i t o x i n  t e s t s *
T e s t in g  o f  D ip h th e r ia  T o x in s  and T e tan u s  
T o x in s*
W ith th e  t e s t i n g  o f  th e s e  s u b s ta n c e s  a  p r e l im in a r y  p o te n c y  
t e s t  was co n d u c ted  to  d e te rm in e  th e  e x a c t  M*L*D* p e r  c .c*  A f te r  
t h i s  d e te r m in a t io n  d i l u t i o n s  w ere made in  e x a c t ly  th e  same m anner 
a s  i n  th e  c a se  o f  th e  a n t i t o x i n s f e x c e p t in  th e  f i n a l  d i l u t i o n s  
one M*L.D. was c o n ta in e d  i n  2 c .c*  The m ethod o f  i n j e c t i o n  i n to  
g u in e a  p ig s  was th e  same a s  t h a t  u sed  w ith  a n t i t o x i n s  and th e  
r e s u l t s  w ere i n t e r p r e t e d  i n  t h e  same manner* A nim als u se d  in  
th e  t e s t i n g  o f  te ta n u s  t o x in s  and a n t i t o x i n s  w ere on th e  av erag e  
100 ©us. h e a v ie r  in  w e ig h t th a n  th o s e  u sed  in  d i p h th e r ia  to x in  
and a n t i to x in *  The w e ig h ts  w ere w i th in  th e  l i m i t s  o f  350 and 
380 gram s i n  o r d e r  to  be used*
54
RESCTLTS OF POTENCY TESTS. 
D ip h th e r ia  A n t i to x in  P ro to c o la *
P ro to c o l  A.
D ip h th e r ia  a n t i t o x i n .
A n t i to x in  h e ld  a t  4 0 °F . f o r  3.6 d a y s , and s u b s e q u e n tly  
in c u b a te d  f o r  14 days a t  37°C .
U n its  p e r  c . c .  -  1300
S ta n d a rd  to x in  p o te n c y  -  0 .1 6 2  L p lu s  dose  d i lu t e d  to  2 .2 7  c . c .
Serum w ith o u t  any p r e s e r v a t i v e  -  an im al #1 d ie d  7 days a f t e r  i n j e c t i o n ,
showed d i p h t h e r i t i c  l e s i o n s ;
an im al #2 d ie d  7 d ay s  a f t e r  i n j e c t i o n ,  
showed d i p h t h e r i t i c  l e s i o n s .
an im al #3 d is c h a rg e d ,  no l e s i o n s ;  
a n im a l #4 d ie d  in  5jjr d a y s , l e s i o n s .
an im al #5 d ie d  7 d a y s , d i p h t h e r i t i c  l e s i o n s ;
an im al #6 d ie d  9 d a y s , d i p h t h e r i t i c  le s io n s , .
anim al  #7 d ie d  7 d a y s , d i p h t h e r i t i c  l e s io n s ;
an im al #8 d ie d  7 d a y s , d i p h t h e r i t i c  l e s io n s .
an im al #9 d ied  8 d a y s , d i p h t h e r i t i c  l e s io n s ;
animFtl #10 d ie d  8g- d a y s ,d i p h t h e r i t i c  l e s io n s .
Serum p lu s  Qw5^ p h e n o l -
Serum p lu s  0.4% t r i k r e s o l  -
Serum p lu s  1 -5000  p a r t s  
m etaphen
Serum p lu s  1 -5000 p a r t s  
m ercurophen
The L p lu s  dose  o f  s ta n d a rd  to x in  a d m in is te re d  in  t h i s  ex p erim en t 
was j u s t  a  l i t t l e  low .
55
P rotoco l B.
D ip h th e r ia  a n t i t o x i n .
A n t i to x in  h e ld  a t  40°F* f o r  16 d ay s  and  th e n  in c u b a te d  
f o r  22 d a y s  a t  37°C.
U n its  p e r  c .c*  -  1300
S ta n d a rd  to x in  p o te n c y  -  0 .1 6 5  L p lu s  dose in  2 .2 7  c .c *
Serum w ith o u t any  p r e s e r v a t i v e  -  an im al #1 d ie d  3g- d a y s , d i p h t h e r i t i c  symptoms;
a n im a l #2 d ie d  4 d a y s , d i p h t h e r i t i c  symptoms*
a n im a l #3 d ie d  5 days a f t e r  i n j e c t i o n  w ith  
symptoms;
an im al #4 d ie d  4 d ay s a f t e r  i n j e c t i o n  w ith  
symptoms.
a n im a l #5 d ie d  6 days a f t e r  i n j e c t i o n  w ith  
sym ptom s;
an im al #6 d ie d  5 days a f t e r  i n j e c t i o n  w ith  
symptoms.
<i
an im al §7 d ie d  4 days a f t e r  i n j e c t i o n ;  sym ptans 
an im al d ie d  4 days a f t e r  i n j e c t i o n ;  symptoms
Serum p lu s  0*5% p h e n o l -
Serum p lu s  0*4$ t r i k r e s o l  -
Serum p lu s  1 -5000  p a r t s  
me ta p  hen
Serum p lu s  1-5000  p a r t s  
m ercurophen an im al #9 d ie d  5 days a f t e r  in je c tio n ;sy m p to m s  
aniTBRl #10 d ie d  5rg days a f t e r  in jec tion .;sym ptom s
56
P rotoco l C.
D ip h th e r ia  a n t i to x in *
A n t i to x in  h e ld  a t  40°F . f o r  16 d a y s , and th e n  in c u b a te d  
f o r  39 days a t  37°C .
U n its  p e r  c . c .  -  300*
S ta n d a rd  to x in  p o te n c y  -  0*165 L p lu s  dose*
Serum w ith o u t p r e s e r v a t iv e  
Serum p lu s  0*5% p h e n o l -  
Serum p lu s  0*4% t r i k r e s o l  *
Serum p lu s  1-5000 p a r t s  
m etanhen
Serum p lu s  1-5000  p a r t s  
m ercurophen
an im al #1 d ie d  in  4 d a y s ; d i p h t h e r i t i c  l e s io n s  
an im al #2 d ie d  5 d a y s ; d i p h t h e r i t i c  l e s i o n s .
an im al #3 d ie d  in  4 d a y s ; d i p h t h e r i t i c  l e s i o n s ,
an im al #4 d ie d  in  4 d a y s ; d i p h t h e r i t i c  l e s i o n s .
an im al #5 d ie d  i n  5 d a y s ; d i p h t h e r i t i c  l e s i o n s ,
an im al #6 d ie d  i n  6 d a y s ; d i p h t h e r i t i c  le s io n s ,
an im al #7 d ie d  4g d a y s ; d i p h t h e r i t i c  l e s i o n s ,  
an im al #8 d ie d  5 d ay s; d i p h t h e r i t i c  l e s i o n s .
animal #9 d ie d  in  5 d a y s ; d i p h t h e r i t i c  l e s i o n s ,  
an im al #10 d ie d  i n  5 d a y s ; d ip h t h e r i t i c  l e s i o n s . <
57
RESULTS OF POTENCY TESTS
OF DIPHTHERIA TOXIN.
P r o to c o l  D.
D ip h th e r ia  to x in .
The to x in  was h e ld  a t  40°F , f o r  16 d ay s and th e n
in c u b a te d  f o r  16 days a t  37 ° c .
M,L,D. p e r  c , c ,  -  450
Serum w ith o u t  p r e s e r v a t i v e -  an im al #1 d ie d in 5 d a y s , d i p h t h e r i t i c sym ptom s;
anim al #2 d ie d i n 4 d a y s , d i p h t h e r i t i c symptoms.
Serum p lu s  O.Sy^ p h e n o l - a n im al //5 d ie d i n 8 d a y s , d i p h t h e r i t i c sym ptom s;
a n im a l H d ie d i n 7 d a y s , d i p h t h e r i t i c symptoms.
Serum p lu s  0*4# t r i k r e s o l  - an im al #5 d ie d i n 5 d a y s , d i p h t h e r i t i c symptoms;
an im al #6 d ie d i n 7 d a y s , d i p h t h e r i t i c syrup t a n s .
Serum p lu s  1 -5000  p a r t s
m etaphen - an im al #7 d ie d in 7 d a y s , d i p h t h e r i t i c sym ptom s;
an im al #8 d ied in 7 d a y s , d i p h t h e r i t i c symptoms.
Serum p lu s  1-5000 p a r t s
m ercurophen an im al #9 d ie d in 8*•days, d i p h t h e r i t i c symptoms;
an im al #10 d ie d in 7 d a y s , d i p h t h e r i t i c symptoms.
58
P rotoco l E.
D ip h th e r ia  to x in .
The to x in  w as h e ld  a t  40°F . f o r  16 days and th e n  in c u b a te d  
f o r  28 days a t  37°C.
p e r  c .c *  ™ 430
Serum w ith o u t  p r e s e r v a t iv e  
Serum p lu s  0*5^6 p h e n o l 
Serum p lu s  0*4% t r i k r e s o l  «
Serum p lu s  1 -5000  p a r t s  
m etaphen
Serum p lu s  1-5000 p a r t s  
m ercurophen
an im al d ie d 7 d a y s , d i p h t h e r i t i c sym ptoms;
an im al #2 d ie d 5 d a y s , d i p h t h e r i t i c symptoms.
an im al d ie d 5 d a y s , d i p h t h e r i t i c l e s i o n s ;
anim al #4 d ie d 5 d a y s , d i p h t h e r i t i c l e s i o n s .
a n im a l #5 d ie d 8 d a y s , d i p h t h e r i t i c symptoms;
anim al #6 d ie d 61g- days , d i p h t h e r i t i c symptoms.
an im al #7 d ie d  4 days a f t e r  i n j e c t i o n ,  d ip h ­
t h e r i t i c  symptoms; 
an im al #8 d ie d  7 days a f t e r  i n j e c t i o n ,  d ip h ­
t h e r i t i c  symptoms.
flnimfll #9 d ie d  6 days a f t e r  i n j e c t i o n ,  d ip h ­
t h e r i t i c  symptoms;
an im al #1C d ie d  7 d ays a f t e r  i n j e c t i o n ,d i p h ­
t h e r i t i c  symptoms.
59
RESULTS OF POTENCY TESTS
OF TETANUS ANTITOXIN
P ro to c o l  F .
T e ta n u s  a n t i t o x i n .
A n t i to x in  k e p t  a t  a  te m p e ra tu re  o f  4 0°F . f o r  24 d a y s , 
th e n  in c u b a te d  f o r  7 d ay s a t  37°C*
U n its  p e r  c . c .  -£ £ 5
S ta n d a rd  to x in  p o te n c y  -  0*00065 M .L.D.
Serum w ith o u t
p r e s e r v a t iv e  -  animal. # 1 , 4  d ay s -  symptoms ( t e t a n i c )  s u rv iv e d ;
an im al # 2 , 4  d ay s -  symptoms w s u rv iv e d .
Seram p l a s  0*5%
p h e n o l -  a n im a l # 3 , 4  days -  symptoms -  s u r v iv e d ;
an im al # 4 , 4 d ay s  -  s l i g h t  sym ptoms, s u r v iv e d .
Seram  p lu s  0 .
t r i k r e s o l  -  an im al # 5 , 4  d ay s -  symptoms, s u rv iv e d ;
a n im a l # 6 , 4  days -  sym ptom s, su rv iv e d *
Serum p lu s  1-5000
p a r t s  m e tap h en -an im a l # 7 , 4 days -  sym ptoms, s u rv iv e d ;
a n im a l # 8 , 4 d ays -  S ev e re  symptoms, s u rv iv e d .
Serum p lu s  1-5000
p a r t s  m ercurophen—a n im a l $ 9 , 4  d ay s  -  s e v e re  symptoms, s u rv iv e d ;
an im al # 1 0 ,4  days -  s e v e re  symptoms, s u rv iv e d .
60
P rotoco l G*
T e ta n u s  a n t i to x in *
A n t i t o i i n  k e p t  a t  a  te m p e ra tu re  o f  40°F* f o r  39 
d a y s , th e n  in c u b a te d  f o r  14 days a t  37°C.
U n its  p e r  c* c* -  500
S ta n d a rd  to x in  p o te n c y  -  0*00063 M*L.D*
Serum w ith o u t p r e s e r v a t iv e  - an im al
a n im a l
# 1 ,
# 2 ,
d ie d
d ie d
5 d a y s , 
4  d a y s ,
t e t a n i c
t e t a n i c
symptoms;
symptoms*
Serum p lu s  0*5$  p h e n o l - a n im a l
an im al
# 3 ,
# 4 ,
d ie d
d ie d
5 d a y s , 
5 d a y s ,
t e t a n i c
t e t a n i c
sym ptoms; 
symptoms.
Serum p l u s  0*4^  t r i k r e s o l  - gnim al
a n im a l
# 5 ,
# 6 ,
d ie d
d ie d
4 ^  days 
6 d a y s ,
, t e t a n i c  symptoms 
t e t a n i c  symptoms*
Serum
p a r t s
p lu s  1-5000  
m etaphen  - an im a l
a n im a l
# 7 ,
# 8 ,
d ie d
d ie d
5 d a y s ,
6 d a y s ,
t e t a n i c
t e t a n i c
symptoms;
symptoms*
Serum
p a r t s
p lu s  1-5000  
m ercu rophen  - an im a l
an im al
#*»
#10
d ie d
,d ie d
4 d a y s ,
5 d a y s ,
t e t a n i c
t e t a n i c
symptoms; 
symptoms •
RESULTS OF POTENCY TESTS OF
TETANUS TOXIN,
P r o to c o l  H.
T e ta n u s  to x in *
T o x in  h e ld  a t  a  te m p e ra tu re  o f  40°F* f o r  24 d a y s , 
th e n  in c u b a te d  f o r  14 d ay s  a t  37°C*
M.L.D. p e r  c*c* -  10000*
Serum w ith o u t  p r e s e r v a t i v e  - a n im a l
an im al # 2 ,
d is c h a r g e d ,
d is c h a rg e d ,
no
no
symptoms;
sym ptom s.
Serum p lu s  0*5# p h e n o l - an im a l
a n im a l
# 3 ,
H ,
d is c h a rg e d ,
d is c h a rg e d ,
no
no
symptoms;
symptoms.
Serum p lu s  0*4# t r i k r e s o l  - an im al
an im al
# 5 ,
# 6 ,
d is c h a rg e d ,
d is c h a rg e d ,
no
no
symptoms;
symptoms*
Serum p lu s  1 -5000  
p a r t s  m etaphen  - an im al
an im al
# 7 ,
# 8 ,
d is  c h a rg e d , 
d is c h a rg e d ,
no
no
symptoms; 
synptom s.
Serum p lu s  1 -5000  
p a r t s  m ercu rophen  - an im al
an im al #10
d is c h a rg e d , 
, d is c h a rg e d ,
no
no
symptoms;
symptoms*
S u b seq u e n t p o te n c y  t e s t s  w ere made upon t h i s  serum  i n  M*L»Dt s ,  
o f  5000 , 1000 , and 100 and found  th a t  no symptoms w ere evidenced* The 
te t a n u s  to x in  c o m p le te ly  l o s t  i t s  p o te n c y  upon in c u b a tio n  a t  37 C*
62
DISCUSSION*
The t e s t s  w ere so  d e v is e d  t h a t  i f  t b s  b i o lo g i e s  had n o t 
l o s t  t h e i r  p o te n c y , th e  t e s t  a n im a ls  i n j e c t e d  w ith  th e  L p lu s  dose  
o f  to x in  sh o u ld  d ie  i n  a p p ro x im a te ly  f o u r  d a y s . H ow ever, i n d iv id u a l  
g s i& ea  P ig s  v a ry  i n  t h e i r  power o f  re s p o n s e  to  an  a n t i t o x i n  and 
to x in  and t h i s  c a u se d  a  v a r i a t i o n  i n  th e  d e a th  r a t e  o f  a n im a ls  on 
i d e n t i c a l  d o sa g e s ; , a s  shown by  a  re v ie w  o f  th e  p r o t o c o l s .  I f  
th e  b io lo g ic  f a c t o r  o f  i n d iv i d u a l  r e s i s t a n c e  i s  ta k e n  i n t o  con­
s i d e r a t i o n  i n  th e  i n t e r p r e t a t i o n  o f  th e  r e s u l t s  th e  fo l lo w in g  
c o n c lu s io n s  seem  t o  be  w a r ra n te d .
D ip h th e r ia  a n t i t o x i n  when p re s e rv e d  w ith  p h e n o l,  t r i k r e s o l ,
me tap  h e n , and m ercu rophen  showed no lo g *  o f  im m unizing power fro m
ot h a t  o f  th e  u n p re s e rv e d  serum  when in c u b a te d  a t  37 C. f o r  s i x t e e n ,  
tw en ty -tw o , and t h i r t y - n i n e  d a y s .
The d i p h t h e r i a  to x in  showed no d e c re a s e  i n  p o te n c y  from  t h a t  
o f  th e  u n p re s e rv e d  to x in  a f t e r  s ix te e n  and tw e n ty -e ig h t  days in c u b a t io n  
a t  37°C .
T e tan u s  a n t i t o x i n  r e t a in e d  i t s  in m u n iz in g  pow er f o r  th e  
p e r io d s  s tu d ie d ;  se v en  and f o u r te e n  d a y s .
I n c u b a t io n  a t  37°C. f o r  f o u r te e n  d ay s caused  a  com plete  l o s s  
o f  p o te n c y  i n  th e  c o n t r o l  and p re s e rv e d  s e r a  o f  te ta n u s  t o x in .
A c lo s e  a n a ly s i s  o f  th e  p o te n c y  t e s t s  showed s im i la r  r e s u l t s  
w ith  no d e f i n i t e  e v id e n c e  o f  l o s s  o f  p o te n c y  due to  th e  a c t io n  o f  any 
o f  th e  c h e m ic a l s u b s ta n c e s  u sed  a s  p r e s e r v a t i v e s .
63
HI STOPHATO LOGIC STUDY OF THE EFFECTS 
OF METAPHEN AND MERCUROPHEN UPON THE TISSOES.
Th© a b i l i t y  o f  c e r ta in .  M e rc u r ic  s a l t s  to  produec© a 
n e p h r i t i s  h a s  been  known f o r  y e a rs*
I n  I8 6 0  Pavy^ in d u c e d  an e x p e r im e n ta l  n e p h r i t i s  
w ith  m ercury* He c o n s id e re d  th e  M a lp h ig ian  b o d ie s  to  be 
th e  s e a t  o f  an  i n j u r y  and a ls o  n o te d  th e  n e c r o s i s  o f  
tu b u la r  e p ith e liu m *  H einke^*2  ^c o n s id e re d  t h e  t o x ic  a c t io n  
o f  b i c h lo r id e  o f  m ercu ry  in  t h e  k id n ey  to  b e  due t o  ta s o m o to r  
p a r a l y s i s ,  w i th  r e s u l t a n t  th ro m b o s is  and in f a r c t io n *  A t a
03)l a t e r  d a te  A se h o ff  r e p o r te d  in  d e t a i l  th e  r e n a l  p a th o lo g y  
o f  m e rc u r ic  e h lo r id e /_ jfo £ c h nc o & i s t e d  i n  s w e l l in g ,  h y a l in e  v a c u o la  
t i o n ,  and n e c r o s i s  o f  th e  t u b u la r  e p ith e liu m *
The p r e s e n t  s tu d y  w as u n d e rta k e n  to  l e a r n  i f  th e  m e r c u r ia ls  
me ta p  hen  and m ercurophen  p ro d u ced  any t i s s u e  changes in  25$ 
Maximum T o le ra te d  Doses* T h is  q u a n t i t y  o f  e ac h  su b s ta n c e  
was chosen  a s  i t  p e rm its  a  w ide m arg in  o f  s a f e t y  in  th e  
p r e s e r v a t io n  o f  th e  b io lo g ie s *
P rocedure*
F or t h i s  s tu d y  a lb in o  r a t s  were u se d  a s  th© e x p e rim e n ta l 
an im als*  One hund red  and f i f t y  p e r  c e n t  and 25 p e r  c e n t
64
o f  th e  Maximum T o le r a te d  D oses o f  m etaphen , m ercu ro p h en , 
and m e rc u r ic  c h lo r id e  w ere a d m in is te r e d  in tra m u s c u la r ly *  
These d o sa g e s  w ere u se d  in  o r d e r  t h a t  th e  l e s io n s  p ro d u ced  
by a  to x ic  d o se  and  sub—t o l e r a t e d  d o se  m ig h t be lea rn ed *  
M ercu ric  c h lo r id e  was a l s o  employed so t h a t  a  c o m p a ra tiv e  
s tu d y  o f  th e  l e s io n s  p ro d u ced  b y  m etaphea  and m ercurophen  
m ig h t be made*
The an im a ls  w ere  k i l l e d  a t  v a ry in g  le n g th s  o f tim e  
in  an a t te m p t  to  o b se rv e  th e  v a r io u s  s ta g e s  o f  th e  l e s io n s  
p ro d u ced . The h i s t o lo g i c  s tu d y  w as l im i t e d  to  th e  h e a r t ,  
l i v e r ,  s p le e n ,  and k id n e y s . No g r o s s  l e s i o n s  w ere ob­
se rv ed  i n  th e  o th e r  o rg a n s . The fo l lo w in g  d e t a i l e d  
p r o to c o ls  show th e  f in d in g s  i n  e a c h  t e s t  a n im a l.
M ercu rophen .
150 p e r  c e n t  Maxi mum T o le r a te d  Dose.
Rat # 1 .
D ied  on t h i r d  d a y . P o s t  m ortem  a u t o l y s i s  d id  no t 
p e n a i t  a  s tu d y  o f  t h e  t i s s u e s *
R at # 2 .
D ied th r e e  days a f t e r  in je c t io n *  P o s t  m ortem  ob­
s e r v a t io n s :
S p le en  -  e n la rg e d  
H e a r t  -  n o rm a l 
L iv e r  -  n o rm a l
65
K idney  — num erous p a l s  y e llo w  f l e c k s  i n  th e  c o rtex *  
M ic ro sc o p ic  f in d in g s :
H e a r t  m usc le  — some a re a s  o f  t r a n s v e r s e  fra g m e n ta tio n *  
C ross  s t r i a t i o n  i s  g e n e r a l ly  w e l l  p re se rv e d *  Some e v id e n c e  
o f  i n t e r s t i t i a l  f i b r o s i s .
S p le en  ** engorged  w i th  b lo o d  w ith  c o n s id e r a b le  num bers 
o f  ly m phocy tes  i n  t h e  p u lp .
L iv e r  -  engo rged  w ith  b lo o d . The p e r ip h e r y  o f  th e  
p o r t a l  v e in  show s some f i b r o s i s .  The n u c le i  a r e  w e ll  
s ta in e d *  R e la t i v e ly  few l i v e r  c e l l s  i n  t h e  p e r i - p o r t a l  
a re a s  a re  f i l l e d  w ith  an a l l  f a t  d r o p l e t s .
K idney — i n  th e  o u t e r  zone o f  th e  c o r te x  th e  e p i th e ­
l i a l  c e l l s  i n  some tu b u le s  a p p e a r  c lo u d y , o th e r s  o x y p h il  
and g r a n u la r ,  o th e r s  show c o a g u la t io n  n e c r o s i s ,  some tu b u le s  
a re  c a l c i f i e d ,  some s w o lle n , and i n  o th e rs  th e  e p i th e l iu m  
i s  desquam ated* The g lo m e ru li  i n  g e n e ra l  ap p ea r q u i te  n o rm al, 
an o c c a s io n a l  m e  show ing p o o r n u c le a r  s ta in in g *  T here  
i s  m o d era te  i n t e r  tu b u la r  c o n g e s t io n . The s t r a i g h t  c o l l e c t in g  
tu b u le s  a re  n o t  much in v o lv e d ,  a  few show g ra n u la t io n  and a  
few c a l c i f i e d  p a tc h e s .  F a t  d r o p le t s  a re  d e m o n s tra te d  in  
th e  e p i th e l iu m  o f  th e  p ro x im a l c o n v o lu ted  tu b u le s*  S e v e ra l 
p a tc h e s  o f  f i b r i n o i d  d e g e n e ra t io n  w ere found in  th e  o u te r  
and th e  in te r m e d ia te  zones o f  th e  c o n v o lu ted  tu b u le s .
66
B at # 3 .
D ied  th r e e  d a y s  a f t e r  i n j e c t i o n .  P o s t  mortem o b s e r -  
v a t io n s :
H e a r t  — norm al
L iv e r  -  norm al
S p le en  — e n la rg e d ,  b lu i s h —b la c k  c o lo r
K idney -  showed a  few  in h ite  sp e c k s  i n  th e  c o r t i c a l  su b ­
s ta n ce *
M ic ro sco p ic  f in d in g s :
H e a r t  -  a  few a r e a s  o f  t r a n s v e r s e  f r a g m e n ta t io n ,  o th e rw is e  
normal*
L iv e r  -  no le s io n s *
S p leen  -  th e  M a lp h ig ia n  b o d ie s  a r e  en la rg ed *  The p u lp  
i s  engorged  w ith  b lood  and c o n ta in s  a  c o n s id e ra b le  amount o f  
h e m o sid e rin  and  many sm a ll  lym phocytes*
K idney -  i n  th e  o u te r  zone o f  th e  c o r te x  th e  c o n v o lu ted  
tu b u le s  a r e  s w o lle n , th e  e p i th e l iu m  i n  sane  g ra n u la r  and i n  
o th e r s  desquam ated* T here  a r e  s e v e r a l  l a r g e  a re a s  o f  hem orrhage 
and a g e n e r a l iz e d  s m a ll  lym phocyte  i n f i l t r a t i o n *  The g lo m e ru li  
a re  no rm al i n  a p p e a ra n c e . C a l c i f i c a t i o n  i s  seen  in  a  few o f  
th e  c o n v o lu te d  tu b u le s*  The e p i th e l iu m  o f  th e  c o l l e c t in g  tu b u le s  
i s  g r a n u la r  and sw ollen*  T here  i s  seme i n t e r s t i t i a l  hem orrhage 
and sm a ll  lym phocy te  i n f i l t r a t i o n .  T here  i s  a  com plete  ab sen ce  
o f  c a s ts *
67
R at # 4 .
D ied  t l i r e 6 d a y s  a f t e r  i n j e c t i o n .  P o s t  m ortem  o b se rv a ­
t io n s :
H e a r t  -  norm al
L iv e r  -  norm al
S p le en  -  e n la rg e d
K idney — th e  c o r te x  i s  sw o lle n  and shows p a le  y e llo w  
p a tc h e s .
M ic ro sco p ic  f i n d in g s :
H e a r t  -  no rm al
L iv e r  -  c o n g e s te d . T h ere  a re  sm a ll p a tc h e s  o f  g r a n u la r  
n e c r o t ic  l i v e r  t i s s u e  show ing no n u c le a r  s t a i n i n g ,  b o rd e re d  
by sma l l  lym phocy tes and s i t u a t e d  n e a r  th e  p o r t a l  v e n u le s .  L iv e r  
c e l l s  i n  th e  p e r i - p o r t a l  a r e a s  a re  f i l l e d  w ith  sm a ll f a t  
d r o p le t s .
K idney -  i n  th e  c o r te x  some tu b u le s  a r e  sw o lle n  and 
show c o a g u la t io n  n e c r o s i s ,  w h ile  o th e r s  a re  g r a n u la r .  Some 
tu b u le s  show co m p le te  desq u am atio n  and b re a k in g  down o f  
th e  e p i th e l iu m  to  th e  basem ent membrane. C a lc i f i e d  tu b u le s  
a re  found  th ro u g h o u t th e  c o r t e x .  The m a jo r i ty  o f  th e  
g lo m e ru li  a r e  n o rm a l, s e v e r a l  c o n ta in  some g r a n u la r  ex u d a te  
i n  th e  c a p s u la r  s p a c e s .  Some o f  th e  s t r a i g h t  c o l l e c t in g
68
"tubules a r e  sw o lle n  and  o th e r s  g r a n u l a r ,  w h ile  a few  con­
t a i n  h y a l in e  c a s ts *  L arge  a r e a s  o f  hem orrhage a r e  found 
w i th in  th e  xone o c c u p ie d  by  H e n le f s lo o p s  and th e  c o l l e c t i n g  
tu b u le s*  A m o d e ra te  amount o f  f i b r i n o i d  d e g e n e ra t io n  i s  
se en  fi n  $he fconvolu ted  tu b u le s  in  th e  in te rm e d i a te  2oh0t 
V ery sm a ll f a t  d r o p le t s  a r e  d e m o n s tra te d  in  th e  b a s a l  
p o r t io n  o f  th e  e p i t h e l i a l  c e l l s  o f  th e  c o n v o lu te d  tu b u le s  
i n  th e  in te r m e d ia te  zone*
R at #5*
D ied  f i v e  days a f t e r  in je c t io n *  P o s t mortem o b se rv a ­
t io n s :
H e a r t  -  no rm al 
L iv e r  -  no rm al 
S p le en  -  norm al
K idney -  th e  c o r te x  i s  sw o lle n  and shows p a le  y e llo w  
p a tch es*
M ic ro sc o p ic  f in d in g s :
H e a r t m usc le  -  norm al
S p leen  -  M a lp h ig ia n  b o d ie s  a r e  v e ry  much en la rg ed ^  
made up o f  and b o rd e re d  by num erous sm a ll lym phocy tes. T here 
i s  a  m o d era te  am ount o f  i n t e r s t i t i a l  f i b r o s i s ,  d i f f u s e  lympho­
c y te  i n f i l t r a t i o n ,  a  few r e t ic u lu m  c e l l s ,  and a  number o f
69
p lasm a  c e l l s  w i th in  th e  pu lp*
l i v e r  -  th e  l i v e r  shows a  few  l i v e r  c e l l s  i n  th e  
p e r i - p o r t a l  a r e a s  f i l l e d  w ith  sm a ll  f a t  d ro p le ts *
K idney  -  C u te r  zone o f  th e  c o r te x  shows a r e a s  o f  
hem orrhage. Some tu b u le s  show com plete  c o a g u la t io n  
n e c r o s i s , o th e r s  a r e  sw o lle n  and g r a n u la r ,  w h ile  o th e r  
c o n v o lu ted  tu b u le s  show d esq u am atio n  o f  th e  e p i th e l iu m  
and f i l l i n g  o f  th e  lumen w ith  c e l l u l a r  d e b r i s ,  T here  
i s  no c a l c i f i c a t i o n *  A few  g lo m e ru li  show lym phocy tes 
w ith in  Bowman's c a p s u le s .  The s t r a i g h t  tu b u le s  a r e  t h i n  
w a lle d , a few show c lo u d y  s w e l l in g .  O th e rw ise , th e  
c o l l e c t in g  tu b u le s  a p p e a r  n o rm al.
R at #6 .
D ied f i v e  days a f t e r  i n j e c t i o n .  P o s t mortem o b s e r ­
v a t io n s :
H e a r t  -  no rm al 
S p leen  -  norm al 
L iv e r  -  norm al
K idney — th e  c o r te x  shows sma l l  y e llo w  p a tc h e s . 
M ic ro sco p ic  f in d in g s :
H e a r t  m usc le  -  norm al
S p leen  -  m o d era te  amount o f  b lo o d . The p u lp  shows i n t e r ­
s t i t i a l  f i b r o s i s  and d i f f u s e  lym phocyte and p lasm a c e l l  i n f i l ­
t r a t i o n *
70
L iv e r  -  m o d e ra te  amount o f  c o n g e s t io n . I n  th e  
p o r t a l  a r e a s  t h e r e  i s  an a c c u m u la tio n  o f  e n d o th e l& & & ^ e lls . 
T here  i s  s l i g h t  c lo u d y  s w e l l in g  o f  some o f  th e  h e p a t ic  
c e l l s •
K idney — i n  th e  o u te r  zone o f  th e  c o r te x  t h e r e  i s  com­
p l e t e  c o a g u la t io n  n e c r o s i s  o f  th e  m a jo r i ty  o f  th e  c o n v o lu te d  
tu b u le s .  I n  th e  in te r m e d ia te  zone some tu b u le s  show com ple te  
desquam ation  o f  th e  e p i th e l iu m , o th e r s  a r e  g r a n u la r .  T here  
i s  no e v id e n c e  o f  c a l c i f i c a t i o n  b u t c o n s id e ra b le  i n t e r s t i t i a l  
f i b r o s i s  i s  s e e n . The lim b s  o f  H e n le 's  lo o p s  show a  few 
p a tc h e s  o f  c lo u d y  s w e l l in g .  The c o l l e c t i n g  tu b u le s  show 
no l e s io n s .
25 p e r  c e n t  Maximum T o le r a te d  D ose.
Rat #7 .
K i l le d  f i v e  d ay s a f t e r  i n j e c t i o n .  P o s t  mortem o b se rv a ­
t i o n s :
H e a rt -  n o rm al 
S p leen  -  no rm al 
L iv e r  — norma 1 
K idney -  n o rm a l 
M ic ro sc o p ic  f i n d in g s :
H e a r t  -  no rm al
S p le en  -  p u lp  engorged  w ith  b lo o d  and c o n ta in s  many
71
lym phocy tes and p lasm a c e l l s #
K idney -  i n  th e  o u te r  zone o f  th e  c o r te x  th e  c o n v o lu te d  
tu b u le s  i n  a  few  a r e a s  w ere q u i t e  g r a n u la r ,  o t h e r s  w ere 
sw o lle n , and  a  few  showed p a r t i a l  d e sq u am atio n  o f  th e  e p i t h e ­
lium# The g lo m e ru li  a p p e a re d  norm al# Among th e  s t r a i g h t  
tu b u le s  t h e r e  was lym phocyte  i n f i l t r a t i o n  and some f ib r o s i s #
L iv e r  -  th e  p e r i - p o r t a l  c o n n e c tiv e  t i s s u e  showed a  
heavy  i n f i l t r a t i o n  w ith  sm a ll  lym phocy tes and p lasm a c e l ls #
B at # 9 .
K i l le d  se v en  days a f t e r  in je c t io n #
A ll o rg a n s  norm al on b o th  g ro s s  and h i s t o lo g i c  exam ination#  
Rat #15 .
K i l le d  tw e lv e  days a f t e r  i n j e c t i o n .
A l l  o rg a n s  norm al on b o th  g ro s s  and h i s t o lo g i c  exam ination#  
Rat #8#
K i l le d  f i f t e e n  days a f t e r  i n j e c t i o n .
at t o rg an s  norm al on b o th  g ro s s  and h i s t o lo g i c  e x am in a tio n . 
R at # 1 1 .
K i l le d  s e v e n te e n  d a y s  a f t e r  i n j e c t i o n .
a h  o rg a n s  n o rm al on b o th  g ro s s  and h i s to lo g ic  e x am in a tio n . 
R at #12#
K i l l e d  tw e n ty -o n e  days a f t e r  i n j e c t i o n .
H e a r t ,  s p le e n ,  and l i v e r  n o rm al.
72
K idney  — i n  th e  o u te r  zone o f  th e  c o r te x  some o f  th e  
p ro x im a l c o n v o lu te d  tu b u le s  a re  s w o lle n ;  th e  o rg a n  i s  
norm al i n  o th e r  r e s p e c t s .
M etaghen .
150 p e r  c e n t  Maximum T o le r a te d  B ose .
B at # 1 6 .
D ied th r e e  d ay s  a f t e r  i n j e c t i o n .  P o s t  m ortem  o b se rv a ­
t io n s :
H e a r t  -  no rm al 
S p le e n  -  e n la rg e d  
L iv e r  -  no rm al
K idney  -  num erous y e llo w  sp eck s s c a t t e r e d  th ro u g h o u t 
th e  c o r te x .
M ic ro sc o p ic  f in d in g s ;
H e a r t  -  p e r i v a s c u la r  f i b f o s i s ,  w ith  a  m o d era te  amount o f  
i n t e r s t i t i a l  lyngphocyte i n f i l t r a t i o n .
S p le en  -  en g o rg ed  w ith  b lo o d . M a lp h ig ian  b o d ie s  e n la rg e d . 
I n f i l t r a t i o n  w ith  lym phocy tes and a  few p lasm a c e l l s  in  th e  
pu lp *  C o n s id e ra b le  d e p o s i t io n  o f  h e m o s id e r in .
L iv e r  -  s l i g h t  c lo u d y  s w e ll in g  o f  th e  h e p a t ic  c e l l s  
w ith  r e l a t i v e l y  few  l i v e r  c e l l s  in  th e  p e r i - p o r t a l  a re a s  
f i l l e d  w ith  sm a ll f a t  d r o p l e t s .
73
K idney  -  i n  th e  o u t e r  zone o f  th e  c o r te x  some tu b u le s  
show o x y p h il  g r a n u le s ,  o th e r s  a r e  sw o lle n , o th e r s  show 
com plete d esq u am atio n  o f  th e  e p i th e l iu m  and  s t i l l  o th e r s  a r e  
much d i l a t e d  and  c o n ta in  c a s ts *  The g lo m e ru li  a s  a  w hole 
a re  normal , a  few  a re  hyperetm lc, and  c o n ta in  g r a n u la r  e x u d a te  
w ith in  th e  cap su le s*  The s t r a i g h t  c o l l e c t i n g  tu b u le s  show 
a m odera te  amount o f  c lo u d y  s w e ll in g  in  th e  d i s t a l  p o r t io n .  
Small f a t  d r o p l e t s  a re  d e m o n s tra te d  i n  th e  b a s a l  p o r t io n  
o f  th e  e p i t h e l i a l  c e l l s  o f  th e  c o n v o lu te d  tu b u le s  i n  th e  
o u te r  zone*
Rat #20 .
D ied th r e e  d a y s  a f t e r  in je c t io n *  P o s t  mortem o b se rv a ­
t io n s :
H e a r t  -  norm al
S p le e n  -  g r e a t l y  sw o lle n
L iv e r  -  no rm al -
K idney  -  th e  e n t i r e  c o r te x  shows y e llo w is h  d e p o s its *  
M ic ro sco p ic  f in d in g s :
H e a r t  -  norm al
S p le e n  -  th e  M a lp h ig ia n  b o d ie s  a re  v e ry  much e n la rg e d , 
and p o o r ly  d e f in e d .  The p u lp  i s  engorged  and c o n ta in s  much 
h e m o s id e r in . T here  a r e  many lym phocyte and p lasm a c e l l s  in  th e  
p u lp .
74
L iv e r  -  i n  th e  p e r i - p o r t a l  zones t h e r e  i s  c lo u d y  
s w e ll in g  o f  th e  h e p a t ic  c e l l s  w hich a r e  f i l l e d  w ith  
sm a ll  f a t  d ro p le ts *  M oderate  f i b r o s i s  o c c u rs  a b o u t th e  
b i l e  d u e ts  i n  t h e  p e r i - p o r t a l  zones*
K idney — i n  th e  o u t e r  zone  o f  th e  c o r te x  a  m a jo r i ty  
of th e  c o n v o lu te d  tu b u le s  a r e  g r a n u l a r  and  sw ollen*  Some 
show no n u c le a r  s t a i n i n g , same o x y p h il  g r a n u la t io n  o f  
th e  e p i th e l iu m , and s t i l l  o th e r s  co m p le te  desquam ation  o f  
th e  e p i th e l iu m , f i l l i n g  t h e  lum en w ith  c e l l u l a r  d e b r i s .
T here  i s  a  m o d era te  amount o f  i n t e r s t i t i a l  f i b r o s i s *  The 
g lo m e ru li  a r e  g e n e r a l l y  w e l l  p r e s e r v e d ,  a  few show h y p erem ia . 
The c o n v o lu te d  tu b u le s  i n  th e  in te r m e d ia te  zone c o n ta in  a  
few c a s t s  b u t  th e r e  i s  no  c a l c i f i c a t i o n *  The c o l l e c t in g  
tu b u le s  a re  t h in  w a lle d  and c o n ta in  a  few h y a lin e  c a s ts *
Rat # 1 9 .
D ied  f i v e  days a f t e r  i n j e c t i o n .  P o s t  mortem o b s e rv a t io n s :  
H e a r t  -  norm al 
L iv e r  -  norm al 
K idney  -  norm al 
M ic ro sc o p ic  f in d in g s :
H e a r t  m u sc le  — norm al
S p le e n  -  M a lp h ig ian  b o d ie s  a re  e n la rg e d , and c o n ta in  
num erous ly m p h o cy tes  and p lasm a  c e l l s ,  ih e  p u lp  i s  engorged
75
and c o n ta in s  h e m o s id e r in  and num erous ly m phocy tes  and p lasm a  
c e l ls *
L iv e r  -  t h e  v e n u le s  a re  c o n g e s te d  w ith  b lo o d  and b ro k en  
down e ry th r o c y te s *  R e la t i v e ly  few h e p a t i c  c e l l s  i n  th e  p e r i ­
p o r t a l  a r e a s  c o n ta in  sm a ll  f a t  d ro p le ts *
S id n e y  -  i n  t h e  o u te r  zone o f  th e  c o r te x  a l l  th e  tu b u le s  
a re  sw o lle n 9 some show g r a n u la r  d e g e n e ra t io n ,  o th e r s  show th e  
e p ith e l iu m  t o  he m o s tly  d e sq u a m a te d , and  o th e r s  c o n ta in  h y a l in e  
c a s t s • A few g lo m e ru li  show f i b r o s i s  a b o u t th e  p e r ip h e ry  
o f  Bowman*s c a p s u le s .  No l e s io n s  a re  found  in  th e  s t r a i g h t  
c o l l e c t in g  tu b u le s .
R at #18 .
D ied f i v e  d ay s a f t e r  i n j e c t i o n .  P o s t  mortem o b se rv a ­
t io n s :
H e a r t  -  no rm al 
S p le en  -  norm al 
L iv e r  -  norm al
K idney -  a  few sm a ll y e llo w  p a tc h e s  i n  th e  c o r te x  
IS ic ro sc o p ic  f in d in g s :
H e a r t  m u sc le  -  norm al
S p le e n  -  th e  L ia lp h ig ian  b o d ie s  show p o o r s t a i n i n g ,  a re  
e n la rg e d  a  num ber show f in e  c o lla g e n  f i b r i l s  p e rm e a tin g
76
t h e i r  s t r u c tu r e *  The p u lp  i s  r i c h  in  b lo o d , c o n ta in s  
num erous ly m p h o cy tes  and much h em o sid e rin *  
l i v e r  -  norm al
K idney  -  i n  th e  o u te r  zone o f t h e  c o r te x  some o f  
th e  c o n v o lu te d  tu b u le s  show o x y p h il  g r a n u la r  d e g e n e ra t io n ,  
o th e r s  a re  sw o lle n , o th e r s  c a l c i f i e d  and  o th e r s  c o n ta in  
c a s t s ,  T here  i s  no c o a g u la t io n  n e c r o s i s .  I n  th e  in te r m e d ia te
zone th e r e  i s  c a l c i f i c a t i o n  o f  many t u b u le s ,  w h ile  many o th e r s
show p a r t i a l  d e sq u am a tio n  o f  th e  e p i th e l iu m . The g lo m e ru li  
a s  a T iiole a r e  n o rm a l, a  few show ing p a r t i a l  anem ia . The 
s t r a i g h t  c o l l e c t i n g  tu b u le s  a r e  som etim es a t r o p h ie d ,  and 
c o n ta in  a  few  c a s t s .  T h ere  i s  a  m o d era te  amount o f  
f i b r in o i d  d e g e n e ra t io n  i n  th e  o u te r  and in te r m e d ia te  zones 
o f  th e  c o r t e x .
Bat #17 .
D ied f i v e  d ay s a f t e r  in je c t io n *  P o s t  mortem o b s e rv a t io n s ;  
H e a r t  -  norm al 
L iv e r  -  norm al 
S p le e n  -  norm al 
K idney — norm al 
M ic ro sc o p ic  f in d in g s :
Heart -  normal
77
L iv e r  — normal.
S p le e n  -  norm al
K idney  -  th e  c o n v o lu te d  tu b u le s  i n  th e  o u te r  zone 
o f  th e  c o r te x  show, some sw o lle n , o th e r s  g r a n u la r ,  o th e r s  
p a r t i a l l y  n e c r o t i c ,  an d  o th e r s  p a r t i a l l y  desquam ated .
The g lo m e ru li  a r e  norm al w ith  th e  e x c e p tio n  o f  a  few  t h a t  
c o n ta in  a  g r a n u la r  e x u d a te  w i th in  th e  c a p s u la r  s p a c e s .
The s t r a i g h t  c o l l e c t i n g  tu b u le s  a re  n o rm al. W ith in  th e  
c o r te x  a re  s e v e r a l  s m a ll  f o c i  o f  f i b r i n o i d  d e g e n e ra t io n  o f  
th e  e p i th e l iu m .
H at # 2 1 .
-^ied sev en  days a f t e r  i n j e c t i o n .  P o e t  m ortem  o b s e rv a ­
t io n s :
H e a r t  -  norm al 
S p le e n  -  e n la rg e d  
L iv e r  -  norm al
K idney — a  few y e llo w  p a tc h e s  w ith in  th e  c o r te x  
M ic ro sc o p ic  f in d in g s :
H e a r t  -  i n t e r s t i t i a l  i n f i l t r a t i o n  w ith  a  few lympho­
c y te s .
S p le e n  -  en g o rg ed . The p u lp  c o n ta in s  c o n s id e ra b le  hamo-
s i d e r i n .
Liver — normal
78
K idney  -  th e  c o n v o lu te d  tu b u le s  in  th e  o u te r  zone/
^ 'fagw M a e  sw o lle n , o th e r s  g r a n u la r ,  and  a  few  c o n ta in  c a s t s .  
The d i s t a l  c o n v o lu te d  tu b u le s  show p a r t i a l  e p i t h e l i a l  d e s ­
quam ation  in  one s n a i l  a r e a .  T h ere  a r e  a  number o f  f o c i  
o f  i n t e r s t i t i a l  i n f i l t r a t i o n .  A few g lo m e ru li  show g r a n u la r  
ex u d a te  w i th in  th e  c a p s u le s .  H e n le ’ s  lo o p s  and th e  s t r a i g h t  
c o l l e c t i n g  tu b u le s  a r e  n o rm a l.
M etaphen .
25 p e r  c e n t  Maximum T o le r a te d  D ose.
R at #27.
K i l l e d  f i v e  d a y s  a f t e r  i n j e c t i o n .
No g ro s s  p o s t  m ortem  l e s i o n s .
M ic ro sco p ic  f i n d in g s :
H e a r t  -  norm al
S p leen  -  M a lp h ig lan  b o d ie s  e n la r g e d .  P u lp  engorged  
w ith  b lo o d .
L iv e r  -  no rm al
K idney  -  t h e  o u te r  z cn e  o f  th e  c o r t e x  shows some sw o lle n  
c o n v o lu ted  tu b u le s*
Rat # 2 5 .
K i l l e d  tw e lv e  d ays a f t e r  i n j e c t i o n .
No g ro s s  p o s t  mo ite m  l e s i o n s .
79
M ic ro sc o p ic  f in d in g s :
H e a r t  -  no im al 
L iv e r  -  norm al 
S p le e n  -  norm al 
K idney -  norm al 
Bat #25 .
K i l le d  tw e lv e  days a f t e r  i n j e c t i o n .
No g ro s s  p o s t  mortem l e s i o n s .
M ic ro sco p ic  f in d in g s :
L iv e r  -  norm al
S p le en  -  en g o rg ed  w ith  b lo o d .
H e a r t  m u sc le  -  no rm al 
K idney -  norm al 
Bat #26 .
K i l le d  n in e t e e n  d ay s  a f t e r  i n j e c t i o n .  P o s t  m ortem  
o b s e rv a t io n s :
H e a r t  -  no rm al 
L iv e r  -  no rm al 
S p leen  -  norm al 
K idney -  norm al 
M ic ro sc o p ic  f i n d in g s :
Liver -  normal
80
S p le e n  — norm al 
H e a r t  m u sc le  -  no rm al 
K idney  -  norm al 
Bat #84 .
K i l le d  n in e te e n  days a f t e r  in je c t io n *
B oth  th e  g r o s s  and h i s t o l o g i c  e x a m in a tio n s  showed th e  
h e a r t , l i v e r ,  s p le e n ,  and  k id n e y s  t o  be n o rm a l.
Rat #22 .
K i l le d  tw e n ty - tw o  d a y s  a f t e r  i n j e c t i o n .  P o s t  mortem 
o b s e rv a t io n s :
H e a r t  -  norm al 
S p leen  -  norm al 
l i v e r  -  norm al 
K idney  -  norm al 
M ic ro sco p ic  f in d in g s :
L iv e r  -  norm al 
H e a r t  m usc le  -  norm al 
S p leen  -  no rm al
K idney -  p a tc h e s  o f  i n t e r s t i t i a l  f i b r o s i s  among th e  p ro x im a l 
c o n v o lu te d  tu b u le s  o f  th e  c o r te x .
81
M e rc u ric  C h lo r id e *
150 p e r  c e n t Maximum T o le r a te d  Dose*
R at # 1 5 a . ' * i t
D ied th r e e  days a f t e r  i n j e c t i o n ;  p o s t  m ortem  a u to ly —
sis*
R at #13*
Died, f i v e  days a f t e r  i n je c t i o n *  P o e t  m ortem  o b s e rv a ­
t io n s :
H e a r t  -  norm al 
S p leen  -  e n la rg e d  
L iv e r  -  e n la rg e d
K idney  -  num erous s m a ll  y e llo w  g r a n u le s  in  th e  c o rte x *  
M ic ro sco p ic  f in d in g s :
H e a r t m u sc le  -  sm a ll p a tc h e s  o f  g r a n u la r  d e g e n e ra t io n  
o b sc u r in g  t h e  c r o s s  s t r i a t i o n  o f  th e  m u sc le  f i b r e s  i n  s e v e r a l  
a re a s*  S l ig h t  p a tc h y  lym phocyte  i n f i l t r a t i o n  among th e  
m id c le  f ib r e s *
S p leen  -  th e  M a lp h ig ia n  b o d ie s  a re  p o o r ly  d e f in e d ,  
made up o f  lym phocy tes and p lasm a c e l l s *  The p u lp  i s  engo rged  
w ith  b lo o d  and c o n ta in s  much h e m o s id e r in , num erous lym phocy tes
and a  few  p lasm a  c e l l s *
K idney -  i n  th e  o u te r  zone o f  th e  c o r te x  th e  c o n v o lu te d  
tu b u le s  a r e  a l l  s w o lle n , i n  same to  th e  p o in t  o f  o c c lu s io n .  A
82
few tu b u le s  show g r a n u la r  d e g e n e ra t io n .  I n  th e  in te r m e d ia te  
zone th e  tu b u la r  e p i th e l iu m  i s  more g r a n u l a r ,  som etim es 
p a r t i a l l y  desquam ated . The m a jo r i ty  o f  th e  g lo m e ru li  
a re  n o u n a l, a  few show ing h y p e rem ia . Among th e  s t r a i g h t  
tu b u le s  th e r e  i s  lym phocy te  i n f i l t r a t i o n  and p r o l i f e r a t i o n  
o f  young f i b r o b l a s t s .
R at #14 .
D ied seven  days a f t e r  i n j e c t i o n .  P o s t  m ortem  o b s e rv a ­
t io n s :
At th e  s i t e  o f  i n j e c t i o n  th e r e  i s  a  l a r g e  a r e a  o f  
n e c ro s is  i n  th e  m u sc u la r  t i s s u e .
H e a r t  -  norm al
S p leen  -  g r e a t l y  e n la rg e d
L iv e r  -  no rm al
K idney — th e  c o r te x  i s  c lo u d y  and s l i g h t l y  sw o lle n . 
M ic ro sco p ic  f in d in g s :
H e a r t  m u sc le  -  s l i g h t  i n t e r s t i t i a l  lym phocyte  i n f i l t r a ­
t io n .
S p le en  -  t h e  p u lp  i s  engorged  w ith  b lo o d . T here  i s  a 
heavy  d e p o s i t  o f h e m o s id e r in  and an i n f i l t r a t i o n  w ith  sm a ll  
ly m p h o cy tes . The M a lp h ig ian  b o d ie s  a r e  e n la rg e d .
83
L iv e r  -  many h e p a t ic  c e l l s  show c lo u d y  sw e llin g *
T h ere  a r e  r e l a t i v e l y  few  l i v e r  c e l l s  in  th e  p e r i —p o r t a l  
a r e a  f i l l e d  w ith  m in u te  f a t  d ro p le ts *
K idney  -  th e  c o n v o lu te d  tu b u le s  i n  th e  o u te r  zone o f  
th e  c o r te x  show v a ry in g  p h a se s  o f  n e c ro s is *  Some a re  
sw o lle n  and g r a n u l a r ,  some show com ple te  c o a g u la t io n  n e c r o s i s ,  
and o th e r s  c a l c i f i c a t i o n *  I n  th e  in te rm e d ia te  zone th e r e  
i s  more e x te n s iv e  c o a g u la t io n  n e c r o s i s  th a n  in  th e  o u te r  
zone* I n  many tu b u le s  th e  e p i th e l iu m  i s  d esquam ated , 
o t h e r  tu b u le s  a r e  c a l c i f i e d ,  and  s t i l l  o th e r s  c o n ta in  
c a s t s .  T h is  a r e a  shows some i n t e r s t i t i a l  f i b r o s i s *  The 
g lo m e ru li  a p p ea r n o im a l o r  o c c a s io n a l ly  hyperem ic* The 
s t r a i g h t  c o l l e c t in g  tu b u le s  a r e  normal*
R at #15 .
D ied se v e n  d a y s  a f t e r  i n j e c t i o n .  P o s t  mortem o b se rv a ­
t i o n s :
H e a r t  -  no rm al 
L iv e r  — norm al 
S p leen  -  norm al
K idney -  c o r te x  c o n ta in e d  num erous y e llo w  p a tc h e s .  
M ic ro sc o p ic  f in d in g s :
Heart -  normal
64
S p le e n  — shows e n la rg e m e n t o f  t h e  M a lp h ig ia n  c o rp u s­
c l e s .  The s p le e n  p u lp  c o n ta in s  a m o d era te  amount o f  b lo o d  
end some p ig m e n t.
L iv e r  -  t h e r e  i s  c lo u d y  s w e l l in g  o f  th e  h e p a t ic  c e l l s  
a b o u t th e  p o r t a l  v e in s  w ith  c o m p a ra tiv e ly  few c e l l s  f i l l e d  
w ith  sm a ll f a t  d r o p l e t s .
K idney  -  i n  th e  o u te r  zone o f  th e  c o r te x ,  some o f  th e  
tu b u le s  show o x y p h il  g r a n u la t io n ,  o th e r s  a re  sw o lle n , o th e r s  
desquam ated  and o th e r s  show c o a g u la t io n  n e c r o s i s .  T here 
i s  some c a l c i f i c a t i o n  o f  a  few o f t h e  tu b u le s .  S e v e ra l  a r e a s  
show f o c i  o f  sm a ll lym phocyte i n f i l t r a t i o n  and f i b r o s i s .
The g lo m e ru li  a r e  n o rm al. A few c o l l e c t in g  tu b u le s  c o n ta in  
c a s t s .
25 p e r  c e n t  Maximum T o le ra te d  D ose.
R a t #31 .
K i l le d  seven  days a f t e r  i n j e c t i o n .
The g ro s s  and m in u te  p a th o lo g icQ l  f in d in g s  in  th e  
h e a r t ,  l i v e r ,  s p le e n ,  ahd k id n e y s  a r e  n e g a t iv e .
R a t #29 .
K i l le d  seven  d ay s  a f t e r  i n j e c t i o n .
G ross and m ic ro sc o p ic  pafcliologlc f in d in g s  in  th e  h e a r t ,  
l i v e r ,  s p le e n ,  and k id n e y s  a r e  no rm al.
85
R a t # 3 0 .
K i l le d  tw e n ty -o n e  days a f t e r  i n j e c t i o n .
G ross and h i s t o l o g i c  f in d in g s  in  th e  h e a r t ,  l i v e r ,  
s p le e n ,  and  k id n e y s  a re  n e g a t iv e .
R a t # 2 8 .
K i l le d  tw e n ty -o n e  days a f t e r  i n j e c t i o n .
G ro ss  and  h i s t o l o g i c  f in d in g s  i n  th e  h e a r t ,  l i v e r ,  
s p le e n ,  and k id n e y s  a re  norm al.
86
D is c u s s io n .
The a d m in i s t r a t io n  o f  150# o f  th e  Maximum T o le ra te d  D oses 
o f  m etap h en , m ercu ro p h en , and m e rc u r ic  c h lo r id e ,  p roduced  t i s s u e  
i n j u r i e s  w hich  w ere  e s s e n t i a l l y  i d e n t i c a l  in  c h a r a c te r  and in  
d e g re e . The changes p roduced  by a l l  th r e e  m e rc u r ia ls  a re  
th e r e f o r e  d e s c r ib e d  i n  common a s  fo llo w s ;
The h e a r t  g e n e r a l ly  showed no d e f i n i t e  p a th o lo g ic  changes.
I n  seme a n im a ls  th e r e  was t r a n s v e r s e  f ra g m e n ta tio n  o f  th e  mus­
c le  f i b r e a ,  i n  a  few , i n t e r s t i t i a l  f i b r o s i s  and i n f i l t r a t i o n  w ith  
ly m p h o cy tes .
The s p le e n  was u s u a l ly  e n la rg e d  and engorged  w ith  b lo o d . The 
M a lp h ig ia n  c o rp u s c le s  w ere e n la rg e d  and  som etim es c o n ta in e d  plasm a 
c e l l s  a s  w e ll  a s  ly m p h o cy tes . The p u lp  g e n e r a l ly  c o n ta in e d  la r g e  
am ounts o f h e m o s id e r in  and o f t e n  many lym phocy tes and a  few  p lasm a 
c e l l s .
I n  m ost o f  t h e  a n im a ls  when any h i s t o lo g i c  change was n o ted  in  
th e  l i v e r ,  i t  com prised  o n ly  c lo u d y  s w e ll in g  o f  th e  h e p a t ic  c e l l s  
i n  th e  p e r i - p o r t a l  a r e a s .  R e la t iv e ly  few o f  th e s e  c e l l s  c o n ta in e d  
em ail f a t  d r o p l e t s .  I n  one in s ta n c e  where th e  a n im a l su rv iv e d  
f o r  a  lo n g e r  p e r io d  o f  t im e ,  t h e r e  was p e r i - p o r t a l  i n f i l t r a t i o n  
w ith  lym phocy tes and p lasm a  c e l l s  and some f i b r o s i s .
The k id n e y s  r e v e a le d  th e  most c o n s ta n t  and co n sp icu o u s 
c h an g es. A ty p ic a l  t o x ic  n e p h ro s is  was produced  in  each  i n s t a n c e .  
The g lo m e ru li  g e n e r a l ly  w ere n o t in v o lv e d , o c c a s io n a l ly  s e v e ra l
8?
a p p e a re d  h y p e re m ic , o th e r s  p a r t i a l l y  anem ic , and in f r e q u e n t ly  
a  g r a n u la r  e x u d a te  was fbund in  th e  c a p sv ila r  sp a c e s  o f  o th e rs *
The c o n v o lu te d  tu b u le s  w ere a l t e r e d  m ost, th e  changes v a ry in g  
o n ly  i n  i n t e n s i t y  and  m o s tly  governed  by th e  l e n g th  o f  tim e  
th e  an i mal  su rv iv ed *  The l e s io n s  were c h a r a c te r iz e d  by 
c lo u d y  s w e l l in g ,  c a s t s ,  o jJyph il^ . g ran u la r^  and f a t t y  d e g en e ra ­
t i o n ,  e p i t h e l i a l  d esq u am atio n , c o a g u la t io n  n e c r o s i s ,  and c a l -
-o icf  dt'*op^U' t e a t U /  h ( i i ' ; '  :
c i f i c a t i o n .  I n t r a c e l l u l a r  f i b r i n ,  and sm a ll d r o p le t s  o f  f a t  w ere
A /.
found  i n  t h e  c o n v o lu te d  tu b u le s  in  th e  o u te r  and in te rm e d ia te  
zones*
The s t r a i g h t  c o l l e c t i n g  tu b u le s ,  aid th e  lo o p s  o f  H enle 
u s u a l ly  e scap ed  se v e re  in ju ry *  As a  g e n e ra l  r u l e  th e  changes 
w ere c o n fin e d  t o  m odera te  c lo u d y  s w e l l in g ,  o r  a  th in n in g  o f  
th e  w a lls*  A nim als s u rv iv in g  f i v e  d ay s  o r  more som etim es showed 
ly m p h o c y tic  i n f i l t r a t i o n  and th e  developm ent o f  young f ib ro u s  
t i s s u e  in  th e  i n t e r s t i c e s  o f  th e  t i  ssue*
M e rcu ric  c h lo r id e  p roduced  n e c r o s i s  o f  th e  m u sc u la r  t i s s u e  
a t  th e  s i t e  o f i n j e c t i o n  in  two a n im a ls . T h is  c o n d it io n  was 
n o t  found i n  an y  o f  th e  m etaphen and m ercurqphen r a t s .
A s tu d y  of h i s t o l o g i c a l  changes p roduced  by th e  a d m in is t r a t io n  
o f  25$ o f  th e  jfayttwrm T o le r a te d  Dose o f  m etaphen , m ercurophen , 
and m e rc u r ic  c h lo r id e ,  showed t i s s u e  changes in  o n ly  one an im al —
88
a  r a t  i n j e c t e d  w ith  m ercu ro p h en . The k id n e y s  showed c loudy  
s w e l l in g  o f th e  c o n v o lu te d  tu b u le s  and ly m p h o cy tic  i n f i l t r a t i o n  
and  f i b r o s i s .  A l l  o th e r  t e s t  a n im a ls  showed no a l t e r a t i o n s  o f  
th e  t i s s u e s  s tu d ie d .
The r e s u l t s  o b ta in e d  in  th e s e  e x p e rim e n ts  seem to  j u s t i f y  th e  
c o n c lu s io n  t h a t  150$ Maximum T o le ra te d  D oses o f  m etaphen , m ercu ro ­
p h e n , and m e rc u r ic  c h lo r id e  p ro d u ced  t i s s u e  changes w hich w ere 
e s s e n t i a l l y  id e n t i c a l*  L e s io n s  o f  th e  h e a r t  m u sc le , s p le e n ,  
and l i v e r ,  w ere  n o t  c o n s ta n t  and when d e m o n s tra te d  a t  a l l  were 
o f  m in o r im p o rta n c e . The k id n e y  showed th e  m ost c h a r a c t e r i s t i c  
c h a n g e s ; nam ely , a  t o x ic  n e p h ro s is*
I t  was n o t  p o s s ib le  to  d i f f e r e n t i a t e  th e  l e s io n s  p ro d u ced  
by  th e  t h r e e  m e r c u r ia ls  s tu d ie d  from  one a n o th e r .  The d e g re e  
o f  t i s s u e  i n j u r y  p ro d u ced  depended a p p a re n t ly  on th e  amount o f  
m ercury  a d m in is te r e d  and th e  l e n g th  o f  s u r v iv a l .  One f o u r th  o f 
th e  Maximum T o le r a te d  Dose p ro d u ced  no e v id e n t  h i s to lo g ic  changes*
89
SUMMARY*
o
Y a tre n  and h e x y lr e s o r s in ^ t l  (1 -1000) do n o t have s u f f i c i e n t  germ i­
c id a l  power to  p e rm it t h e i r  u se  in  s m a ll  enough q u a n t i t i e s  f o r  th e  
p r e s e r v a t io n  o f  b i o lo g i e s .
C o l lo id a l  c h lo r o - c r e s o l , 25%, and c o l l o i d a l  c h lo r o - c r e s o l ,  50%, 
c o l l o i d a l  c h lo ro - th y m o l, 25%, and c o l l o i d a l  c h lo ro - th y m o l, 50%, 
b e c a u se  o f  t h e i r  d e s t r u c t iv e  a c t io n  i n  p r e c i p i t a t i n g  th e  album inous 
s u b s ta n c e s  to  such  a  m arked d e g re e  a re  u n s u ite d  f o r  b io lo g ic  p r e s e r v a ­
t i v e s .
I n t r a m u s c u la r  i n j e c t i o n s  i n  r a t s  o f  1% s o lu t io n s  o f  m etaphen 
and m ercurophen  w ere a p p ro x im a te ly  14 t im e s  more to x ic  th a n  pheno l 
and t r i k r e s o l .  I n  serum , m ercurophen  h a s  an app rox im ate  g e rm ic id a l  
p o te n c y  40 tim e s  g r e a t e r ,  and m etaphen 25 tim e s  g r e a t e r  th a n  p h en o l 
and t r i k r e s o l *
The in c r e a s e d  g e rm ic id a l  power o f th e  m e rc u r ia ls  c au se s  them  to  be 
l e s s  to x ic  th a n  p h e n o l and t r i k r e s o l ,  when u se d  a s  b io lo g ic  p r e s e r v a t iv e s .
N e i th e r  m etaphen  no r m ercurophen  p r e c i p i t a t e  p r o t e in s  a t  t h e i r  
g e rm ic id a l  s t r e n g th  w h ile  b o th  p h e n o l and t r i k r e s o l  do*
The u se  o f  p h e n o l and t r i k r e s o l  in  th e  s p in a l  c a n a l i s  p r o h ib i te d  
by  ex trem e  to x ic  a c t io n  upon th e  r e s p i r a t o r y  and vasom otor c e n te r s .
Serums p r e s e rv e d  w ith  m etaphen and m ercurophen when i n je c t e d  in to  th e  
s p in a l  c a n a l  had no e f f e c t  upon th e  b lo o d  p re s s u re  o r  r e s p i r a t o r y  move­
ment s .
I t  was d e m o n s tra te d  t h a t  th e  ty p e  o f  t i s s u e  in ju r y  p roduced  by
90
m etaphen  and  m ercurophen  i s  e n t i r e l y  dep en d en t upon th e  q u a n t i ty  
o f  m ercu ry  a d m in is te re d *  T w e n ty -f iv e  p e rc e n t  Maximum T o le ra te d  Doses 
p ro d u c e  no h i s t o l o g i c  changes*
As f a r  a s  c o u ld  he  d e te rm in e d  n e i t h e r  p h e n o l, t r i k r e s o l ,  
m e tap h en , n o r  m ercurophen  d e c re a se d  th e  p o te n c y  o f  t o x in s  and a n t i ­
to x in s *
CONCXJtJSIONS.
1* Of th e  b io lo g ic  p r e s e r v a t iv e s  s tu d ie d  th e  m e r c u r ia l  m ercurophen 
a p p e a rs  t o  b e  th e  m o st s a t i s f a c t o r y  w ith  m etaphen a s  second choice*
Zm F u r th e r  s t u d i e s  on th e  a d a p t a b i l i t y  o f  m e r c u r ia ls  a s  b io lo g ic  
p r e s e r v a t i v e s  seem w a rran ted *
i