Am' Ip%' ^gi7m'  J  Chemical  Reactions  of  Diphtheria  Antitoxin. 
The  determinations  of  the  antitoxic  values  were  made  by  the 
firms  from  which  the  preparations  were  obtained  and  controlled, 
when  any  variation  was  suspected.  The  toxin  was  made  by  the  Cut- 
ter Laboratory  and  its  L  +  dose  determined  there,  but  was  checked 
with  standard  antitoxin  obtained  from  Dr.  G.  McCoy  of  the  Hy- 
gienic Laboratory. 
In  these  experiments  usually  only  three  guinea  pigs  were  used 
for  each  test.  The  injections  were  made  subcutaneously  in  the  mid- 
abdominal  region  and  each  injection  had  a  volume  of  4.5  Cc.3  All 
evaporations  were  done  in  vacuo  between  500  and  55 0  C,  and  prep- 
arations which  were  not  used  immediately  were  usually  kept  in  an 
ice  box.  Instead  of  filtering,  solutions  were  obtained  by  centrif- 
ugalizing  at  4,000  revolutions  a  minute. 
The  guinea  pigs  weighed  about  250  Gm.,  although  owing  to  diffi- 
culty in  obtaining  a  proper  supply,  guinea  pigs  below  250  Gm.  were 
often  used.  At  the  autopsy  of  those  dying  after  the  injections,  en- 
largement and  hemorrhages  into  the  suprarenal  glands,  hemorrhages 
in  the  gastric  mucosa  and  signs  of  local  irritation  were  the  only 
macroscopic  changes  looked  for  and  found  No  histological  exami- 
nations were  made. 
Some  of  globulin  preparation  IV  was  evaporated  to  dryness 
in  vacuo,  and  the  flask  jarred  with  a  vibrator  for  varying  intervals 
of  time  from  July  20  to  July  31,  191 4,  then  put  aside  till  February, 
1917,  but  no  signs  of  crystallization  have  appeared. 
Fifteen  Cc.  of  globulin  II  were  shaken  with  400  Mg.  cholesterin 
and  centrifugalized.    The  fluid  retained  its  full  antitoxic  value. 
After  precipitation  of  antitoxin  with  aluminium  hydroxide4  the 
filtrate  of  globulin  preparation  IV  became  inactive. 
Five  Cc.  of  globulin  preparation  I  were  evaporated  to  dryness 
and  then  twice  extracted  at  room  temperature  with  20  Cc.  acetic 
ether  c.p.  The  undissolved  portion  possessed  about  the  antitoxic 
value  of  the  original,  showing  the  antitoxin  to  be  insoluble  in  acetic 
ether. 
Five  Cc.  of  globulin  preparation  I  were  evaporated  to  dryness 
then  extracted  with  20  Cc.  n/10  NaOH  and  after  a  few  minutes 
neutralized  with  20  Cc.  n/10  HC1.  Even  3  times  the  theoretical 
amount  which  should  neutralize  one  L  -f-  dose  failed  to  protect. 
Evidently  n/10  NaOH  injures  this  antitoxin. 
The  same  amount  of  this  globulin  was  evaporated  and  treated 
3  Rosenau,  M.  J.,  Hyg.  Lab.  Bull.  21,  1905. 
4Joum.  Amer.  Chem.  Soc,  1913,  p.  820. 
