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19ZO 


THE  CHEMICAL  NATURE  OF  THE  ANTIGENIC 
SUBSTANCES  IN  BACILLUS  COLI 


DISSERTATION 

Presented  in  Partial  Fulfillment  of  the  Requirements  for  the 

Degree  of  Doctor  of  Philosophy  in  the  Graduate 

School  of  the  Ohio  State  University 


BY 

EDWARD  EVERETT  HALE  BOYER,  B.  Sc.,  M.  Sc. 


The  Ohio  State  University 
1920 


'      '    :-    '•  '.:V\.:  :.  •  ;• 


THE  CHEMICAL  NATURE  OF  THE  ANTIGENIC 
SUBSTANCES  IN  BACILLUS  COLI 


DISSERTATION 

Presented  in  Partial  Fulfillment  of  the  Requirements  for  the 

Degree  of  Doctor  of  Philosophy  in  the  Graduate 

School  of  the  Ohio  State  University 


BY 

EDWARD  EVERETT  HALE  BOYER,  B.  ScM  M.  Sc. 


The  Ohio  State  University 
1920 


I 

- 


EDWARDS  BROTHERS 


THE  CHEMICAL  NATURE  OF  THE  'A'WTTiGE^O,  ^B^'AK 
IN  BACILLUS  COLI 


Introduction. 

Certain  substances  when  taken  into  the  body  paren- 
terally  will  cause  the  production  and  appearance  of 
protective  substances  within  the  body  fluids.   The  pro- 
tective substances  we  call  antibodies.   That  which  calls 
forth  the  production  of  antibodies  is  known  as  antigen. 
When  antigens  are  allowed  to  enter  into  contact  with 
their  respective  antibodies,  either  within  the  animal 
body  or  in  a  test  tube,  definite  changes  are  observed; 
thus  we  have  manifestations  of  these  changes  in  the  phe- 
nomena of  agglutination,  precipitation,  complement  fix- 
ation and  other  serological  reactions. 

But  of  what  do  these  changes  consist?  Are  they  true 
chemical  reactions  which  follow  the  usual  laws  of  chemis- 
try, or  are  they  due  to  alterations  in  surface  energy  and 
molecular  attraction  in  a  physical  or  physico-chemical 
sense?  And  what  is  the  nature,  chemically,  of  the  react- 
ing substances? 

% 

Most  authorities  state  that  all  antigens  are  protein 
substances.   Such  statements,  as  will  be  shown  later,  are 
based  to  a  considerable  extent  upon  prejudice  and  precon- 
ceived notions  which  are  not  entirely  borne  out  by  exact 
experimental  procedure.   The  substances  most  generally 
used  as  antigens  are  body  fluids  (blood  serum),  body 
cells,  and  bacterial  bodies.   These  substances,  in  the 
dried  state,  are  composed  largely  of  protein  and  mineral 
salts,  with  a  great  predominance  of  the  former.   Fats  and 
lipoidal  material  are  often  present  in  such  small  amounts 
as  to  escape  detection  unless  large  quantities  of  the 
native  material  are  used  in  the  analysis.   Since  the  pro- 
teins seem  to  be  the  important  constituent  of  substances 
used  as  antigens,  and  since  such  substances  may  be  used 
with  equal  results  after  the  salts  have  been  removed  by 
dialysis  or  other  method,  it  was  but  natural  to  consider 
the  proteins  as  antigenic  principles. 

A  few  investigators  have  endeavored  to  demonstrate 
the  value  of  fats  and  lipoids  in  serological  work.   Thus, 
Jobling  and  Peterson  (1)  showed  that  when  bacteria  were 
injected  into  the  blood  stream  they  absorbed  lipoidal 
material  which,  ordinarily  acts  as  the  anti-enzyme  con- 
stituent.  Noguchi  (2)  showed  that  the  tetanolysin  frac- 
tion of  the  tetanus  toxin  was  neutralized  by  cholesterin 
and  alcoholic  extracts  of  blood  serum.   Muller  (3) 

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demonstrated  that  the  action  of  tetanolysis  (which  is  an 
antigen)  has  to  do  with  lipoid  substances,  and  that 
alcohol- soluble  lipoids  inhibit  the  action  of  tetano ly- 
sis; thus  confirming  Noguchi's  findings.   Landsteiner  (4) 
found  that  ether  extracts  of  red  blood  corpuscles  were 
capable  of  neutralizing  tetanolysin.   He  also  showed  that 
ether- soluble  lipoidal  substances  were  involved  in  serum 
hemolysis.   Bang  and  Forsmann  (5)  found  that  hemolytic 
activity  was  due  to  lipoid  substances  in  the  stroma  of 
the  red  blood  cells.   These  lipoid  substances  were  ob- 
tained by  extraction  of  the  cells  with  ether.   Upon 
analysis  they  found  the  extracted  material  to  be  composed 
of  lecithin,  cholesterin,  a  phosphatid  and  a  cerebroside. 
This  material,  when  used  as  an  antigen  and  injected  into 
susceptible  called  forth  the  production  of  hernolytic 
amboceptors.   Working  with  various  serological  reactions 
Kyes  (6)  (7)  demonstrated  that  lecithin  was  the  ingre- 
dient which  played  the  part  of  complement.   Sachs  (8) 
studying  hemolytic  reactions,  determined  the  importance 
of  lecithin  in  hemolysis.   Pick  (10)  obtained  a  precipo- 
tinogen  by  trypsin  digestion  of  egg  albumin,  and  he  was 
unable  to  demonstrate  protein  in  such  an  antigen.   Some 
workers  confirmed  this  finding,  while  others  were 
unsuccessful. 

The  results  obtained  by  these  workers  have  not  gone 
unchallenged.   Nor  does  their  work  necessarily  indicate 
anything  concerning  the  chemical  nature  of  antigens.   It 
has  been  of  value,  however,  in  demonstrating  the  fact 
that  there  is  a  relation  between  fats  and  lipoids,  and 
antigens.   As  to  what  this  relation  consists  of  has  been 
quite  obscure,  but  the  evidence  is  strongly  in  favor  of 
the  theory  that  serological  reactions  are  manifestations 
of  changes  in  surface  tension  and  molecular  attraction, 
and  not  true  chemical  reactions  according  to  Ehrlich's 
idea. 

The  chief  objection  to  the  antigenic  nature  of  fats 
and  lipoids  has  been  that  the  investigators  overlooked 
the  possibility  of  a  protein  constituent  being  present. 
Thus  the  other  extract,  or  other  extracted  material, 
might  have  contained  protein  material  unsuspected  by  the 
workers.   Furthermore,  the  antigens  may  have  been  not  in 
a  pure  lipoid  state,  nor  in  a  pure  protein  state,  but 
rather  in  the  condition  of  a  complex  conjugated  lipo- 
protein  which  perhaps  was  taken  up  by  the  solvent.   Thus, 
in  their  studies  on* anaphylaxis,  Pick  and  Yamanouchi  (11) 
used,  as  antigens,  alcoholic  extracts  of  horse  serum. 
This  extract  was  evaporated  and  redis solved  until  it  gave 
a  negative  biuret  test.   They  were  successful  in  using 


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such  an  antigen  to  produce  anaphylaxis.   In  a  similar 
manner  Bogomolez  (12)  used  alcohol  and  ether  extracts  of 
egg  yolks  to  produce  anaphylactic  phenomena.   It  is 
known,  furthermore,  that  the  solubilities  of  proteins 
and  other  substances  may  be  profoundly  altered  by  the 
presence  of  lipoids.   The  existence  of  a  protein- free 
antigen,  therefore,  was  not  established  until  the  recent 
work  of  Warden  (to  be  considered  further  on). 

There  is,  however,  one  exception,  namely  the 
protein- free  antigen  of  Ford  and  Abel  (9).   These  men, 
studying  the  poisons  of  Amanita  phalloides,  found  that 
the  toxic  principle  was  an  active  glucoside.   Their  find- 
ings have  never  been  refuted. 

Those  who  favor  the  protein  theory  of  the  nature  of 
antigens  are  prone  to  criticise  all  experiments  which 
attribute  fats  and  lipoids  a  role  in  antibody  production. 
The  one  chief  critical  point  lies  in  the  face  that  the 
lipoidal  antigens  have  not  been  satisfactorily  proven  to 
be  protein- free.   On  the  other  side,  however,  is  it  not 
fair  to  inquire  if  the  so-called  protein  antigens  are 
fat- free?  Take  practically  any  native  antigen  for  use  in 
antibody  production,  and  we  must  concede  that  there  is 
as  definite  an  amount  of  lipoid  as  there  is  protein;  and 
merely  because  of  the  proportionately  greater  quantity 
of  protein  is  no  reason  at  all  to  assume  the  role  of  the 
latter  as  antigen. 

Numerous  investigators,  assuming  that  a  given  sub- 
stance was  an  antigen,  have  endeavored  to  separate  the 
protein  material  and  to  determine  what  particular  frac- 
tion of  the  protein  served  in  the  capacity  of  antigen. 
In  the  beginning  they  find  that  the  substance  under  con- 
sideration is  a  true  antigen.   They  then,  as  a  rule,  pro- 
ceed to  separate  the  various  protein  constituents  either 
by  fractional  precipitation  or  by  enzymotic  digestion. 
And  finally  they  arrive  at  a  point  in  the  analysis  beyond 
which  antigens  are  not  found.   In  reviewing  this  v/ork  one 
is  surprised  to  find  in  how  few  experiments  the  fat  and 
lipoid  constituents  have  been  eliminated.   It  is  possible 
that  these  lipoids  were  sufficiently  bound  to  the  pro- 
teins as  to  be  precipitated  along  with  them;  and  due  to 
subsequent  procedure,  during  the  separation  of  the  vari- 
ous, fractions,  the  fats  were  liberated  and  discarded,  or 
remained  in  combination  with  higher  fraction.   If  the 
antigenic  principle  was  lodged  in  the  fatty  portion,  this 
theory  would,  -of  course,  account  for  the  results  ob- 
tained.  Similarly,  in  analyses  by  means  of  enzymotic 
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lipase.   Or  the  presence  of  aatabolic  materials  may  even 
produce  a  change  in  the  configuration  of  the  fat  mole- 
cule.  There  is  no  doubt  that  some  fat -free  proteins  are 
antigens,  but  before  we  eliminate  fats  and  lipoids  from 
the  class  of  antigens,  we  must  first  eliminate  them  from 
the  various  protein  substances  used  as  antigens. 

Although  it  would  seem  that  some  pure  proteins  are 
antigens,  it  would  also  be  equally  apparent  that  other 
pure  proteins  are  not  antigens.   Starin  (13),  for 
instance,  working  with  a  purified  gelatin,  was  unable  to 
demonstrate  antigenic  function.   It  has  been  suggested 
that  antigenic  properties  of  proteins  rest  with  the  aro- 
matic radicals  attached  to  the  amino  acids.   Perhaps 
these  radicals  have  some  affinity  for  fats  and  lipoids, 
so  that  in  the  absence  of  such  radicals  there  is  also  an 
absence  of  fats;  i.e.,  during  the  process  of  fractional 
analysis  those  portions  which  bear  the  aromatic  radicals 
are"  split  off  with  fats,  and  the  lower  fractions,  being 
fat-free,  are  non- antigenic.  The  value  and  importance 
of  the  fatty  acids  in  treatment  of  diseases  due  to  acid 
fast  bacteria  have  been  demonstrated  for  several  years. 
Thus  Chaulmoogra  oil,  sodium  salts  of  the  fatty  acids  of 
cod- liver  oil  are  used  in  treatment  of  tuberculosis  and 
leprosy  (14).  Walker  and  Sweeney  (15)  showed  that  the 
fatty  acids  obtained  from  Chaulmoogra  oil  are  specifi- 
cally bactericidal  for  acid-fast  micro-organisms,  but 
not  for  non-acid  fast  micro-organisms.   Similarly, 
Hollman  and  Dean  (16)  demonstrated  the  theraputic  value 
of  esters  of  the  fatty  acids  of  Chaulmoogra  oil  in  the 
treatment  of  leprosy.   These  findings  lend  strength  to 
the  theory  that  fats  are  intimately  concerned  in 
immunological  reactions. 

The  most  interesting  work  which  has  been  done  on 
the  antigenic  nature  of  fats  is  found  in  the  recent 
experimental  data  contributed  by  Warden.   This  investi- 
gator has  been  studying  the  problem  from  the  experimen- 
tal standpoint  during  a  period  of  several  years.   In 
1915  he  suggested,  as  a  result  of  his  studies  on  the 
relation  between  bacterial  fats  and  proteins,  that  some 
fats  are  in  a  lipoidal  combination  with  protein  and  are 
not  hydrolysed  until  the  nitrogen  portion  is  thoroughly 
broken  up  (17).   In  the  same  year,  reporting  further 
results,  using  the  gonococcus  as  the  source  of  his 
material,  he  found  that  the  organic  nitrogen  of  the 
gonococcus  did  not  seem  to  be  altogether  available  as 
antigen.   The  fats  of  the  gonococcus,  chemically  iso- 
lated, possessed  a  much  higher  antigenic  power  (18), 
Later,  (19)  (20),  it  was  found  that  the  fats,  as 


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glycerol  estus,  did  not  cause  as  great  a  degree  of 
antigen  reaction  when  used  in  serological  tests  as  did 
the  usual  gonococcus  antigen;  but  by  saponif ication  of 
the  fats  with  subsequent  .isolation  of  pure  nitrogen- free 
fatty  acids,  the  serological  tosts  gave  better  results 
when  such  fatty  acids  were  used  as  antigens.   Continuing 
along  this  line  of  investigation,  Warden  (21)  determined 
that  the  neutral  fats  of  the  gonococcus  were  of  little 
value  when  used  as  antigens  in  complement-fixation  tests, 
but  very  excellent  and  specific  results  were  obtained  by 
using  the  fatty  acids,  or  still  better,  the  alkaline 
salts  of  the  fatty  acids.   Due  to  such  findings  he  con- 
cluded that  the  important  factors  in  such  reactions  were 
not  only  the  empirical  chemical  constitutions  of  the 
respective  antigens,  but  that  the  molecular  configuration 
was  as  significant  and  as  specific.   It  has  been  shown, 
furthermore,  by  the  same  author  (22)  that  if  the  fats  are 
added  to  a  "solution"  of  colloidal  cholesterol  the  degree 
of  dispersion  of  the  molecular  aggregates  of  the  antigens 
is  greatly  increased,  thus  presenting  more  surface  and 
producing  a  more  active  and  sensitive  antigen.   This 
finding,  of  course,  adds  confirmation  to  our  ideas  con- 
cerning the  colloidal  reactions  in  immunology.   By  simi- 
lar methods  the  same  author  demonstrated  the  antigenic 
nature  of  the  fats  from  Bacillus  typhosus,  Bacterium 
anthracis,  pneumococci,  streptococci,  and  red  blood  cells 
(23) (24).   These  fats  were  used  not  only  as  antigens  in 
experiments  "in  vitio",  but  were  injected  into  suitable 
animals,  with  subsequent  production  of  specific  anti- 
bodies.  Moreover,  the  antibodies  obtained  were  not  only 
specific,  but  were  also  protective  against  a  dose  of 
homologous  bacteria  which  was  fatal  for  a  control  animal. 

The  careful  work  of  Warden  sheds  a  vast  amount  of 
light  on  the  question  of  chemical  composition  of  antigens. 
His  results  have:  not  yet  been  confirmed,  nor  have  they 
been  disproved.   No  one  will  deny  that  certain  fat- free 
pure  proteins  cannot  be  used  as  antigens.   But  no  one, 
on  the  other  hand,  can  maintain  that  all  antigens  are 
protein. 

The  object  of  this  work  is  to  study  the  chemical 
nature  of  the  antigen  or  antigens,  of  Bacillus  coli;  to 
determine  whether  such  antigens  are  protein,  fats,  carbo- 
hydrates, salts,  or  a  mixture  of  two  or  more  of  these; 
and  to  analyse,  if  possible,  the  antigen  in  order  that  we 
may  know  of  what  it'  is  composed.   The  experimental 
methods  and  data  are  set  forth  in  the  succeeding  pages. 


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EXPERIMENTAL  WORK. 

The  strain  of  Bacillus  coli  which  was  selected  was 
taken  from  a  stock  culture.  To  confirm  the  identity  of 
the  organism  it  was  subjected  to  the  following  tests; 

a.  The  organism  was  a  small  rod- shaped  organism, 
with  morphology,  as  regards  the  usual  criteria,  typical 
of  Bacillus  coli. 

b.  It  was  negative  to  Gram's  method  of  staining. 

c.  It  produced  abundant  acid  and  gas  when  grown  in 
nutrient  broth  containing  one  per  cent  lactose.   It  pro- 
duced acid  and  gas  also  in  dextrose  and  sucrose  broth. 

d.  It  did  not  liquefy  gelatin. 

e.  It  was  readily  agg3.utinated  by  high  dilution 
(up  to  1:2500)  of  B.  coli  antiserum. 

In  order  that  a  sufficiently  large  quantity  of 
material  be  obtained,  the  cultures  were  grown  in  liter 
flasks.   About  thirty  flasks  were  used  at  one  time.   To 
each  flask  was  added  ten  grams  of  peptone,  four  grams  of 
sodium  chloride  and  one  liter  of  tap  water.   This  titra- 
table  acidity  of  such  medium  varied  for  each  lot,  but 
usually  fell  between  1.5  to  2.0$  acid,  using  phenolphtha- 
lein  as  indicator.   This  comparatively  strong  acid  re- 
action was  not  adjusted,  because,  as  the  Bacillus  coli 
produces  abundant  ammonia  in  sugar- free  protein  media, 
the  ammonia  gradually  neutralizes  the  acids  present. 
This  particular  species  usually  thrives  in  a  medium  whose 
initial  reaction  is  not  greater  than  2.5$  acid.   On  the 
other  hand,  its  growth  ceases  when  the  reaction  reaches 
an  alkalinity  of  2.5$.   It  is  obvious,  therefore,  that 
with  a  comparatively  strong  initial  acid  reaction,  the 
longer  will  be  the  time  interval  before  the  maximum  alka- 
line reaction  is  reached;  and  presumably,  therefore,  the 
greater  will  be  the  number  of  bacteria  produced;  and  it 
is  a  large  number  of  bacteria  that  is  necessary  for  the 
work.   The  flasks  were  not  plugged  with  cotton,  but  were 
capped  with  three  thicknesses  of  wrapping  paper.   They 
were  then  sterilized  in  the  autoclave  for  two  hours  at 
fifteen  pounds  pressure.   Then  they  were  allowed  to  stand 
at  room  temperature  for  a  few  days,  at  the  end  of  which 
time  were  discarded  any  flasks  which  revealed  the 
presence  of  bacterial  growth.   The  paper  caps  were  next 
washed  with  bichloride  of  mercury  solution  1:1000.   The 
flasks  were  then  inoculated  with  a  broth  culture  of  the 


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Bacillus  coll.   The  inoculations  were  made  by  the  use  of 
a  hypodermic  syringe  and  needle.   The  broth  culture  was 
aspirated  into  a  sterile  syringe;  then,  after  inserting 
the  needle  diagonally  through  the  paper  caps,  a  few  drops 
were  forced  into  the  flasks.   This  method  has,  to  commend 
it,  the  advantage  that  the  flasks  are  never  opened  for 
inoculation  after  having  been  sterilized.   The  inoculated 
flasks  were  then  incubated  at  37°C.  for  about  ten  days. 

In  order  to  separate  the  bacteria  from  the  fluid, 
the  contents  of  the  flasks  were  run  through  a  Sharpies 
laboratory  "supercentrifuge".   This  machine  is  very  simi- 
lar to  the  ordinary  cream  separator  in  construction  and 
mechanism,  but  is  run  by  steam  or  compressed  air.   Suffi- 
cient force  is  obtained  to  completely  separate  out  all 
particles  in  suspension. 

This  material  was  washed  with  sterile  physiological 
sodium  chloride  solution  and  recentrifuged.   The  sediment 
consists  of  bacteria  with  precipitated  salts,  sulphides, 
and  perhaps  other  amorphous  material.   Most  of  these 
foreign  substances  are  easily  removed  by  filtration 
through  cotton. 

The  remaining  bacteria  were  now  subjected  to  saponi- 
fication  in  potassium  alcoholate,  at  a  temperature  of 
100°C.  for  one  hour.   The  resultant  material  was  then 
acidified  with  dilute  hydrochloric  acid.   It  was  expected 
that  this  procedure  would  yield  a  definite  layer  of  fatty 
acids  which  could  be  removed,  but  such  proved  not  to  be 
the  case.   Instead,  there  was  a  mass  of  solid  material, 
including  fatty  acids,  proteins  and  salts,  in  a  state  of 
very  fine  suspension,  which  could  not  be  separated  by 
gravity  or  by  centrifugal  force  at  three  thousand  revolu- 
tions per  minute.   It  was  then  decided  to  extract  the 
fatty  acids  with  ether.   After  shaking  up  with  ether  it 
was  found  that  only  about  ten  percent  of  the  ether  could 
be  recovered  and  that  only  when  comparatively  large 
amounts,  were  used.   Most  of  the  ether  became  a  part  of 
the  suspensoid  mass.   The  entire  mass  was  then  trans- 
ferred to  a  ten- inch  porcelain  evaporating  dish,  and 
evaporated  at  37°C.  for  several  days,  until  the  moisture 
content  was  very  low  (too  low  to  support  the  previous 
suspension).   The  residual  mass  was  then  shaken  up  with 
ether  and  the  latter  separated,  removed  and  evaporated 
at  room  temperature.   The  residue  consisted  of  a  very 
minute  amount  of  crystalline  and  amorphous  material, 
presumably  fatty  acids.   The  amount  was  considered  too 
small  to  work  with. 


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Another  thirty  liter  mass  was  prepared  as  in  the 
above  experiment.   The  bacteria  were  washed  and  separated 
by  the  same  method,  but  instead  of  being  saponified,  they 
were  treated  directly  with  ether.   This  ether-bacteria 
mixture  was  allowed  to  stand,  with  frequent  shaking,  for 
six  days.   The  ether  was  then  pipetted  off.   The  residue 
was  again  treated  with  ether  in  the  same  manner.   These 
two  ether  extracts  were  then  evaporated.  The  residue 
consisted  of  brownish-colored  fats,  some  of  which  ap- 
peared crystalline,  other  amorphous.   This  material  was 
washed  with  water,  then  taken  up  with  ether,  the  ether 
removed  and  evaporated.   The  weight  of  the  resultant  fats 
amounted  to  approximately  0.4  gram.   The  fats  were  then 
saponified  with  potassium  alcoholate  at  100°C.   After 
acidifying  with  hydrochloric  acid,  and  cooling,  a  defin- 
ite layer  of  fatty  acids  was  obtained.   The  alcohol  was 
evaporated  to  nearly  dryness  and  the  fatty  acids  extract- 
ed with  ether.   This  ethereal  extract  was  concentrated  to 
dryness  and  the  fatty  acids  taken  up  with  alcohol.   This 
was  treated  with  sodium  carbonate  to  convert  the  fatty 
acids  into  the  sodium  salts.   The  sodium  salts  were  then 
drystallized  out  and  redissolved  in  100  cc.  of  alcohol. 
This  constitutes  the  fatty  antigen. 

It  is  desirable,  at  this  point,  to  examine  the 
antigen  to  see  if  any  protein  material  is  present. 
Several  different  tests  were  used  as  follows: 

a.  The  biuret  test.   To  three  cubic  centimers  of 
antigen  was  added  an  equal  amount  of  strong  potassium 
hydroxide  solution.   The  mixture  was  well  shaken  and 
treated  with  a  few  drops  of  very  dilute  copper  sulphate 
solution.   Absence  of  color  change  indicated  absence  of 
protein. 

b.  The  Kjeldahl  method,   10  cc.  of  the  antigen  were 
mixed  with  20  cc.  of  concentrated  sulphuric  acid.   0.2 
gram  of  copper  sulphate  was  added  and  the  material  was 
boiled  gently  for  ninety  minutes.   It  was  then  cooled  and 
diluted  to  250  cc.  with  distilled  water,  then  neutralized 
with  hydroxide,  adding  a  slight  excess  of  the  alkali. 
The  material  was  then  distilled,  the  distillate  being 
collected  in  25  cc.  of  N/10  sulphuric  acid.   When  one- 
half  the  liquid  had  passed  over,  the  process  was  stopped. 
The  acid  solution  was  then  neutralized  with  tenth-normal 
sodium  hydroxide,  using  congo  red  as  an  indicator.   An 
equal  amount  of  alkali  was  necessary  to  neutralise  the 
acid,  thus  indicating  the  absence  of  nitrogen  in  the 
antigen. 


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c.  The  ninhydrin  test.   To  ten  cubic  centimeters  of 
antigen  was  added  0.2  cc.  of  a  one  per  cent  aqueous 

solution  of  ninhydrin  (triketophydridene  hydrate).   The 
mixture  was  boiled  for  exactly  one  minute  after  the 
appearance  of  the  first  bubbles  on  the  side  of  the  tube. 
No  color  change  could  be  detected,  even  after  cooling 
and  standing  for  four  hours.   This  indicates  the  absence 
of  alplia  amino  acids. 

d.  The  cyanide  test.   About  three  cubic  centimeters 
of  the  antigen  was  evaporated,  and  the  residue  fused  with 
metallic  sodium.   The  fused  mass  was  placed  in  a  small 
amount  of  distilled  water,  boiled  and  filtered.  To  the 
filtrate  was  added  a  few  drops  of  fenous  sulphate,  fenie 
chloride  and  hydrochloric  acid.   Absence  of  a  blue  colora« 
tion  indicated  an  absence  of  nitrogen. 

It  is  evident  that  we  are  dealing  with  a  nitrogen- 
free  substance. 

The  original  bacterial  cells,  after  being  doubly  ex- 
tracted with  ether,  were  then  suspended  in  500  cc.  of 
sterile  physiological  sodium  chloride  solution.   For 
preservation,  sufficient  carbolic  acid  was  added  to  make 
a  O.5$  solution.   The  greater  part  of  the  solids  in  this 
mass  are  supposedly  bacterial  proteins  and  for  experi- 
mental purposes  the  mass  is  called  the  protein  antigen. 
Obviously  it  is  a  bacterial  antigen  minus  the  fats. 

For  use  as  a  control  there  was  next  prepared  a 
suspension  in  physiological  saline,  of  a  twenty-four  hour 
agar  culture  of  the  same  strain  of  B.  Coli,   This  was 
made  up  with  0.5$  phenol  and  it  constitutes  the  B.  coli 
of  antigen. 

We  now  have  three  distinct  antigens: 

1.  Sodium  salts  of  the  fatty  acids  of  B.  coli. 

2.  B.  coli  after  being  extracted  with  ether. 

3.  A  simple  suspension  of  B.  coli. 

It  is  the  purpose  now  to  determine  whether  or  not 
these  antigens  are  true  antigens;  i.e.,  whether  or  not 
they  give  proper  antigenic  reactions  "in  vitro",  "in 
vivo",  or  in  both.   To  this  end  the  antigens  were  inject- 
ed into  suitable  animals,  at  certain  intervals,  in  order 
to  obtain  antibodies. 

The  fatty  antigen  was  diluted  with  physiological 
salt  solution,  approximately  one  part  of  the  alcoholic 
solution  to  five  parts  of  the  saline.   This  gives  a  very 


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10 

opalescent  suspension.   The  protein  antigen  was  used 
without  further  dilution,  as  was  also  the  control  B.  coli 
a'ntigen.   The  course  of  the  immunation  is  given  in  the 
following  proctocals: 

Table  #1.   Rabbit  injected  with  fat  antigen. 


Time 

Dose 

Weight  in 

Remarks 

interval 

in  cc. 

grams 

0.25 

1770 

Prompt  recovery  from  injection 

5  days 

0.5 

1800 

»      M      i»       M 

6  days 

0.75 

1960 

Mild  signs  of  shock 

6  days 

1.00 

2040 

Prompt  recovery 

12  days 

0.00 

2100 

Animal  bled 

Table  #2.  .Rabbit  injected  with  protein  antigen 


Time 

Dose 

Weight  in 

Remarks 

interval 

in  cc  . 

grams 

0.25 

1630 

Moderate  shock. 

5  days 

0.50 

1635 

"       " 

6  days 

0.50 

1680 

Severe 

6  days 

0.50 

1695 

Moderate 

12  days 

0.00 

1730 

Animal  bled 

This  animal  was  the  third  to  be  used  for  protein 
injections;  the  other  two  died  immediately  after  the 
initial  injection,  thus  demonstrating  the  possibility  of 
a  toxic  fraction  present  in  the  protein  mass. 


Table 


Rabbit  injected  with  B.  coli  antigen 


Time 

Dose 

Weight  in 

Remarks 

interval 

in  cc. 

grams 

0.5 

1690 

Prompt  recovery 

5  days 

1.0 

1700 

ti      ti 

6  days 

1.0 

1700 

ii      it 

6  days 

1.0 

1850 

it      it 

12  days 

0.0 

1910 

Animal  bled 

The  injections  were  all  given  by  the  intravenous 
method,  using  the  prominent  veins  in  the  ears  as  the 
sites  of  injection. 


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11 

Twelve  days  after  the  last  Injection  each  rabbit  was 
anesthetized  with  ether  and  by  the  use  of  a  sterile 
syringe  and  needle  about  forty  cubic  centimeters  of  blood 
were  aspirated  from  the  heart.   The  blood  was  placed  in 
sterile  glass  tubes,  which  were  then  placed  in  the  ice- 
box until  the  serum  separated  from  the  clot.   The  clear 
serum  was  then  pipetted  off  into  sterile  tubes  and  an 
equal  amount  of  glycerol  was  added  for  preservative. 

Each  of  the  above  antisera  was  then  tested  against 
the  three  different  antigens.   The  tests  used  were  as 
follows: 

a.  Complement  fixation 

b.  Agglutination 

c.  Precipitin 

d.  Anaphylaxis 

a.  The  complement  fixation  test.   In  testing  the 
antigens  and  antibodies  by  the  method  of  fixation  of 
complement,  guinea-pig  serum  diluted  with  ten  parts  of 
physiological  saline  was  used  for  complement.   The  anti- 
human-rabbit  hemolytic  system  was  used;  i.e.,  the  blood 
serum  of  rabbits  which  were  immunized  against  human  red 
blood  corpuscles  was  used  as  hemolytic  antibody.  The 
following  table  sets  forth  the  procedure  for  obtaining 
the  hemolytic  antibody: 

Table  #4.   Rabbit  injected  with  hemolytic  antigen. 


Time  interval 

Dose  in  cc. 

Weight 

Remarks 

2.0  cc. 

1680 

Prompt  recovery 

4  days 

3.0  cc. 

1725 

U          11 

4  days 

4.0  cc. 

1755 

it       it 

5  days 

5.0  cc  . 

1790 

ti       H 

10  days 

0.0  cc. 

1835 

Animal  bled 

The  animal  was  bled  and  the  serum  was  obtained  and 
preserved  in  a  manner  similar  to  that  previously  des- 
cribed.  The  hemolytic  system  was  titrated  as  follows: 
the  antigens  consisted  of  a  five  per  cent  suspension  of 
washed  blood  cells;  the  complement  was  guinea  pig  serum 
diluted  with  ten  parts  of  salt  solution.   The  hemolytic 
antibody  was  then  diluted  with  twenty  parts  of  salt 
solution  and  titrated  according  to  the  following  table: 


.jor^ni   dsfil    orftf 
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-  ni  bf^OBlq  aerfrf  eiew'rfolrfw  teocfjLrd  csfilg  ellisrfs 
T  .cJolo  e/ij  raoi":  bectaiaqse  nrjjiea  9.r£J  lictnir  xocf 
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o  .ro-ss 


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~^?> 

>c   glq  sonijj^  BBW    ^rtf?  "  o   9/i^    ;a.Cj 

i 

• 


Table  #5. 

Tube  #1 
-  .   #2 

Complement  0.2cc. 
n        tt 

antigen  0.2  cc. 
ii 

•H  Antibody 
0.5.cc.+ 
"  0.10 

No  henolysis 
it     n 

#3 

ii        n 

n 

"  0.15 

Partial 

#4 

n        n 

M 

11  0.20 

Hemolysis 
Complete  " 

#5 

n        ii 

u 

11  0.25 

11       U 

#6 

n        n 

M 

"  0.30 

n    n 

#7 

n        n 

II 

11  0.35 

n    u 

#8 

it        n 

It 

"  0.40 

M    u 

t 
C  r(  #9 

o  o( 

n  i(#10 
s 

M        it 
n        u 

M 
It 

n  _______ 
"  0.4 

No  hemolysis 
u     n 

The  unit  of  antibody,  therefore,  was  0.2  cc.   Using 
this  unit  of  antibody  and  varying  the  amount  of  comple- 
ment, the  unit  of  complement  was  determined.  Two  units 
of  complement  were  used  in  running  the  complement-fixation 
test,  also  two  units  of  hemolytic  antibody.  Each  bacter- 
ial antigen  was  nob  tested  against  each  of  the  three  anti- 
sera.   The  results  of  such  tests  are  shown  in  the  follow- 
ing proctocols:   (The  preliminary  titrations  of  antigens 
and  antibodies  are  purposely  omitted  because  they  vary 
with  each  lot  and  merely  tend  to  confuse  the  reader. 

Table  #6.   Protein  Antigen  and  Homologous  Antiserum. 


Tube 

56°  for  30' 

37°  for  30' 

37°  for  30' 

Result 

1 
2 
3 

Antiserum 

IT-"  H 

Antigen  &  Complement 

•fhemolytic  system 

Partial 
hemolysis 
Complete 

u      u 

Antigen  & 

it                      M 

s% 

l» 

t,                       .. 

V^L 

It 

•i                       n 

H 

0   w 

.. 

ii                       if 

3% 

P,.o  " 

n 

. 

n                       n 

•^      ! 

v* 

^0   " 

t» 

I* 

„ 

Jt 

.,VL     \          r> 

^•Tv    /  T     J 

» 

kO  ' 

" 

n                       M 

.- 

iplx  <* 

;       .  uselqmoo   erf^ 
5      .xbodiun 

3fi3    lO    ff.0i39 


'lo 


brus   xboc 

sew   -Jn^fnolqraoo   'lo    ;.?i.nt/   ori?    t 
ni   beet/  9ri°«t7  ^fismalqr^oo 
orl  lo   Svtinjj  owrf  osZfl    Xci 
bod  sec1    joii  ee-\v  negl-lriP 
rioi/c    lo   i.iltrr.O'i    eriT      .B 
arfT)      :elcon^ooiq 


' 


. 
o 


'Oo  10!     YS 

imjnalqrftoO  ^  n^si^rfA 


.j 


13 


Table  #7.      Antigen  and  Anti-coli  Serum. 


Tube 

56°  for  30  » 

37°  for  30' 

37°  for  30* 

Result 

1 
2 
3 

Antiserum 
it 

+Antigen  &  Complement 
ii 

+Hemolytic  system 
M         n 

Complete 

hemolysis 

n     u  "" 

Table  #8.   Protein  Antigen  and  Ant  i  -fat  Serum. 

Tube 

56°  for  30  » 

37°  for  30  « 

37°  for  30' 

Result 

1 
2 

Antiserum 
ii 

+  Antigen  &  Complement 
it 

+Hemolytic  system 
n       n 

Hemolysis 
n 

Table  #9.   Protein  Antigen  and  Normal  Serum 

Tube 

56°  for  30' 

37°  for  30' 

37°  for  30' 

Result 

1 

Serum 

+  Antigen  &  Complement 

+Hemolytic  system 

Hemolysis 

2 

ii 

it 

n       it 

n 

Table  #10.   B.Coli  Antigen  and  Homologous  Antiserum. 

Tube 

56°  for  30' 

37°  for  30' 

37°  for  30' 

Result 

1 
2 
3 

Antiserum 
ti 

+  Antigen  &  Complement 
ii 

+Hemolytic  system 
ii       M 

n       n 

No  hemolysis 

Complete  " 
n     n 

it          ir 

Table  #12.  B  Coli  Antigen  and  Anti-protein  Serum 

Tube 

56°  for  30' 

37°  for  30» 

37°  for  30' 

Result 

1 
2 
3 

Antiserum 
ii 

+  Antigen  &  Complement 
it 

Hemolytic  system 
ii       ii 

n       u 

Complete 

hemolysis 
n    M 

n    » 

ii          ii 

•t<-»?j  tctnA 
'OS 


.8%- 


ivTornsh-f   rfrt  •  ;?•: >1  TrcO   v    ns' 


«0«  10!  °V5 


A   ft.:     : 

•06    1. 
[••rrrtoO 


•  f 


«0£   ic'l  Ovio 


•A  4 


mw:  ': 


n£»-:>  /  ^ riA   i  I oO .  9      ,2 1-^   o  I  cr/?T 

iol  c 


Ian?' 


«?.J 


14 


Table  #13.  B  Coli  Antigen  and  Normal  Serum 


Tube 

56°  for  30» 

i 

37°  for  30  « 

37°  for  30  » 

Result 

1 
2 
3 

Serum 
ii 

+  Antigen  &  Complement 
u 

•fHemolytis  system 
n        n 

n        n 

Complete 

hemolysis 
u     n 

n     u 

ii         n 

Table  $14.   Fat  Antigen  and  Hemologous  Ant  i  serum 

Tube 

56°  for  30' 

37°  for  30* 

37°  for  30  » 

Result 

1 
2 
3 

Antiserum 
it 

•f  Antigen  &  Complement 
u 

+Hemolytic  system 
n       u 

n       n 

No  Hemolysis 

Complete  " 
n      n 

n          n 

Table  $15.   Fat  Antigen  and  B.  Coli  Antiserum 

Tube 

56°  for  30' 

37°  for  30' 

37°  for  30' 

Result 

1 

Antiserum 

+  Antigen  &  Complement 

+Hemolytic  system 

No  hemolysis 

2 

3 

ii 

n 

u       u 
n       n 

Complete  " 
it      it 

ti           n 

Table  $16.   Fat  Antigen  and  Protein  Antiserum 

Tube 

56°  for  30» 

37°  for  30* 

37°  for  30' 

Result 

1 
2 
3 

Antiserum 
it 

+Antigen  &  Complement 
+     " 

fHemolytic  system 

+ 
u       n 

Complete 

hemolysis 
n     it 

u     ti 

ii         ii 

Table  $17.   Fat  Antigen  and  Normal  Serum 

Tube 

56°  for  30  » 

37°  for  30' 

37°  for  30» 

Result 

1 
2 

3 

Serum 
ii 

Antigen  &  Complement 
n 

Hemolytic  system 
n       u 

n       u 

Complete 
hemolysis 

It        II 

n      ii 

,  u          fi 

H  brrjs  nsgr.htA    jfi'? 


' 


: 


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J      •           r          I  J.  -  '          -1.   V  1  I  '*.  1 

H 

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If 

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II                                      II 

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15 

In  the  preceding  (16-17)  protocols  all  tubes  marked 
#2  are  the  serum  controls,  used  to  show  that  the  serum 
itself  will  not  fix  complement.   Similarly  the  #o  tubes 
are  antigen  controls. 

The  Bacillus  coli  antigen,  when  used  with  a  hemolo- 
gous  antiserum,  (table  10)  gives  complete  fixation  of 
complement.   The  same  antigen  when  used  with  the  ant i- fat 
serum,  (table  11)  gives  similar  results;  but  when  used 
with  the  anti-protein  serum,  (table  12)  one  fails  to 
obtain  a  positive  reaction.   These  findings  confirm  the 
idea  that  fats  may  act  as  antigens  and  lend  proof  to  the 
specificity  of  the  reaction.   The  protein  antigen,  when 
used  with  its  homologous  antiserum  (table  6)  gave  results 
which  were  nearly  completely  positive,  but  it  is  worthyof 
note  that  the  reaction  was  not  as  clear  cut  or  as  satis- 
factory as  the  previous  tests.   When  used  with  the  other 
antisera  (tables  7-9)  there  was  no  complete  fixation. 
Thus  there  is  evidence  that  the  fats  are  true  specific 
antigens  and  that  the  protein  material  plays  a  minor  role, 
if  any. 

B.  Agglutination  tests.   Although  the  presence  of 
agglutinins  probably  does  not  indicate  protective  ability, 
as  Bordet  (26)  showed  that  horse  serum  clumps  tetanus 
bacilli;  yet  the  horse  is  very  susceptible  to  tetanus, 
nevertheless,  the  experimental  production  of  agglutinins 
in  the  serum  of  immunized  animals  is  very  indicative  of 
a  specific  reaction. 

The  sera  obtained  from  the  immunized  animals  were 
tested  against  the  B.  coli  antigen.   Ten  drops  of  serum, 
in  various  dilutions,  were  added  to  ten  drops  of  antigen. 
The  tubes  were  well  shaken  and  incubated  at  37°C.  for  one 
hour.   The  results  are  recorded  as  follows: 


Complete 
agglutination 


Partial 
agglutination 


No 
agglutination 


Table  #18.   B.  Coli  Antigen  with  Heraologous  Antiserum 


Dilution  of 

Serum 

1:100 

1:200 

1:300 

1:400 

1:500 

1:600 

1:700 

1:800  1:1200 

Result 

* 

•j- 

-;- 

+ 

+ 

+ 

+ 

/j  n  -r'v    ^at.     i^;i*    1:1  on    ~.i:If  IsaO   -jrfT 

•  •  •-•      •>  £»-.••  '-  v     (  C\  r     •*  fff  <"•  -I  \        "ff  if^ip  r  "'TT  R     p  i 

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oajj  rrerfw  #$$l:3rrB   tMnnc   oriT      .dnr<m9lq 
^1  UP.  91  isllffiis    af/vl;    (II   efcted)    ,; 
91    olcfect)    trrri,"ief-   nto^oTq-i^n^   e.rfj   rfrtiw 
.'-'  v  B 


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c  -/•:!£  3)  tnurf.9£l^nB  cuogoloriori  acfl  -rf^iw  boeu 
rcf  tovioieoq  ^Idtfsl^moo  Y^'1^0^  Qi^w  rfolrfw 
o  Tfislo  SB  ^on  ssw  rioictojsot  erfo  cfsrW  o^on 
boe.t/  noriV'  .ecfseJ  L-woiveiq  sifcf  cs  Y'10^0-6^ 
alqrnoo  or:  BBW  oporto  (G-V  seldB^)  BioBJt^fiB 

o^£   set  i?":   9ii-i   d-crfJ   eonsbive  r,i  e'larf^   s 
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1  Jt 


jia   ^^Br»ii-:ij   cforr 
aqmulo  n:ir'ij«!    ©sior!  J 
GJ    elo't.vqect^fs   if-^ov 
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16 


Table  #19.  B.  Coli  Antigen  with  Antifat  Serum 


Dilution  of  Serum 
Result 

1:100 

4 

1:200 

4 

1:300 
•f 

1:400 

4 

1:500 

+ 

1:600 

i 

1:700 

1:800 

Table  #20.   B.  Coli  Antigen  with  Antiprotein  Serum 


Dilution  of  Serum 
Result 

1:100 

1:200 

1:300 

1:400 

1:500 

1:600 

1:700 

Table  #21.  B.  Coli  Antigen  with  Normal  Serum 


Dilution  of  Serum  ; 
Result 

1:50 

1:100 

1:200 

1:300 

1:400 

1  :  500 

1:600 

A  control  suspension  of  the  B.  coli  antigen  did  not  show 
agglutination* 

Comparison  of  the  results  by  use  of  the  complement 
fixation  and  agglutination  tests  give  a  very  striking 
picture.   We  find  that  the  fat  antigen  (table  19)  gives 
rise  to  specific  antibodies  and  that  the  protein  antigen 
(table  20)  is  of  negligible  importance  in  this  respect. 

C.  The  Precipitin  Reaction.   This  test  has  to  do 
with  the  precipitation  of  solid  matter  out  of  solution  of 
invisible  "colloidal  suspension".   Obviously  the  B.  coli 
and  protein  antigens  cannot  be  used,  but  the  fat  antigen 
is  suitable  for  the  reaction.  The  most  desirable  antigen 
is  one  which  is  perfectly  clear  and  transparent.   Clarity, 
in  a  fat  suspension,  depends  upon  the  state  of  the  fat 
particles,  so  that  the  greater  the  degree  of  dispersion, 
the  greater  the  clarity.   A  perfectly  clear  antigen  was 
not  obtained,  but  a  sufficient  degree  of  dispersion  was 
made  to  run  the  tests.   Ten  drops  of  serum  was  layered 
under  ten  drops  of  antigen,  and  after  the  incubation  at 
37°C  for  fifteen  minutes,  the  following  results  were 
obtained. 


Table  #22.      Fat   Antigen  and  Homologous   Ant  i  serum 

Dilution  of  Serum 

1:25 

1:50 

1:75 

1:100 

1:125 

1:150 

1:175 

Result 

+ 

+ 

4- 

+ 

± 

- 

- 

!_L    * 


r,   f  ,-• 

\    J50/19S    Ifc    ffCK 


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1   -  i    -  i    : 

** 

v  i 

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?.   lo 


^to  noaarrio 


dl^ilgsn 


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• 


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ts.tfrfsiffl  £)iloe  lo 

lvdO      ."ffoicneqej 

ircf     b^ct    scf  ^oon 


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17 


Table  #23.   Pat  Antigen  and  Anti-coli  Serum. 


Dilution  of  Serum 
Result 

1:25 

+ 

1:50 
+ 

1:75 

+ 

1:100 

± 

1:125 

1:150 

1:175 

Table  #24.   Pat  Antigen  and  Antiprotein  Serum 


Dilution  of  Serum 
Result 

1:25 

1:50 

1:75 

1:100 

1:125 

1:150 

1:175 

Table  #25.   Fat  Antigen  and  Normal  Serum 


Dilution  of  Serum 
Result 

1:25 

1:50 

1:75 

1  :  100 

1:125 

1:150 

1:175 

+  =  Complete  precipitation 

*  =  Partial        " 

-  =  No  " 

The  results  are  very  indicative  of  a  specific  pre- 
cipitatin  reaction,  in  which  the  fats  play  the  most 
important  part.   Too  much  weight,  however,  should  not  be 
attached  to  these  reactions  of  precipitins  because  in 
some  cases  (preliminary  titrations)  spontaneous  reaction 
occurred.   Such  reactions  also  frequently  occurred  after 
the  fifteen  minute  incubation  period.   This  was  probably 
due  to  the  unstable  dispersoid  phase  of  the  antigen,  so 
that  it  was  very  susceptible  to  the  reactions  of  the 
various  sera. 

D,  Anaphylactic  Reactions.   It  was  found  by  trial 
that  1.0  cc.  of  a  1:10  dilution  of  the  fatty  antigen  was 
usually  fatal  to  guinea  pigs  when  injected  directly  into 
the  heart,  but  the  pigs  could  withstand  the  same  amount 
of  a  1:20  dilution.   This  amount,  therefore,  was  used  as 
the  "toxic"  dose.   The  animals  were  sensitized  by  intra- 
cardiac  injections  of  1.0  cc.  of  a  1:500  dilution.   They 
promptly  recovered  from  the  effects  of  the  injections  and 
gradually  gained  in  weight.   Tv/elve  days  after  the  sensi- 
tizing dose  the  pigs  received  the  "toxic"  dose.   The 
results  are  recorded  as  follows: 


I   001:1 


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18 


Table  #26.   Anaphylactic  Reactions. 


Temperature 

Symptoms 

Before  injection 

102.  2°F 

5  rain,  after  " 
10   "     "    ft 
15   "     "    " 

100.4 
100.0 
'  99.6 

Slight  motor  paralysis  of 
hind  legs 
Increased  motor  paralysis  of 
hind  legs 
Dyspnoea 

20   "     "    " 

99.0 

25   "     "    " 

99.8 

Paralysis  decreasing 

30   "      "    " 

100.4 

Dyspnoea  disapeared 

35   "      "    " 

101.0 

Paralysis  disappeared 

A  normal,  non- sensitized  animal  was  then  injected 
with  the  "toxic"  dose.   The  results  were  as  follows: 


Table  #27 

Temperature 

Symptoms 

Before   injection 

100.8 

None 

5  min.  after   " 

99.2 

n 

10   "     "     " 

99.4 

: 

15   "     « 

99.8 

M 

20   "      "     " 

100.0 

II 

25   "      "     " 

100.4 

" 

30   "      M     " 

100.4 

;  »   •  '  '  ::. 

35   "      "     " 

100,4 

M 

These  experiments  were  repeated  three  times  and  the 
results  were  practically  the  same  with  each  set.   The 
sensitized  animals  always  showed  a  marked  sub-normal 
temperature,  with  paralysis  of  the  hind  legs.   The  acted 
as  if  they  were  trying  to  "huddle  up"  and  the  hair  "stood 
out".   The  underlying  principles  of  anaphylaxis  are  as 
yet  very  obscure,  but  it  is  very  doubtful  that  proteins 


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are  essential  for  sensitization.   Either  the  fats  may 
split  off  a  molecule  that  is  toxic,  thus  giving  rise  to 
the  reaction  according  to  Vaughn1 s  idea,  or  the  reaction 
may  be  due  to  variations  in  surface  energy  and  molecular 
attraction. 

So  far,  then,  we  have  prepared  an  antigen  from  the 
fats  of  Bacillus  coli  and  have  demonstrated  the  ability 
of  this  antigen  to  cause  the  production  of  various  sero- 
logical  reactions  when  injected  into  suitable  animals. 
Our  interest  now  turns  toward  the  chemical  composition  of 
the  antigen  and  the  attempt  is  made  to  analyse  the  fatty 
mass . 

In  the  previous  preparation  of  the  fatty  antigen 
about  0.4  gram  of  fat  was  obtained  from  thirty  litres  of 
broth  culture.   In  order  to  obtain  a  larger  amount  of 
material  two  hundred  litres  of  broth  were  made  and  inocu- 
lated as  before.   The  bacilli  were  collected  in  the  same 
manner.   The  bacteria  mass  was  then  transferred  to  a 
litre  flask  and  covered  with  ether.   The  flask  was  then 
fitted  with  a  reflux  condenser  and  heated  to  50°C  on  an 
automatic  electric  water-bath.   The  heat  was  applied  con- 
tinuously for  a  period  of  forty-eight  hours.   At  the  end 
of  this  time  the  flask  and  contents  were  cooled  and  the 
ether  was  pipetted  off  into  another  flask.   This  latter 
flask  was  tightly  stopped  and  placed  in  the  icebox.   The 
bacterial  residue  was  extracted  again  in  the  same  manner 
and  the  ether  extract  was  added  to  the  first  lot.   A 
total  of  five  extractions  was  made.   The  mixed  ether  from 
the  five  extractions  was  then  evaporated  under  reduced 
pressure  (obtained  by  attaching  an  ordinary  filter  pump), 
at  a  temperature  of  25°C.   The  remaining  fatty  mass  was 
then  treated  with  potassium  alcoholate.   The  flask  was 
fitted  with  a  reflux  condenser  and  the  fats  were  saponi- 
fied at  a  temperature  of  90°C.   The  flask  and  contents 
were  cooled  and  acidified  with  hydrochloric  acid.   The 
flask  was  then  attached  to  the  reflux  condenser  and  the 
fluid  was  evaporated  at  70°C  under  reduced  pressure.   The 
concentrated  mass  of  fatty  acids  was  then  taken  up  with 
ether.   The  ethereal  solution,  containing  the  fatty 
acids,  was  pipetted  off  and  the  ether  evaporated  at  room 
temperature  under  reduced  pressure.   The  mass  of  residual 
fatty  acids  weighed  2.873  grams.   A  portion  of  these 
acids  was  fluid  at  30°C,  but  the  remainder  were  solid  up 
to  about  65°C.   The  acids  were  then  subjected  to  distil- 
lation in  steam.   The  distillate  was  treated  with  solid 
sodium  chloride  ,and  then  shaken  up  with  ether.   The  ether 
layer  was  removed  and  evaporated  in  vacuo  at  room  temper- 
ature.  There  was  a  very  slight  trace  of  residue,  too 


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small  to  weigh  with  any  degree  of  accuracy.   This  repre- 
sents the  volatile  constituent. 

The  remaining  mass  was  redissolved  in  alcohol  and 
made  slightly  neutral  by  adding  potassium  alcoholate, 
using  phenolphthalein  as  indicator.   The  so3.ution  was 
diluted  with  distilled  water  to  about  100  cc.  30  cc.  of 
a  ten  per  cent  solution  of  lead  acetate  was  diluted  to 
150  cc.  with  water  and  boiled.   The  hot  solution  was  run 
into  the  soap  solution,  constantly  shaking  the  latter  so 
that  the  lead  soaps  would  adhere  to  the  sides  of  the 
flask.   The  flask  was  then  filled  with  hot  water  and  then 
allowed  to  cool.   The  lead  salts  all  adhered  to  the 
glass,  leaving  a  clear  supernatant  fluid  which  was  de- 
canted off.   The  soap  was  then  shaken  up  with  ether  at 
37°C.   The  ethereal  solution  was  then  cooled  and  filtered. 
The  filtrate  contains  the  lead  salts  of  the  liquid  fatty 
acids.   This  filtrate  was  then  shaken  with  twenty  per 
cent  hydrochloric  acid,  to  decompose  the  lead  salts.   As 
the  fatty  acids  are  set  free  from  the  lead  salts,  they 
are  taken  up  by  the  ether.   The  ethereal  layer  was  re- 
moved; washed  with  water  until  the  washings  were  free 
from  acid;  and  the  ether  then  evaporated.   The  remaining 
liquid  fatty  acid  weighed  1.021  gram.   This  acid  solidi- 
fied when  cooled  on  ice  and  melted  at  13°C.   The  neutra- 
lization value  was  then  found  as  follows:   1.021  grams 
were  neutralized  by  34.9  cc.  of  N/10  KOH.  This  is  equiva- 
lent to  34.1  cc.  per  gram  of  acid,  or  191  grams  KOH.   The 
neutralized  acids  were  now  acidified  with  20%  hydro- 
chloric acid  and  shaken  out  with  ether.   The  ether  was 
then  evaporated  and  the  iodine  value  was  obtained  as 
follows  (Hubb's  process): 

The  iodine  solution  is  prepared  by  dissolving  13.5 
grams  iodine  in  250  cc.  of  95$  alcohol,  and  by  dissolving 
15  grams  of  mercuric  chloride  in  250  cc.  of  95$  alcohol 
and  mixing  these  two  solutions.   A  standardized  solution 
of  sodium  thiosulphate  was  prepared  by  dissolving  24 
grams  of  the  salt  in  one  liter  of  water.   0.2  gram  of 
iodine  and  1.0  gram  of  potassium  iodide  are  dissolved  in 
about  50  cc.  of  water.   This  solution  is  titrated  to 
neutrality  by  the  thiosulphate  solution,  using  starch 
solution  as  indicator.   It  was  found  that  15,4  cc .  of  thi- 
sulphate  solution  was  equivalent  to  0.2  gram  of  iodine. 
The  1.021  gram  of  fatty  acid  is  dissolved  in  10  cc.  of 
chloroform  and  50  cc.  of  iodine  solution  added.   The 
solution  was  allowed  to  stand  for  fifteen  hours.   20  cc. 
of  10$  potassium  iodide  solution  was  added,  and  the 
volume  made  up  to  500  cc.  with  water.   This  solution  was 
then  titrated  with  the  thiosulphate  solution.   It  was 


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found  that  0.863  gram  of  iodine  was  absorbed.   100  grams 
of  the  fatty  acid,  therefore,  would  absorb  84.6  grams  of 
iodine,  the  iodine  value,  then,  being  84.6.   The  neutra- 
lization value  of  oleic  acid  is  198;  its  melting  point  is 
14°C;  and  its  iodine  value  90.   We  are  dealing  with  an 
unsaturated  liquid  fatty  acid,  whose  melting  point  is 
13°C;  neutralization  value  191;  and  iodine  value  84.6. 
Oleic  acid  is  an  unsaturated  liquid  fatty  acid  with  the 
above  given  constants.   The  unknown  approaches  very 
closely  to  oleic  acid  in  these  three  values,  and  it  is 
permissible,  under  the  circumstances,  to  consider  such 
unknown  as   oleic  acid. 

The  remaining  insoluble  lead  soaps  were  now  decom- 
posed with  hydrochloric  acid  and  the  liberated  fatty 
acids  were  extracted  with  ether.   The  ethereal  portion 
was  pipetted  off,  washed  with  water,  and  the  ether 
evaporated.   The  residue  weighed  1.84  grams.   The  iodine 
value  was  2.3,  due,  probably,  to  admixture  of  the  un- 
saturated acid  previously  described.   We  are  dealing, 
therefore,  with  two  or  more  saturated  fatty  acids,  since 
it  was  found  that  about  one-half  of  the  mass  melted  at  a 
temperature  of  about  32°C,  while  the  remaining  portion 
did  not  melt  until  heated  to  a  temperature  of  68°C.   The 
saturated  fatty  acids  which  have  melting  points  between 
30°C  and  35°C  are  confined  to  one  member,  capric  acid. 
The  saturated  acids  which  melt  between  65°C  and  70°C  are 
confined,  likewise,  to  one  member,  stearic  acid.   To  sep- 
arate these  acids,  when  one  has  such  a  very  small  amount 
of  material,  is  hardly  a  feasible  procedure.   Nor  can  one 
determine  the  analysis  by  the  melting  points,  since  such 
acids  form  entectic  mixtures  which  often  have  a  melting 
point  higher  or  lower  than  any  of  the  individual  con- 
stituents (27) . 


SUMMARY 

The  Bacillus  coli  has  been  divided  chemically  into 
two  parts,  one  of  a  protein  nature,  the  other  fatty.   The 
protein  portion  alone  is  not  an  antigen  in  the  sense  that 
it  will  give  rise  to  the  specific  antibodies  of  the 
bacillus  coli. 

The  fatty  portion,  on  the  contrary,  contains  the 
substances  essential  for  the  production  of  specific  anti- 
genie  reactions. 


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It  has  been  analysed  to  show  the  following  approxi- 
mate constitution: 

a.  Volatile  fats,  trace 

b.  Oleic   acid 

c.  Capric  acid 

d.  Stearic  acid 


CONCLUSIONS. 

The  specific  antigens  of  the  Bacillus  coli  are 
chemical  entities  bound  up  in  the  fats  of  the  organism. 

The  proteins  of  the  organism  are  not  concerned  in 
the  specificity  of  the  antigens. 

The  fats  of  the  antigens  consist  of  volatile, 
saturated  and  unsaturated  acids. 


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BIBLIOGRAPHY. 


1.  Jobling  &  Peterson,  Quotes  from  Zinsser,  "Infection 


2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 
10. 
11. 

12. 

13. 

14. 
15. 
16. 
17. 
18. 
19. 
20. 


22. 
23. 
24. 
25. 

26. 
27. 


and  Resistance'1,  1918,  p.  39. 
Noguchi,  H.,  Univ.  Pa.  Med.  Bull.,  Nov.  1902. 
Muller,  Centrabbl.  f.  Bakt.,  34,  1903,  p.  562. 
Landsteiner,  "  ,  33,  1905,  p.  318. 

Bang  &  Porsmann         "   ,  40,  1906,  p.  151. 
Kyes,  Berl,  klin.  Wchnschi,  1902,  pp.  856  &  918. 

1904,  p".  494. 

Sachs,  Wien,  1905,  p.  901. 

Ford  oc  Abel,  Jour.  Biol.  Chem.,  1902,  2,  p.  273. 
Pick,  E.,  Kolle  u.  Wassermann  Handbuch  Vol.  2. 

u.  Yamanouchi,  Zeitche.  f.  Gumuntatschforch, 
I,  1909. 
Bogomolez, 

5,  1910. 

Star in,  W.,  Jour.  Infec.  Dis.,  1918,  25,  p.  139. 
Editorial,  Jour,  Am.  Med.  Assoc.  1919,  73,  p.  609. 
Walker  &  Sweeney,  Jour.  Infec.  Dis.,  1920,  p.  256. 
Hollmann  oc  Dean,  Jour.  Cut.  Dis.,  1919,  57,  p.  367. 
Warden,  C. C. ,  Jour.  Infec.  Dis.,  1915,  16,  p.  426. 

Jour.  Am.  Med.  Assoc.,  19T5,  65,  p. 2080. 

1917,  53,  p.  432. 
&  Schmidt,  L.E.,  Jour.  Lab.  &  C3.£n.  Med., 

1916,  1   p. 333. 
Jour.  Infec.  Dis.,  1918,  22,  p.  133. 

1918,  33,  p.  504. 

1919,  3?,  p.  285. 
1919,  35,  p.  399. 

Vaughn,  V.,  Protein  Split  Products,  Lea  &  Pebiger, 

1912. 

Bordet,  J.,  Am.  de  1'inst.  Pasteur,  1896,  10,  p.  193. 
Lewkowitsch,  Chemical  Technology  of  Oils,  Fats  & 

Waxes.  3rd  ed.  vol.  1. 


it  u 


AUTOBIOGRAPHY 

I,  Edward  Everett  Hale  Boyer,  was  born  in  Lynn, 
Massachusetts,  February  15,  1893.   I  received  all  of  my 
secondary  school  education  in  the  public  and  high  schools 
of  the  City  of  Lynn;  my  undergraduate  education  at  the 
Massachusetts  Agricultural  College,  from  which  I  obtained 
the  Degree  of  Bachelor  of  Scisnce  in  1916.   I  pursued 
graduate  studies  at  the  Ohio  State  University,  from  which 
I  obtained  the  Degree  of  Master  of  Science  in  1917  and 
the  Degree  of  Doctor  of  Philosophy  in  1920. 


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Binder 
Gaylord  Bros. 

Makers 

Stockton,  Calif. 
PAT.  JAN.  21.  1908 


56394 1 


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