144  The  Opsonins  and  Bacterial  Vaccines. 
manufacture  of  Koch's  old  tuberculin,  the  glycerin  must  be  removed 
by  repeated  washing  with  water  and  finally  with  1-5  per  cent,  salt 
solution.  The  washed  culture  is  worked  up  in  a  mortar  and  cen- 
trifugated  until  the  clumps  are  practically  all  thrown  down,  and  the 
cloudy  layer  or  emulsion  is  removed. 
The  emulsions  must  be  of  uniform  density.  Wright  computed 
the  number  of  germs  in  a  given  volume  by  counting,  but  McFarland 
and  L'Engle  devised  an  apparatus  which  is  called  a  nephelometer, 
consisting  essentially  of  mixtures  of  BaS04,  put  up  in  sealed  tubes, 
which  correspond  to  solutions  containing  from  I  to  10  per  cent,  of 
BaCl2,  which  serve  as  standards.  The  turbidity  of  the  emulsion  is 
compared  in  similar  layer  with  the  standard  tubes  of  BaS04.  They 
found  that  the  tube  containing  "  5  per  cent,  of  BaCl2  corresponds  to 
the  most  useful  bacterial  suspension."  The  permanency  of  the 
emulsions  varies  a  good  deal.  Suspensions  of  the  gonococci  should 
be  used  at  once,  staphylococci  within  two  days,  etc.,  while  the  emul- 
sion of  tubercle  germs  may  be  employed  indefinitely. 
II.  WASHED  WHITE  BLOOD-CORPUSCLES. 
The  finger  or  lobe  of  the  ear  is  punctured  as  in  making  a  blood 
count,  and  the  blood  is  allowed  to  drop  directly  into  normal  saline, 
•85  per  cent,  solution,  containing  about  I  per  cent,  sodium  citrate  ; 
this  decalcifies  the  blood  and  prevents  clotting.  The  corpuscles  are 
completely  precipitated  in  the  centrifuge,  and  then  repeatedly  washed 
with  -85  per  cent,  saline  and  centrifugated,  until  all  traces  of  the 
citrate  and  serum  are  removed.  After  the  final  precipitation  the 
saline  solution  is  withdrawn,  and  the  thin  upper  grayish  layer  of  the 
sediment,  the  leucocytic  cream,  consisting  for  the  most  part  of 
washed  white  blood  cells,  is  removed. 
111.  THE  SERUM. 
Blood  is  obtained  in  the  usual  way,  but  is  collected  in  small,  bent, 
glass  tubes,  which  can  be  readily  held  in  the  centrifuge  and  the 
serum  separated.  In  obtaining  normal  serum,  care  must  be  exer- 
cised in  selecting  a  healthy  subject,  or,  what  is  better,  obtain  serum 
from  the  mixed  blood  of  several  normal  persons'  "  Pool." 
IV.  STAIN. 
With  the  exception  of  Malta  fever  and  tubercle  bacilli,  Leish- 
man's  stain,  consisting  of  eosin  and  methylene-blue,  in  combination, 
