Double  Staining.  —  The  films,  after  suitable  fixation  obtained  by
immersion  in  absolute  alcohol,  alcohol  and  ether,  or  by  heating  as  pre-
viously described,  are  treated  first  with  an  acid  stain  followed  by  a  basic
dye,  rinsed  in  water,  dried,  and  mounted.  A  staining  fluid  containing
acid  and  basic  coloring  principles  may  be  employed  for  this  purpose.
Most  of  the  methods  of  double  staining  are  not  suitable  for  differentiating
all  forms  of  blood-cells,  as  certain  histological  elements  remain  unstained.
Neutrophilic  granules  are  as  a  rule  not  colored,  and  therefore  neutrophilic
myelocytes  cannot  be  distinguished  from  large  mononuclear  leucocytes.
If  only  the  nuclear  structures  are  to  be  studied,  double  staining  is  of  service,
but  is  nevertheless  inferior  to  Leishman's  stain  and  similar  methods.
Plehn^s  stain  has  the  following  formula:

Saturated  aqueous  solution  of  methylene  blue 60  c.c.
One-half  per  cent,  eosin  solution  (in  75  per  cent,  alcohol) 20  c.c.
Distilled  water 40  c.c.
Twenty  per  cent,  solution  of  caustic  potash 5-1  c.c.
Specimens  are  fixed  in  absolute  alcohol  for  from  three  to  five  minutee,
stained  with  Plehn's  solution,  washed  in  water,  dried,  and  mounted.  This
mixture  stains  malarial  parasites  blue  and  eosinophilic  granules  red.
Eosin  and  Methylene  Blue, — A  convenient  plan  consists  in  treating
the  fixed  smear  with  a  solution  consisting  of  eosin  .5  part  in  70  per  cent.
alcohol  100  parts,  for  a  minute  or  two;  wash  the  cover  in  water,  and  then
counterstain  with  a  half-saturated  solution  of  methylene  blue  or  Delafield's
ha?matoxylin  solution  for  a  half  to  one  minute.  The  specimen  is  then
rinsed  in  water,  dried  between  bibulous  paper  or  in  the  air,  and  mounted.
