66  Standardisation  of  Heart  Tonics.      { February  Pi9i™' 
The  solution  to  be  injected  should  not  contain  more  than  10  per 
cent,  alcohol  and  the  dilution  should  be  made  with  physiological  salt 
solution  (0.85  per  cent.  NaCl). 
The  doses  are  calculated  on  the  weight  of  the  frog,  i.e.,  the 
M.  L.  D.  is  the  minimum  lethal  dose,  per  gram  weight  of  frog. 
For  example,  when  the  frogs  are  of  average  resistance  the  M.  L.  D. 
of  Strophanthin  is  0.000,001  gm.  per  gram  weight  of  frog,  i.e.,  for  a 
30  gram  frog  the  lethal  dose  of  Strophanthin  is  .000,03  gm->  which 
should  be  so  diluted  that  this  amount  is  contained  in  approximately 
0.5  c.c.  Several  series  of  tests  are  necessary  to  establish  the  activity 
of  any  sample  of  unknown  strength  and  since  the  frogs  vary  in 
resistance  among  themselves  and  also  because  of  conditions  more  or 
less  beyond  control,  the  standard  Strophanthin  must  be  tested  at  the 
same  time.  When  the  M.  L.  D.  of  sample  and  of  standard  are 
obtained  the  activity  can  readily  be  expressed  in  Heart  Tonic  Units 
(H.  T.  U.)  by  reference  to  a  table. 
In  the  method  just  given  the  observations  are  to  be  made  at  the 
end  of  twenty-four  hours,  hence  the  one-hour  method  has  certain 
advantages  when  several  series  are  desired  on  a  single  sample  as  soon 
as  possible.  When  the  one-hour  method  is  used  it  is  necessary  to  not 
consider  all  frogs  that  have  not  absorbed  the  dose  injected. 
The  One-Hour  Method. 
"  In  this  method  the  frogs  are  secured  and  kept  in  the  manner 
already  described,  weighed,  and  such  a  dose  is  injected  that  the 
heart  will  be  found  in  complete  systolic  contraction  at  the  end  of 
exactly  sixty  minutes.  The  drug,  properly  diluted  so  as  to  make 
a  volume  of  0.5  to  1  c.c,  is  injected  into  the  anterior  lymph  sac  by 
means  of  a  glass  pipette.  Shortly  before  the  hour  is  up  the  frog  is 
pithed,  tied  to  a  frog  board,  and  the  heart  is  exposed  in  the  usual 
manner.  If  the  heart  is  still  beating,  the  dose  has  been  too  small 
and  must  be  increased  in  subsequent  trials.  In  the  first  series  doses 
are  chosen  with  wide  limits,  which  in  a  second  and  third  series  of 
animals  are  narrowed  down  until  the  smallest  amount  of  the  drug 
which  will  produce  systolic  standstill  in  one  hour  is  found.  Usually 
three  series  of  frogs  are  sufficient  to  assay  one  preparation,  but  in 
case  of  any  irregularity  in  the  reaction  of  any  of  the  frogs  a  fourth 
or  even  a  fifth  series  may  be  necessary." 
The  method  of  Focke  17  is  long  and  complicated  and  does  not 
appear  to  have  any  advantage  over  the  other  frog  methods  that  have 
been  described. 
