﻿532 
  Seventh 
  Annual 
  Repokt 
  of 
  the 
  

  

  grown 
  until 
  they 
  will 
  grow 
  no 
  longer 
  — 
  that 
  is, 
  the 
  products 
  re- 
  

   sulting 
  from 
  their 
  multiplication 
  and 
  imparted 
  to 
  the 
  medium 
  

   have 
  inhibited 
  their 
  further 
  development. 
  It 
  is 
  not 
  definitely 
  

   determined 
  just 
  what 
  these 
  products 
  are 
  or 
  just 
  how 
  they 
  are 
  

   elaborated. 
  Briefly 
  stated, 
  the 
  preparation 
  of 
  tuberculin 
  consists 
  

   of 
  the 
  following 
  procedures, 
  viz. 
  : 
  

  

  1. 
  The 
  preparation 
  of 
  the 
  culture 
  medium, 
  distributing 
  it 
  in 
  

   suitable 
  flasks 
  and 
  inoculating 
  it 
  with 
  the 
  growth 
  from 
  a 
  pure 
  

   culture 
  of 
  tubercle 
  bacilli. 
  

  

  2. 
  The 
  flasks 
  are 
  placed 
  in 
  an 
  incubator 
  at 
  a 
  temperature 
  of 
  

   about 
  37° 
  C, 
  where 
  they 
  remain 
  until 
  the 
  growth 
  ceases. 
  The 
  

   length 
  of 
  time 
  necessary 
  to 
  accomplish 
  this 
  depends 
  upon 
  the 
  age 
  

   and 
  condition 
  of 
  the 
  culture 
  from 
  which 
  the 
  inoculations 
  were 
  

   made. 
  From 
  four 
  to 
  ten 
  weeks 
  are 
  usually 
  required. 
  

  

  3. 
  After 
  the 
  maximum 
  growth 
  is 
  attained 
  the 
  cultures 
  are 
  

   sterilized 
  by 
  heat, 
  either 
  by 
  boiling 
  in 
  a 
  closed 
  water-bath 
  or 
  

   heating 
  to 
  a 
  higher 
  temperature 
  in 
  an 
  autoclav. 
  

  

  4. 
  After 
  sterilization 
  the 
  cultures 
  are 
  filtered 
  to 
  remove 
  all 
  of 
  

   the 
  dead 
  bacilli, 
  and 
  then 
  the 
  filtrate 
  is 
  evaporated 
  to 
  the 
  desired 
  

   degree 
  of 
  concentration 
  over 
  a 
  water-bath. 
  

  

  5. 
  The 
  concentrated 
  liquid 
  is 
  passed 
  through 
  a 
  Pasteur 
  or 
  

   Berkefelter 
  filter, 
  standardized, 
  bottled 
  for 
  distribution, 
  and 
  

   labelled 
  with 
  the 
  name, 
  quantity, 
  and 
  size 
  of 
  dose. 
  It 
  should 
  

   be 
  perfectly 
  clear, 
  although 
  the 
  color 
  may 
  vary. 
  If 
  it 
  is 
  cloudy 
  

   it 
  should 
  be 
  rejected 
  when 
  received. 
  

  

  A 
  careful 
  consideration 
  of 
  these 
  various 
  procedures 
  assures 
  one 
  

   that 
  each 
  and 
  every 
  step 
  requires 
  skilled 
  attention. 
  It 
  is 
  logical, 
  

   at 
  least, 
  to 
  presume 
  that 
  if 
  each 
  part 
  in 
  the 
  process 
  is 
  not 
  care- 
  

   fully 
  looked 
  after 
  the 
  product 
  may, 
  to 
  that 
  extent, 
  be 
  unreliable. 
  

   Of 
  the 
  possible 
  errors 
  in 
  its 
  preparation 
  the 
  danger 
  of 
  using 
  too 
  

   young 
  cultures 
  — 
  that 
  is, 
  before 
  the 
  required 
  degree 
  of 
  saturation 
  

   of 
  the 
  bacterial 
  products 
  takes 
  place 
  — 
  is, 
  perhaps, 
  the 
  one 
  most 
  

   apt 
  to 
  occur. 
  For 
  example, 
  if 
  the 
  flasks 
  of 
  bouillon 
  are 
  inocu-r 
  

   lated 
  from 
  an 
  old 
  culture, 
  several 
  wrecks 
  may 
  elapse 
  before 
  the 
  

  

  