﻿84 
  Dr. 
  W. 
  B. 
  Bottomley.. 
  The 
  Effect 
  of 
  Nitrogen- 
  -fixing 
  

  

  Effect 
  of 
  Azotobacter 
  chrooeoccurn. 
  

  

  Two 
  series 
  of 
  five 
  dishes 
  each 
  were 
  arranged 
  ; 
  the 
  dishes 
  in 
  Series 
  I, 
  

   numbered 
  from 
  1 
  to 
  5, 
  each 
  containing 
  250 
  c.c. 
  of 
  Detmer's 
  solution, 
  and 
  

   those 
  in 
  Series 
  II, 
  numbered 
  from 
  6 
  to 
  10, 
  each 
  containing 
  250 
  c.c. 
  of 
  the 
  

   same 
  solution, 
  with 
  the 
  addition 
  of 
  - 
  l 
  grm. 
  of 
  a 
  growth 
  of 
  Azotobacter 
  

   chroococcum 
  in 
  every 
  100 
  c.c. 
  This 
  growth 
  was 
  obtained 
  by 
  making 
  streak 
  

   cultures 
  on 
  agar-mannite 
  plates 
  from 
  a 
  pure 
  colony 
  of 
  the 
  organism, 
  and 
  

   incubating 
  for 
  about 
  14 
  days. 
  At 
  the 
  end 
  of 
  this 
  period 
  a 
  copious 
  brown 
  

   gelatinous 
  growth 
  was 
  obtained 
  over 
  practically 
  the 
  whole 
  surface, 
  which 
  was 
  

   carefully 
  scraped 
  off 
  with 
  a 
  spatula, 
  transferred 
  to 
  a 
  tared 
  beaker, 
  and 
  

   rapidly 
  weighed. 
  The 
  mass 
  in 
  the 
  beaker 
  was 
  covered 
  with 
  distilled 
  water, 
  

   and 
  the 
  whole 
  was 
  raised 
  to 
  a 
  temperature 
  of 
  120° 
  C. 
  for 
  one 
  hour 
  in 
  an 
  

   autoclave. 
  The 
  contents 
  of 
  the 
  beaker 
  were 
  then 
  measured 
  and 
  transferred 
  

   to 
  a 
  well-stoppered 
  bottle, 
  the 
  beaker 
  and 
  measuring 
  cylinder 
  being 
  

   repeatedly 
  rinsed 
  out 
  with 
  measured 
  quantities 
  of 
  distilled 
  water, 
  and 
  the 
  

   washings 
  added 
  to 
  the 
  bottle, 
  until 
  a 
  volume 
  of 
  about 
  10 
  c.c. 
  for 
  every 
  gramme 
  

   of 
  the 
  gelatinous 
  growth 
  had 
  been 
  obtained. 
  The 
  stoppered 
  bottle 
  was 
  placed 
  

   in 
  a 
  shaking 
  machine, 
  and 
  well 
  shaken 
  for 
  six 
  hours, 
  at 
  the 
  end 
  of 
  which 
  time 
  

   the 
  gelatinous 
  mass 
  appeared 
  to 
  be 
  completely 
  broken 
  up 
  and 
  a 
  uniform 
  

   suspension 
  obtained. 
  A 
  few 
  drops 
  of 
  chloroform 
  were 
  added 
  to 
  this 
  to 
  ensure 
  

   sterility. 
  

  

  When 
  required 
  for 
  use, 
  an 
  aliquot 
  portion 
  of 
  this 
  liquid, 
  containing 
  the 
  

   requisite 
  quantity 
  of 
  bacterial 
  growth, 
  was 
  measured 
  out 
  after 
  the 
  bottle 
  had 
  

   been 
  well 
  shaken, 
  transferred 
  to 
  a 
  beaker 
  together 
  with 
  some 
  distilled 
  water 
  

   and 
  heated 
  on 
  a 
  water-bath 
  to 
  a 
  temperature 
  of 
  about 
  80° 
  C. 
  to 
  remove 
  the 
  

   chloroform. 
  When 
  cold, 
  the 
  concentrated 
  Detmer's 
  solution 
  and 
  distilled 
  

   water 
  in 
  sufficient 
  quantity 
  to 
  make 
  up 
  the 
  required 
  volume 
  were 
  added. 
  

   The 
  Detmer's 
  solution 
  was 
  prepared 
  in 
  quantity 
  at 
  one 
  hundred 
  times 
  the 
  

   normal 
  concentration 
  and 
  kept 
  in 
  a 
  stock 
  bottle. 
  These 
  experiments 
  were 
  

   carried 
  out 
  entirely 
  with 
  conductivity 
  water. 
  

  

  Ten 
  similar 
  plants 
  of 
  Zemna 
  minor 
  were 
  counted 
  out 
  into 
  each 
  of 
  the 
  ten 
  

   dishes, 
  and 
  300 
  plants 
  counted 
  out 
  at 
  the 
  same 
  time 
  for 
  a 
  determination 
  of 
  

   their 
  dry 
  weight. 
  The 
  dishes 
  were 
  covered 
  with 
  black 
  paper 
  to 
  the 
  surface 
  of 
  

   the 
  liquid, 
  as 
  described 
  in 
  the 
  previous 
  communication, 
  and 
  the 
  whole 
  set 
  

   placed 
  in 
  a 
  greenhouse. 
  The 
  dishes 
  were 
  protected 
  from 
  dust 
  by 
  a 
  large 
  

   sheet 
  of 
  glass, 
  supported 
  at 
  a 
  height 
  of 
  about 
  2 
  in. 
  above 
  the 
  top 
  of 
  the 
  

   dishes. 
  The 
  culture 
  solutions 
  were 
  changed 
  twice 
  weekly, 
  and 
  the 
  plants 
  

   counted 
  once 
  weekly. 
  At 
  the 
  end 
  of 
  the 
  third 
  week 
  the 
  plants 
  in 
  Series 
  II 
  

   had 
  almost 
  filled 
  their 
  dishes, 
  so 
  the 
  whole 
  set 
  was 
  halved 
  at 
  the 
  weekly 
  

  

  