(e)  The  sections  are   now  stained  red,   and  should  be
placed  in  methylene-blue  and  treated  as  described  before.
(4)  Staining  of  sections  by  Gram's  method.
(a)  Place  the  section  in  alcohol  for  half  a  minute.
viii  CULTIVATION  OF  VIBRIOS  37

(b)  From  the   alcohol  transfer    it   directly   to   filtered
aniline  gentian-violet  and  allow  it  to  stain  for  two  to  ten
minutes.
(c)  Einse  the  section  in  distilled  water.
(d)  Now  place  it  in  Gram's  iodine  solution  till  it  is
black  (about  two  minutes).
(e)  Transfer  it  to  absolute  alcohol  for  a  quarter  to  half

a  minute,  and  thence  to

0f*
(/)  Acid  alcohol  (alcohol  with  3  per  cent  hydrochloric

acid)  for  not  more  than  ten  seconds.
(g)  Again  wash  it  in  alcohol  until  the  section  appears
colourless,  or  if  previously  stained  with  picrocarmine,  until
the  section  again  becomes  red.
(h)  Clear  it  in  xylol  or  turpentine  and  creasote.
(i)  Transfer  it  to  a  slide  with  a  cigarette-paper.
(k)  Mount  it  in  Canada  balsam.
Examine  with  low  and  high  powers  and  -^  in.  oil
immersion :  the  bacilli  appear  dark  blue  on  a
yellowish  or  red  background.
Cholera

Inoculate —

(1)  a  gelatine  tube  (stab),  an  agar-agar  tube  (streak),
and  a  broth  tube  with  Vibrio  cholerse  Asiaticse  ;

(2)  a  gelatine  tube  (stab),  an  agar-agar  tube  (streak),
and  a  broth  tube  with  Vibrio  Finkler-Prior ;

(3)  a  gelatine  tube  (stab),  an  agar-agar  tube  (streak),
and  a  broth  tube  with  Vibrio  Metchnicovi.
38  GENERAL  BACTERIOLOGY  LESSON

Place  the  gelatine  tubes  in  the  cool  incubator  at  22°  C.,
the  others  in  the  warm  incubator  at  38 -5°  C.
Staining  of  Flagella  (Van  Ermengem)

Prepare  the  following  solutions : —

(a)  Osmic  Acid  (2  per  cent  solution)  1  part.
Tannin  (10  to  25  per  cent  solution)  2  parts.
