Skip to main content

Full text of "Laboratory directions in general physiology"

See other formats 


T 


41 
H  26 


LABORATORY  DIRECTIONS 


IN 


GENERAL  PHYSIOLOGY 


BY 

E.  N.  HARVEY 
PRINCETON  UNIVERSITY 


1913 


LABORATORY  DIRECTIONS 


IN 


GENERAL  PHYSIOLOGY 


BY 

E.  N.  HARVEY 
PRINCETON  UNIVERSITY 


1913 


PRINCLTON 

UNIVERSITY 

PRLSS 


PART  I. 

BIOLOGICAL  CHEMISTRY:  PAGE. 

CARBOHYDRATES 9 

Polysaccharides    (Starch,  Dextrine,  Glycogen,   Cellulose) .  .  9 

Monosaccharides   (Dextrose,  Laevulose,  Galactose ) 10 

Disaccharides  (Saccharose,  Lactose,  Maltose) n 

LIPOIDS    12 

Fats  and  Oils    12 

Lipins  (  Lecithin  and  Cholesterin )    13 

PROTEINS 14 

Typical  protein  (egg  albumin) 14 

Derived  albumins  or  albuminates  or  metaproteins 17 

Albumoses  or  proteoses  and  peptones 17 

Plant  proteins    18 

EXTRACTIVES    18 

SALTS 19 

ENZYMES 19 

Hydrolysing   enzymes    ( Inverting,   Diastatic,   Lipolytic   and 

Proteolytic  Enzymes) 23 

Coagulating  enzymes 23 

Oxidizing  and  reducing  enzymes 23 

Special  characteristics  of  enzyme  action 25 

Enzymes  and  metabolism  in  cells 27 

PART  II. 

PHYSICAL  CHEMISTRY  OF  CELLS  29 

A.  SOLUTION  AND  DIFFUSION 29 

a.  General  Phenomena 29 

b.  Semipermeable  Membranes  and  Osmosis 30 

c.  Force  of  Diffusion — Osmotic  Pressure 31 

B.  CELL  PERMEABILITY   33 

C.  SURFACE  TENSION  AND  RELATED  PHENOMENA 35 

a.  The  surface  film 35 

b.  Forms  of  fluids  produced  by  surface  tension 36 

c.  Principle  of  least  or  minimal  surfaces 36 

d.  Internal  pressure  due  to  curved  films 37 

e.  Changes  in  surface  tension  in  fluids 37 

f.  Surface  tension  between  several  substances 38 

g.  Formation  of  films  under  the  influence  of  surface 

tension 39 

h.     Force  of  evaporation 40 

i.     Amoeboid  movement 40 


1).  COLLOIDAL  S<>i.rTiu.\s   41 

;i.      Suspension  colloids 4-' 

It.      Kmulsion  colloids  or  1  lydrophilous  colloids 42 

c.  Swelling  processes    43 

d.  (  >smotic  pressure  of  colloids 43 

e.  Effect  of  salts  on  colloids  in  living  tissues 44 

PART  Ul. 
PHYSK  >!,<  ><iV  (  >K   M<  AT.MKXT   4r> 

I.     MrsCLK    I'llYSIoI.dC.Y     46 

STKIATKD  MrscLK 4^> 

a.      Methods  of  Stimulation 46 

1).      I'haionK-na  of  Contractility  and    Irritability 48 

c.  ( ira])bic  record  of  Contractions 51 

d.  Kftect  of  various  factors  in  Muscle  Contraction 53 

SMOOTH    Mrsru-:   58 

1 1  HART  Mrsru-: 58 

1 1.    Xr.kvi-:  I'IIVSKU.OCV ;S 

a.  Xerve  Fibers   58 

b.  Xerve   Cells.    (     IMiysiology   of   the   Central    Xervous 

System  )    63 

a.      keflexcs 03 

1).     The  brain   MM 

III.   BIOELECTRIC  CI-KKEXTS '>S 

I\".  C'II.IAKV   MOVKMKNT   70 

Y.     1  'KI  >T<  H'l. ASM  1C    l\<  i  r A TH  i\     -  t 

PART  1\'. 

PHYSIOLOGY  OF  XfTklTK  )\    I  Including  Circulation  and 

Respiration  )    7_> 

A.    M  i  i •  \MOUSM    ~2 

\.   I  tolophytic  Metabolism  ~2 

II.    I  lolo/oic   Metabolism    73 

I  '..     (  'l  KM   UL  \  I  '  >K'Y    S^  STI-'.M     So 

I.    Physiology  of  I  I  earl   I'.eat    So 

II.    I  'bysiolo^\   of  I  leart   Muscle S_> 

111.    Inhibition    of    Heart S3 

l\  .    I -'.licet  ol   variou^  factors  on  character  of  rlivthm.  ...  S4 

\.    I'ressure  and  X'elocitv  conditions  in  the  circulation..  S(> 

('.     kl.MMK  \T|o\     SS 

I.    Inspiration  by  luns,rs SS 

I  I.    <  Kidation   in   the   tissues  .  SS 


INTRODUCTION 

The  Laboratory  Directions  in  General  Physiology  contained 
herein  have  been  used  in  the  course  at  Princeton  in  a  somewhat 
modified  form  for  the  past  two  years.  They  have  been  printed  as 
an  easy  solution  of  the  problem  how  best  to  prepare  directions 
for  the  student  in  laboratory  courses.  While  not  attempting 
completeness  in  the  field  of  General  Physiology  they  will  be  found 
to  cover  subjects  of  wider  biological  interest  than  those  considered 
in  the  Physiology  Course  of  the  Medical  School.  Many  of  the 
experiments,  particularly  in  nerve-muscle  physiology,  have  been 
taken  from  Cannon's  "Laboratory  Course  in  Physiology",  Porter's 
"Introduction  to  Physiology",  and  Stewart's  "Exercises  in  General 
Physiology"  and  I  acknowledge  my  indebtedness  to  these  sources. 
I  express  also  my  deepest  thanks  to  Dr.  R.  S.  Lillie  for  many 
suggestions  anil  for  correcting  the  proof.  A  number  of  the  experi- 
ments performed  in  the  General  Physiology  Course  at  the  Univer- 
sity of  Pennsylvania  under  his  direction  are  embodied  in  this  book. 

E.  N.  H. 
June  i,  1913 


GENERAL  DIRECTIONS 

Each  student  will  need  a  notebook  kept  for  this  purpose  alone. 
The  notebooks  will  be  taken  up  and  examined  at  intervals. 

Students  are  to  work  in  pairs  but  each  student  is  to  observe 
the  results  of  every  experiment  himself.  In  some  cases  it  will 
be  necessary  for  the  students  to  observe  experiments  performed  by 
the  instructor.  In  this  case  each  student  is  to  keep  notes  exactly 
as  when  he  performs  the  experiments  himself. 

The  notes  should  include  an  account  of  the  apparatus,  prepara- 
tions made,  etc.,  a  statement  of  what  is  done  in  carrying  out  the 
experiments  or  observations,  a  statement  of  the  results,  and  finally 
of  what  the  experiments  show.  While  the  notes  should  not  be  too 
voluminous,  there  is  much  greater  likelihood  of  erring  on  the  side 
of  making  them  too  brief  than  too  long.  Complete  sentences  should 
always  be  used  and  a  connected  account  given,  that  will  be  perfectly 
intelligible  to  a  reader.  Sketches,  or  better,  diagrams,  of  apparatus, 
figures  of  structures,  etc.,  should  be  given  wherever  possible;  they 
are  more  important  and  take  the  place  of  long  descriptions. 

Any  pertinent  matter  in  the  way  of  explanations,  etc.,  which 
the  student  desires  to  have  on  record  may  be  written  in  the  book, 
but  should  be  distinguished  from  the  results  of  his  own  observa- 
tions, experiments,  and  reasoning  by  being  enclosed  in  parentheses. 
The  instructor  will  then  understand  that  such  matter  is  not  put 
forward  as  original  results. 

Enter  into  each  experiment  in  the  spirit  of  research.  Always 
read  the  directions  carefully  and  obtain  an  idea  of  the  general  mode 
of  procedure  and  purpose  of  the  experiment.  Do  not  call  upon 
the  instructor  at  every  hitch  but  endeavor  to  overcome  the  difficulty 
yourself.  If  you  suspect  the  result  of  your  experiment  incorrect 
consult  the  instructor  before  repeating  it.  Remember  that  the  value 
obtained  from  a  laboratory  course  will  depend  on  your  own  zeal 
and  ingenuity. 

In  Physiology,  as  in  any  experimental  science,  the  best  results 
are  only  obtained  if  the  apparatus  is  in  good  working  condition  and 
clean.  Students  should  never  allow  liquids,  spilled  on  the  table, 
or  apparatus  to  remain  there,  and  before  leaving  the  laboratory, 


UK-  ija--\\are  u-cd  must  be  cleaned  and  instruments  and  reagents 
returned  to  their  proper  place-,  lie  careful  not  to  mix  the  stopper- 
df  reagent  b<»ttle>.  l\e]x)rt  any  breakage  to  the  instructor  at  once. 

For  a  description  of  the  apparatus  used  in  this  course  the  student 
i-  referred  to  the  catalogue  of  the  Harvard  Apparatus  Co.,  makers 
of  physiological  instruments.  Howell's  or  Starling's  Physiology  is 
recommended  for  outside  reading  in  connection  with  both  lectures 
and  laboratory  work. 


8 


PART   I 

BIOLOGICAL  CHEMISTRY 
Some  characteristics  of  the  substances  which   make  up  Organisms 

CARBOHYDRATES 

Chief  of  physiological  importance  are :  polysaccharides 
(CGH10Or,)n,  monosaccharides  (C6H12OG),  and  disaccharides 
(C^H^On) . 

I.     POLYSACCHARIDES. 
A.     STARCH. 

1.  '  Native  Starch.     Mount  a  scraping  from  a  slice  of  potato  in 
water   and    examine   under   the   microscope.      Study   the    structure 
of  the  starch  grains.     Draw.     Run  a  drop  of  dilute  iodine  solution 
under  the  cover-glass.     What  is  the  reaction?     Perform  the  same 
experiment  with  corn-starch,  and  arrow-root  or  some  other  type  of 
starch,  noting  carefully  any  differences. 

2.  Polarization  phenomena.     Demonstration — Starch  grains  ex- 
hibit characteristic  light  and  dark  bands  when  viewed  by  polarized 
light.     Examine  the  starch  under  the  micropolariscope.     Notice  the 
change  in  position  of  the  bands  as  the  analyzer  is  rotated.     Draw 
carefully,  making  the  drawing  large  enough  to  show  details  clearly. 
The  principle  and  descriptions  of  a  polariscope  will  be   found  in 
Carhart's    Physics    ( pp.    323-4 ) .      The    Nicol    prism    in    the    stage 
of  the  microscope  is  the  polarizer;  that  on  the  tube  is  the  analyser. 
Inserting  a  starch  grain  between  these  two  prisms  is  comparable  to 
inserting  a   selenite  plate  between   two   Nicol  prisms,   except   that 
the  starch  grain  is  doubly  refractive  in  crossed  bands.     Read  care- 
fully the  account  given  in  the  Physics. 

3.  Solubility.     Grind  a  little  commercial  starch  in  a  mortar  and 
shake   with   cold  water.      Filter  and   test   the   filtrate   with   iodine. 
Test  solubility  in  boiling  water.     Note  character  of  the   resulting 
solution.     Cool  a  portion  of  a  strong  solution  in  a  test-tube  and 
note  result.     Dilute  and  filter.     To  dilute  starch  paste  add  a  drop 


or  two  of  iodine  solution.  Xote  the  re-ult.  Meat  ;uul  cool  again. 
noting  results.  Try  action  of  i  i  )  alkali  (  io'«  1\<  >I1)  and  (Ji 
acid  M<>',  IK'li  on  the  -tarch  iodide. 

I'..      I  )i:\  i  KI  \K. 

A  product  of  the  boiling  of  -tarch  in  weak  acid-   i  hydroly-i-  i  . 

4.  Xole    the    physical    properties,    taste,    and    solubility    in    water, 
both  h<  >{  and  cold. 

;.      Te-t    with   iodine,  as  under  -tarch. 

C.        <  il.N  lOCKN. 

"Animal  starch."  Kound  in  animal  cell-,  particularly  liver  and 
muscle. 

o.      Kxamine  as  under  dextrine. 
1).     CELLULOSE. 

l-'orms  the  cell-wall-  of  plant  cell-. 

7.  i  ""tii  in  fibre  is  almo-t  pure  cellulose.  Xote  its  insolubility  in 
water  and  alcohol.  Solubility  in  strong  1  lL'1,  11_.S<),,  or  11X<>;: 
Strong  alkali  i  in',  1\(  HI  I?  Does  it  react  with  iodine?  Treat  with 
50^5  H2SP4  and  then  add  iodine.  I\c-ult? 

5.  Malisch    test.      A    te-t     for    carbohydrates    in    general,    even 
tho-e   combined   in   the   protein   molecule.      To  a   few   -tarch   grains 
in  2cc.  water  add  a  drop  of  a-naphthol   solution    (2Of/r    u-naphthol 
in    1^5'  (     alcohol  |    and    an    equal    volume    of    concentrated     1LSO4, 
alowing  the  acid   to  run   down  the  side  of  the  test-tube  and  collect 
in   the  bottom   without   mixing.     At   the  contact   of   sulphuric  acid 
and  starch  -u-pension.  ;i  violet  ring  i  furfurol  )   will   form  indicating 
carbohydrate-. 

9.  Apply  the  above  te-t  to  egg  albumin.  Any  result:  What  do 
\<  m  o  mcludr  ': 

II.     MONOSACCHARIDES.      (CeH12Oa.)      SUGARS. 

A.     I  )i  \  i  KO-I-:    ((ii.rrosK    ok    <ik\n-:    SUGAR.      I'.      GALACTOSE. 


<    .       I  .-i  \  ULOSE. 

Ivxamine  i-ach  as  follo\\  -  : 

ID.  Xote  the  ph\-ical  ]  >ro]  n  rties,  solubility  and  taste.  Make 
about  a  i''  solution  in  hot  \\ater.  Test  -olutioii  a-  follows; 

M.      Add   iodine.     Any  color  reaction? 

i_'.  Moore's  /<-.s-/.  Add  one-  fourth  it-  volume  in',  KOI  I  and 
he.it  gradual!}  to  boiling  point.  XoU-  color  change  and  odor. 

i  .V  I'ower  of  reducing  metallic  oxide-  i-  characteristic  of  many 
-ui;ars.  Sonic  ha\e  al<lehyde  (aldo-c-l.  others  ketone  (ketoses) 

ID 


structure.  Test  the  reducing  power  of  a  simple  aldehyde,  formalde- 
hyde (CHoO)  :  place  a  few  drops  3%  CuSO4  sol.  in  a  test-tube, 
add  a  little  formalin,  then  an  equal  volume  io'/c  KOH.  Note  each 
stage  carefully.  Xow  heat  slowly  to  the  boiling  point.  Result? 
Explain. 

14.  Repeat  the  last  test  with  dextrose.     Trommer's  test:  add  a 
few  drops  3^  CuSO4  to  the  dextrose  solution,  then  half  its  volume 
ioc/o  KOH  and  boil.     Note  the  result  carefully.     Trommer's  test 
is  in  principle  like  Fehling's  test  but  less  delicate. 

15.  Boil  a  little  Fehling's  solution  in  a  test-tube.    Result?    Then 
add  some  sugar  solution  and  boil  again.    Result?     Fehling's  solution 
is  kept  in  two  bottles.     Mix  equal  parts  before  using.     One  con- 
tains CuSO4 ;  the  other  sodium  potassium  tartrate   ( Rochelle  salt) 
and  NaOH.     The  sodium  potassium  tartrate  is  added  to  hold  the 
Cu(OH)2  precipitated  by  the  alkali  in  solution.     Fehling's  test  is 
very  important ;  it  is  used  e.g.  to  detect  the  presence  of  sugar  in 
the  urine  in  cases  of  diabetes.    Be  sure  you  understand  the  reactions 
underlying  the  test. 

16.  'Phenyl-hydrazine  test.     An  important  means  of  distinguish- 
ing different  sugars.  In  a  test-tube  place  a  little  solid  phenyl-hydra- 
zine  hydrochlorate  with  an  equal  bulk  dry  sodium  acetate ;  then  add 
10  cc.  dextrose  solution  and  heat  in  a  water-bath  at   100°   for  one 
hour.     Filter  hot.     Cool  the  solution  and  examine  carefully  under 
the   microscope.      Crystallized   compounds,    Osazones,   are    formed, 
which  are  characteristic  for  each  sugar. 

III.     DISACCHARIDES  (QJH,^). 

SUCROSE,  LACTOSE,  MALTOSE. 

17.  Cane-sugar.    (Saccharose  or  sucrose.}      Note  crystals,  taste 
and  solubility  as  above.     Make  a  i/o  solution.     Apply  Moore's  and 
Fehling's  tests.     Is  it  a  reducing  sugar.'     Why  not? 

18.  Examine  lactose    (milk-sugar),  and   maltose    (malt  sugar), 
a   product  of   starch   hydrolysis,   in   the   same   manner   as    sucrose. 
Note  carefully  any  differences  between  these  disaccharides.    Record 
the  results  of  your  tests  with  the  six  sugars  in  the  form  of  a  table. 

19.  Inversion    of    caue-sugar.      Boil    the    solution    with    a    few 
drops  of  cone.  HC1  for  a  few  minutes.     Cool,  neutralize,  and  apply 
Fehling's  test.    Result?    Explain.    What  sugars  are  formed? 

20.  Formation  of  sugar  from  starcli.     Boil  dilute  starch  paste 
with  a  little  2OC/0  H2SO4  till  the  fluid  is  clear.     Neutralize  and  test 
for  glucose.     Result  ?     Also  test  with  iodine.     Result  ? 

1 1 


_'i.  lla-  starch  reducing  properties  ?  Has  dextrine?  Has  cellu- 
lose?  Test  all  three  with  Kehling's  solution. 

1.1  TO  IDS  (Fats  and  Li  fins) 

I.     FATS  AND  «  )ILS. 

jj.  X«  >tc  the  physical  properties,  differences  in  melting  points, 
etc..  of  fats,  hntter.  beef-tallow  or  lard,  and  olive  oil. 

j%}.  Te>t  solnhilities  of  these  fats  in  (a)  -i^ater,  hot  and  cold; 
I  hi  alcohol,  hot  and  cold;  (c)  chloroform;  (d)  ether.  Xerer  briny 
ether  near  a  flame.  Allow  the  chloroform  to  evaporate  overnight,  in 
a  covered  gla->  ve-sel,  and  note  the  fat  crystals  formed. 

24.  Emulsification.  Shake  a  few  drops  of  olive  oil  with  water 
in  a  tot-tube.  What  happens?  Set  the  tube  aside  for  a  few 
minutes.  What  happens  ?  Xow  repeat,  using  i/v  Na2CO8  instead  of 
water  and  note  the  difference  in  result.  Explain.  Shake  neutral- 
ized olive  oil  in  a  test  tube  with  \'  <  XaL,CO3.  Note  and  explain 
difference  from  ordinarv  olive  oil. 

-'5.  Saponification.  To  some  emulsified  oil  in  a  test-tube  add 
stron//  KOff  and  boil;  formation  of  soap  or  saponification  results. 
Is  the  product  soluble  in  water?  Draw  off  some  of  the  clear  solu- 
tion and  to  it  add  l'aCl_,  solution.  Result?  Explain.  Write  the 
equations  for  these  reactions. 

jo.  To  a  portion  of  clear  soap  solution  add  some  20'  >  1 1,S<  )4  and 
heat.  Xote  the  separation  of  droplets  at  the  surface.  What  is  the 
substance  separated?  Kxplain. 

-•7.  Sal  tin//  mtt  of  soaps.  To  clear  soap  solution  add  XaCl 
crystals  to  saturation.  Xote  the  rlocculent  precipitate.  Remove 
some  of  this,  dissolve  in  water  and  test  for  soap.  Result." 

_JS.  Sudan  111  test  for  fat.  Place  a  drop  of  olive  oil  on  filter 
paper  and  immerse  in  an  alcoholic  solution  of  Sudan  111.  Mien 
wash  quickly  in  alcohol.  Does  the  oil  take  up  the  dye? 

_'<>.  Test  the  solubility  of  fatty  acid  (  oleic  acid)  in  pure  water, 
alcohol  and  chloroform.  Add  some  i'<  \a_,(  <>;  solution  to  oleic 
acid  and  shake.  Result?  Kxplain. 

.y.  I'ressure  of  i/lvcerine  in  fats.  Place  a  little  glycerine  in  a 
test-tube  and  heat  with  some  powdered  acid  potassium  sulphate 
iKMS'),).  Xote  the  characteristic  odor  (acrolein  test  for  gly- 
cerine I.  Repeat  with  olive  oil  or  other  fat  in  a  clean  test-tube. 
l\e-ult?  What  is  the  chemical  composition  of  a  fat? 


II.     LIPINS. 

31.  Cholesterin.      Demonstration    of    the    iodine-sulphuric    acid 
test.      Cholesterin    crystals    are    treated    with     fairly     concentrated 
H2SO4   and  one  drop  of   a  very   dilute   iodinie   solution.  Note   the 
crystalline  form  and  play  of  colors.     Draw. 

32.  Lecithin.       Preparation     from     hen's     egg     yolk.       To     the 
alcoholic  mixture  of   egg  yolk  given  out  add  one-half   its  volume 
of  ether,  shake  and  set  aside  in  a  tightly  stoppered  hottle  till  the 
next  laboratory  period.     Then  filter  into  an  evaporating  dish  and 
evaporate  over  a  water  bath  to  dryness,  taking  care  the  ether  does 
not  catch  fire.     Dissolve  the  residue  in  as  small  an  amount  of  ether 
as  possible  and  add  four  or  five  volumes  of  acetone,  which  precipi- 
tates  the   lecithin.      Collect   on   a   glass   rod,   allow   the   acetone   to 
evaporate  and  use  in  the  following  tests. 

Note  the  physical  properties,  consistency,  color,  etc.,  of  lecithin. 
Is  it  soluble  in  water?  in  alcohol?  in  chloroform? 

33.  Production    of    "inyclin    forms".      Place    a    small    piece    of 
lecithin  in  water  under  a  cover-glass.     Examine  from  time  to  time. 
Note  especially  the  surface.    Draw.    Remove  the  cover-glass  and  try 
drawing  out  the  surface  with  a  glass  rod  or  needle.     Note  the  very 
fine  filaments  formed. 

34.  Presence  of  glycerine  in  lecithin.    Try  the  acrolein  test.     See 
under  Exp.  30. 

35.  Presence    of    nitrogen.      Heat    strongly    in    a    dry    test-tube 
a  small  piece  of  lecithin.     Suspend  in  the  mouth  of  the  tube  a  strip 
of   moistened    neutral    litmus    paper    and    a    strip    of    lead    acetate 
paper.     Is  the  paper  changed  in  color?     Due  to  the   formation  of 
what  substance?     What  would  the  lead  acetate  paper  detect  if  it 
blackened  in  color  ? 

36.  Presence    of   plwsphorns.      Heat    a    small    piece    of    lecithin 
with  a  fusion  mixture  consisting  of  5  parts  KOH  to  i  part  KNO3 
in  a  crucible,  until  colorless.     Cool,  dissolve  in  a  little  warm  water, 
acidify  with  a  few  drops  of  cone.  HNO3  and  add  molybdic  solution. 
A  yellow  precipitate  indicates  the  presence  of  P. 

37.  Presence  of  fatty  acid.     Add  a  small  piece  of  lecithin  to  a 
few  cc.  of  sodium  alcoholate  in  an  evaporating  dish  and  evaporate 
to  dryness.     Take  up  with  water  and  add  CaCL  solution.     A  white 
curdy  precipitate  indicates  fatty  acid.     What  is  this  precipitate? 

Note   that   lecithin   is   like   a    fat    in    many    ways,   yet    differs    in 
important  respects. 


PROTEINS 
I.     TYPICAL  l'l«  ITEIN  •  I -.«  .«  .  ALBUMIN  i. 

tullv    pour   the    white   iif   an    egg   into   an    evaporating 

dish.  i  In-    \.-lks;    they    will    he    collected    hy    the    instructor. 

This    i-    an    approximate!}     u'.     -olntion    of    a    protein    (albumin). 

it-    viscous   con-i-teiicv.      <  ihserve  that    it    ran   he  drawn  out 

into    rather   long   cords    ()r   cylinder-;    compare    with    water    in    this 

;>ecl.  Test  the  reaction  with  litmus  paper;  is  it  acid,  alkaline. 
or  neutral:  <  >f  one-half  of  the  egg-white  make  a  10' ,  -olmion.  To 

this,  place  in  an  evaporating  dish  and  cut  the  egg-white  freely 
with  the  scissors;  this  liU-rates  it  fnun  the  nieiuhranes.  Then  mix 
with  nine  times  its  volume  of  water,  stirring  thoroughly.  A  pre- 
cipitate of  glohuliu.  another  protein,  forms.  Kilter  through  several 
told*  of  wet  linen.  Keep  the  undiluted  half  for  further  u>e. 

\.      Co  VGULATION. 

39.      (  "iif/nliition    />y    heat.      Have    a    water-hath    with    water    at 

hoiling  temperature.      I'm  some  of  the  undiluted  egg-white  and  the 

HP',     egg-white   in    tcst-tuhe-   and    place   in    the    water-hath.      Which 

£lll    Ics   lir-t  ':      \\hai   conclusion   can   he   drawn   as   to   the   ettcct 

of  dilution  on  coagulation  hy  luat  : 

Coagulation  />v  chemicals.  To  5  cc,  of  the  i<>',  solution  add 
a  few  dn»p~  ,.f  ^',  copper  sulphate.  Note  the  coagulation.  I'. sing 
a  new  -pccimen  of  solution  ti<>',  i  at  each  trial,  try  in  the  -ame 
way  mercuric  chloride-  and  lead  acetate,  recording  results.  Trv 
also  strong  nitric,  hydrochloric,  and  sulphuric  acids,  in  the  follow- 
ing wax  :  allow  a  drop  or  two  of  the  acid  to  run  down  the  >ide 
of  the  tuhe  till  it  reaches  the  -olmion.  Tr\  also  <,;',  alcohol,  picric 
acid,  tannic  arid,  phosphotungstic  acid,  and  l\,l;eii  \  i,.  adding  first 
a  little  dilute  acetic  acid  in  the  case  of  the  la-t  four  reagent-. 

ji  Manner  in  rc/nr/i  cinifinlatian  takes  /1/./l-<>.  1  >ip  a  thin  thread 
ot  silk  in  a  $'  ,  s.,lmion  of  copper  sul]ihate  and  lav  the  thread  on  a 
^la--  sljdi  .  hi-iieath  a  cover  glass.  Allow  s,,nie  of  the  K>',  solution 
Jiite  of  ,  gg  !,,  ,-un  under  the  cover  .ula--  while  ol»erving  the 
operalinii  with  the  micro-rope  i  high  |>oweri.  The  alhnmin  ahotit 
the  tlmad  will  hi-  -i-en  to  form  -mall  granule-,  appearing  like  a 
tint-  cloud,  and  the-e  later  run  together  lo  form  an  open  network. 

A     i  ille    of    t  w-  •  111    tin-    pi  i  id 

<   in  M  n  \i.   i  ii  VK  vi  i  i  ui-i  n  - 

I 'lace  a  little  powdered  dry  alhu- 
•i   the  hoitoni  of  a   d>  v   t<--t  -tuhe.  and   in-ert   at   the  mouth   of 


n 


the  tube  a  small  piece  of  moist  red  litmus  paper  and  a  piece  of 
moist  lead  acetate  paper  (strip  of  filter  paper  soaked  in  2f-/(.  lead  ace- 
tate and  dried).  Heat  the  lower  end  of  the  tube,  and  note  the 
characteristic  odor  of  burned  feathers.  If  ammonia  is  evolved 
(showing  the  presence  of  X  and  H  ),  the  litmus  paper  will  turn  blue  ; 
if  sulphur,  the  lead  acetate  will  turn  black,  through  formation  of  lead 
sulphide.  If  the  albumen  chars  black,  the  presence  of  C  may  be 
inferred.  State  the  results  of  your  conclusions  as  to  the  presence 
of  C,  X,  H,  and  S  in  proteid. 
C.  TESTS  FOR  PROTI-: IN. 

43.  Xanthoproteic  reaction.     Dilute  some  of  the  \Qf/c   egg-white 
till  it  is  about  2/r ,   and  place   in  a   test-tube.     Add  a   few  drops 
of   cone,   nitric  acid.      \Yhat   occurs?     Boil.      \Yhat  occurs,   as   to 
color   and   other   changes?     Cool   the   solution   and   add   ammonia. 
Be  careful  that  the  contents  of  the  tube  do  not  shoot  out.     XTote 
the  color  produced    (this  is  the  essential  feature  in  xanthoproteic 
reaction  ).     Describe  and  fix  in  mind  this  reaction. 

44.  Does  gelatin  give  the  xanthoproteic  reaction  ?     Try  in  the 
same  way  a  weak  solution  of  gelatin.     Does  fibrin   (obtained  from 
the  blood  )  ?    Try  this  reaction  also  with  a  small  piece  of  meat,  and 
a  small  piece  of  bone  or  cartilage. 

45.  Millon's  reaction.     To  a  little  of  the  dilute  solution  of  albu- 
min add  a  few  drops  of  Millon's  reagent.     (  This  has  been  made  as 
follows:  dissolve  one  part  of  mercury  in  an  equal  weight  of  [cold] 
nitric  acid.     Then  add  the  solution  to  twice  its  volume  of  water, 
allow   to   stand   some   hours,   then   decant  off  the  liquor   from   the 
sediment. )     Note  the  white  precipitate  formed  when  Millon's  reagent 
is  added  to  the  albumin.     Boil  two  or  three  minutes,  and  observe 
the  result  ? 

46.  Does   gelatin   give    Millon's    reaction?      Does    meat?      First 
boil  a  small  piece  of  meat  to  destroy  the  red  color.     Does  bone? 

47.  The  binrct  test.     To  a  little  of  the  dilute  albumin  solution 
add  an  equal  volume  of   ioc/c   potassium  hydrate  and  add  one  or 
two  drops  (or  more  is  necessary)  of  l/2c/c  copper  sulphate.     Notice 
the  violet  color.     Try  the  biuret  reaction  with  gelatin,  caseinogen 
and  peptone  solutions. 

48.  Adamkiewics's  reaction.     Place  in  a  dry  test-tube  ten  drops 
of    strong    sulphuric    acid    and    20    drops    of    glacial    acetic    acid 
containing  glyoxylic  acid.    To  this  mixture  add  a  little  dried  albumin 
and  warm  slightly.     Notice  the  reddish  violet  color,  due  to  trypto- 
phane.     Try  this  with  some  dry  gelatin. 

15 


4'*.  J  Idler's  ;-/;;./  test.  I 'lace  a  half  inch  of  strong  nitric 
1  in  a  tc-t-tuhc.  1'oiir  UIM)II  thi>.  allowing  it  to  run  gently  down 
thf  -iik-  Hi  the  tul'e.  a  little  "f  the  weak  alhuniin  solution.  -Vote 
the  \\hite  cloud  at  the  junction  of  the  t\vu  -ub-tances  ;  this  is  Hel- 
ler'- tc-t  f->r  alhuniin.  It  U  u-ed  to  te>t  the  presence  <>f  alhuniin 
in  the  urine. 

I  ).      I  >i  \i  I  'KOI  i.i  N 

A. !•!  an  e«|ual  volume  of  m',  XaCl  solution  to  some 
undiluted  white  of  egg  and  place  in  a  collodion  or  parchment 
paper  bag.  or  animal  numhrane.  I 'lace  the  bag  in  a  jar  of  water 
in  such  a  way  that  only  the  lower  part  (covered  hy  the  membrane) 
is  in  contact  with  the  di-tilled  water.  After  two  hours,  observe 
whether  any  considerable  amount  of  the  albumin  has  passed  through 
the  membrane.  I  Mermine  whether  any  has  passed  through  lu- 
te-ting the  water  in  the  jar  by  one  or  two  of  these  tests  given  above. 
Ma-  Xal'l  pa--ed  through?  Te-t  with  _"  ,  AgX<>.  solution. 

\<    I  lo\    o|."    M.CTK  AL    SALTS.       "SALT1NC,    OCT." 

51.  Add  Xal'l  crystals  to  a  solution  of  egg  white  to  saturation. 
Am  precipitation?  Niter  and  test  the  filtrate  for  protein.  Is 
precipitation  complete?  Test  the  residue  on  the  filter  for  protein. 

oti-  the  similarity  to  the  salting  out   of  snap  by    XaCl. 

.-_•.  kepcat  K\p.  51.  u-ing  powdered  (  XI  1,  )._.S<  ),  cry-tals  instead 
of  Xal'l.  \\hich  -alt  i-  more  effective  a-  a  precipitant?  Kgg  albu- 
men i-  purified  by  repeated  -alting  out  with  iXII,i.S<>4,  which 
pucipitate-  completely  tin1  protein. 

At    I  Io\    i  ,|      \(   ||is    AM)    ALKALIKS. 

5.v      I'mtein   is   ain^linteru'.   i.e..    it    \\ill   combine    with   both    acid 
and   alkali.      Use   a    solution   of    Merck's   powdered   egg   albumin    in 
di-tilled    water,    tillered.       Tour    a    few    cc.    into    a    test-tube    and    a 
similar   amount    of    distilled    water   into   another    test-tube.      Add    a 
drop  of   neutral   red   solution   to  both   tube-.      Then   add   very   care- 
fully dmp  b\   drop  froin  a  pipette  n    51)  Xa<  Ml   until  the  neutral  red 
tor    i-    changed    yellow    in    each    tube.      I'mnpare    the    amounts 
uired  in  tin-  albumin  tube  with  the  amounts  required   for  distilled 
\\hal   do  \  ou  conclude   from  this  experiment? 
kepeat  the  ahovi-  but  u-e   i'<    ali/arin  a-  an  indicator  instead 
of  neutral   red  and  n    m    III  1   in-tiad  of   n    50    Xa<)ll.      \\'hat    doe- 
thi  imeiit  -ho\\  ? 


i  > 


II.  DERIVED  ALBUMINS  OR  ALBUMINATES  OR  META- 

PROTEINS. 

55.  Acid     albumin.        To     a      iQc/c>      solution     of      egg-white 
add  an  equal  volume  of  0.2%  HC1  and  heat  in  the  water-bath  to 
about  40°  C.  for  a  few  minutes.     Acid  albumin  is  formed.     Heat 
part  of  this   solution.     Any   coagulation  ?     Color   with   litmus   and 
add  o.2f/c    KOH   from  a  pipette  to  neutralization.     Result?     Add 
more  KOH.     Result? 

56.  Alkali  albumin.     Add  to   iof/c   egg-white  solution  an  equal 
volume  of  0.2%  KOH  and  warm  at  40°  as  before.     Heat  a  part  of 
solution.      Any    coagulation?      Color    with    litmus    and    neutralize 
carefully  with  o.2f/c  HC1.     Result?     Add  more  HC1.     Result? 

57.  Add   ioc/c    KOH    to    undiluted    egg-white    in    a    test-tube. 
Result?     "Lieberkiihn's  jelly"  is  formed  which  is  solid  alkali  albu- 
men and  will  dissolve  on  warming  with  water  at  40°  C.     Try  simi- 
larly glacial   acetic  acid.     Result?      (Acid   and   alkali   albumin   are 
the  first  products  formed  in  the  digestion  of  protein  in  the  stomach 
and  in  the  intestine,  respectively.) 

III.  SIMPLER  PROTEINS.    ALBUMOSES  OR  PROTEOSES 

AND  PEPTONES. 

Use  a  solution  of  U'itte's  "peptone",  prepared  by  dissolving  the 
products  of  a  gastric  digest  (consisting  chiefly  of  albumoses  and 
peptones)  in  distilled  water.  Note  carefully  the  character  of  the 
reaction  in  each  case  for  comparison  with  the  reactions  of  pure 
peptones,  to  be  prepared  by  salting  out  the  proteose.  Test  as 
follows : 

58.  Heat  a  portion  to  boiling.     Any  coagulation? 

59.  Acidulate   with    acetic   acid    and    add   a    little    K4Fe(CN)6. 
Result?     AVarm.     Result?     Cool  as  before.     Result? 

60.  Try    precipitation     with     2c/c     tannic     acid,     95%     alcohol, 
saturated  HgCl,  solution,  3^  CuSO4,  2C/C  lead  acetate,  and  picric 
acid.     Note  in  each  case  the  effect  of  warming  and  cooling  on  the 
precipitate. 

61.  Try   the   Millon    reaction   and   Adamkiewicz    reaction   on    a 
little  dry  peptone  powder. 

62.  Salting  out  of  proteases  and  peptones.     Saturate  a  portion 
of  the  "Witte's  Peptone"  solution  with  solid  (NH4)2SO4  and  filter. 
True  peptones  will  be  separated  from  proteoses  and  be  found  in  the 
filtrate.     Use  the  filtrate   for  the   following  tests. 

1 7 


6r     Tn   the  binrct  test.     Note  carcfullv  tin-  color,  which  is  dis- 
tinctive  for  peptone-.     Try  the  biuret  te-t  mi  the  same  volume  »t   a 
dilute  alliiiinin  -..lution.  in  order  t»  compare  the  color  with  that  given 
li\    peptone-.      Kix  carefully  in   \oiir  mind  the  difference. 
..      Kepcat  experiment  ?<i.      Ke-ult  ? 

Try    al-o    precipitation    l»y    picric    acid,    tannic.    acid.    ()5'< 
:ilo-h"l.  sat.  1 1 -i  1  .  .;'<   CuS<  >,  and  _"  ,    lead  acetate  as  before. 

isihility.  Determine  whether  "Witte's  1'eptone"  solution 
\\ill  ditl'u-c  through  parchment  or  colloilion  within  a  period  of  two 
hours. 

nipare  in  a  tahle  the  reaction-  of  peptone  and  egg  albumin. 

l\  .     PLANT  I'U(  >TEINS. 

I'reparation  of  an  <//M>/;(>/-.S-<>//</'/<'  protein  (i/liadiin  from 
u  heat  tl<  >ur.  hi  -ur  o  -ntain-  chiefly  starch,  gliadin.  glutenin.  albumin. 
-l"bulin.  and  a  prok-.^e.  l;..\amine  some  wheat  flour  under  the 
nik-p '-cope  and  apply  the  Iodine  test.  Result." 

i'S.      Ap]>ly  Millon'v  te-t  to  flour  in  a  test-tube.     l\e-ult? 

<  iliadin  may  be  extracted  by  alcohol.  To  a  -mall  amount  of 
flour  in  a  bottle  add  xnne  (about  y  •  cc.  I  70'  >  alcohol,  and  shake 
at  interval-  during  oiie  hour.  Then  filter  and  u>e  in  the  follouing 

ts. 

70.      |-'.\  ;ipi  >ratc   a    -mall   portion    in   a   di>h   over   the    water   bath, 
the  scales  of  gliadin.     Are  they  -oluble  in  water?     Determine 
tin-  by  -baking  with  water  in  a  te-t   tube,  removing  the  scales  and 
te-ting  the  water  by  the  biuret  te>t. 

-\.     Adil    icx  '      alcohol  in  excess  to  a  small  amount  of  the  70'. 
ale.  'hoi  -nlutioii.     |\e-ult  ? 

7_*.      I 'our    -onie    of    the   alcohol    -olution    of    gliadin    into    water. 
Result: 

7^.      Meat  to  ln.ilin--.      An\    coagnlati'ii? 

7).  that  gliadin  differ-  from  egg  albumin  in  many  important 

pert-.      Yet    if    i-  a   protein.     Try   the   xanthoproteic  and    Millon's 
te-t-    with    -OUH-   dr\    gliadin    -cale-. 

EXTRACTIVES 

'}(.-]•  llii-  hea«l  are  included  a  large  number  of  -nlistance-  -uch 
thi-   alkaloid-.   ghio>-jde-.    ]«igment-.   purin    bases,    urea.    etc..    too 
nunii  rmi-  to  be  con-idered   in  detail   in   the  laboratorv. 


is 


SALTS 

All  living  matter  contains  salts,  chiefly  the  chlorides,  sulphates 
and  phosphates  of  Xa,  K,  Ca,  Mg  and  Fe.  The  role  of  the  salts  in 
the  organism  will  be  considered  under  the  physiology  of  the  various 
tissues. 

EERHIENT  ACTION— ENZYblES 

In  the  preceding  experiments  the  breaking  down  of  biological 
compounds  has  been  studied  from  the  point  of  view  of  pure  chem- 
istry. The  compounds  were  split  by  boiling  acids  or  alkalies.  That 
similar  decompositions  may  be  effected  at  low  temperatures  by 
substances  present  normally  in  living  tissues  is  shown  by  the  fol- 
lowing experiments. 

It  is  very  essential  in  these  experiments  that  the  temperature 
be  as  near  that  indicated  as  possible. 

I.     HYDROLYZING  ENZYMES. 

A.    INVERTING  FERMENTS.    IXVERTASES. 

80.  Iiircrtasc.  Thoroughly  grind  up  one-half  of  a  cake  of  yeast 
with  sand  and  water  in  a  mortar.  Filter.  Test  the  filtrate  for 
sugar  with  Fehling's  sol.  Any  sugar  in  yeast?  Then  mix  equal 
volumes  of  the  yeast  extract  and  cane  sugar  solution  and  keep  at 
40°  for  10  or  15  minutes.  Then  test  with  Fehling's  solution. 
Result  ?  Conclusion  ?  Invertase  may  similarly  be  demonstrated  in 
the  intestinal  mucosa. 

I').     AMYLOLYTIC  OR  DIASTATIC  FERMENTS.     AMYLASES. 

81  A.  Plant  Diastase.  Crush  thoroughly  about  5  gins,  of  ger- 
minating barley  with  sand  in  a  mortar  with  a  little  water.  Filter. 
Then  mix  the  filtrate  with  10  cc.  of  starch  paste,  warm  to  40°,  and 
keep  in  water  bath  at  40°  for  an  hour.  Note  any  change  in  the 
character  of  the  liquid.  Test  with  iodine  and  Fehling's  solution. 
Note  the  taste  of  the  resulting  liquid.  Explain  these  results.  Com- 
pare with  a  control  in  which  the  extract  was  boiled  before  incubating 
with  starch. 

8 1  B.  Saliz'ary  diastase.  Ptyalin.  Collect  a  few  cc.  of  saliva 
into  a  beaker  ( flow  of  the  secretion  may  be  accelerated  by 
chewing  paraffin  )  ;  dilute  the  saliva  with  about  5  volumes  of  water ; 
filter.  Make  the  following  mixtures  :  A.  5  cc.  of  equal  volumes  dilute 
starch  paste  and  saliva  ;  B.  the  same,  using  saliva  that  has  been  pre- 
viously boiled;  C.  the  same  mixture  as  A  -)-  5  drops  iof/f  HC1 ;  D. 

10 


mixture  A  5  drop-  \<  '  ,  l\'  Ml.  Warm  all  four  tube-  to  40"  and 
keep  in  a  water  bath  at  40  for  10  minutes.  Te-t  each  mixture  for 
-larch  i  by  iodine  i  and  t""r  -ngar  i  b\  Kehling's  solution).  Results  : 
I  >ra\v  conclusion-  a-  t"  the  influence  <>f  boiling  and  of  free  acid  and 
alkali  mi  the  acti\ity  of  ptxalin.  Also  try  the  action  of  ptyalin  in 
the  «-.'/(/.  Immerse  a  te-t-iuhe  with  a  mixture  of  -larch  and  saliva 

"ok-d  previously  to  mixing)   in  ice  water  and  test  for  sugar  later. 
What  i-  the  influence  of  teni  pcra/nrc  on  enzyme  action? 

Sj.  fi-cci^itutii'ii.  (.'tilled  I  lor  2  cc.  of  tillered  saliva  in  a  test 
tube  and  add  5  times  its  volume  of  <>5'r  alcohol.  \Vhen  the  white 
precipitate  <  of  ptyalin  and  mucin  )  ha-  settletl.  pour  oft  the  alcohol 
and  dissoke  the  precipitate  in  the  same  amount  of  water  as  the 
\olume  of  alcohol  previously  added.  Take  about  3  cc.  of  this,  add 
an  ei|iial  amount  of  starch  paste  and  place  in  the  water  bath  at 
411  »  Kxamine  at  interval-,  hoe-  the  starch  paste  become  clear? 
Test  with  iodine  solution  and  Fehling's  solution.  Compare  with  the 
preceding  experiment. 

S^.  \/(/-;r\  <>J  Starch  Hydrolysis  :«.•///;  l't\aliii.  Mix  starch  paste 
and  dilute  (i:  loi  tillered  saliva  as  above,  warm  to  4O:.  and 
keep  warm  by  holding  the  tube  in  the  hand.  At  half-minute  intervals 
transfer  with  a  gla--  rod  a  drop  of  the  mixture  to  a  drop  of 
iodine  solution  previously  placed  on  a  white  plate.  Xote  the  pro- 
gressive change  in  color  reaction.  Kxplain.  dive  the  reaction 
which  occurs. 

1  I"' AT-SI'LI  I  I  I  Xt,    OF     Lll'ol.YTir     I-T.RMKXTS. 

*j.  l.iptts,-.  I'-c  artificial  pancreatic  juice  made  by  dissolving 
;ui  extract  ot  paiicrea-  i  commercial  pancreatin  )  in  \<<  Xa..L"(). 
-'  ilutit  in. 

I'lace   in   each  of   two   te-t-tubes   _>   dr  ps   of    neutral   olive   oil 

5  cc.  of  i'     Na2CO  :  warm  and  shake.    Kcsult  ?    To  one  test  tube 

add   5  CC.  artificial   pancreatic    juice:  to  the  other,  the   same,  boiled. 

\\  arm  and  place  both  in  the  water  bath  at  40   .     Kxamine  at  intervals 

minutes.      Does    emulsification    occur    in    either    tube: 

Explain. 

in    each    of    i\\,.    h-st-tubes    _>    dn>p-    of    olive    oil,    and 

-hake.     . \dd  to  ,.m-  tube  5  cc.  jiancreatic  extract;  to  the  oilier   ;  cc. 

pancreatic  extract  ^rtTi«nsly  boileil.      I'lace  at  40     C.     After   I   hour 

•h   tor  -oap  a-  follows;     The  cmul-itied  oil  will  separate  to  a 

I-'"'-  'it  at  the  top  of  the  tube.      l\i  move  some  of  the  relatively 

•i   li«|uid    from  the  bottom  of   the  tube   with  a   long  pipette.      To* 


it  add  a  drop  of  neutral  olive  oil  and  shake.  Does  emulsification 
occur?  CaCl,  cannot  be  used  as  a  soap  test  here  because  pancreatic 
juice  gives  a  precipitate  with  CaCL. 

c.  Milk  test.  Into  each  of  two  test-tubes,  a  and  b,  place  5  cc. 
neutral  milk  and  a  drop  of  litmus  solution.  To  A  add  5  cc.  neutral 
pancreatic  juice;  to  B,  the  same  previously  boiled.  Set  at  40°  for 
40  min.  Does  the  litmus  become  red  in  one  tube?  Why?  Write  the 
reaction. 

85.  Does  pancreatic  juice  hydrolyse  starch?     Perform  an   ex- 
periment to  test  this.     Test  also  hydrolysis  of  cane  sugar. 

86.  Progress  of  fat-splitting  (or  lipolysis).     Racli ford's  experi- 
ment.    Arrange  a  series  of  several  watch-glasses  each  containing 
\c/c   Na,,CO:v     Mix  in  a  test-tube  2  cc.  neutral  olive  oil  and  i   cc. 
pancreatic  extract.     \Yarm  to  40°  ;  shake  the  tube,  then  allow  the  oil 
to  separate,  and  transfer  a  drop  by  a  pipette  to  the  soda-solution  in 
watch-glass    I.     Again   shake   and  after  an   interval   allow   the   oil 
to  separate  as  before  and  place  a  drop  in  watch-glass  2.     Repeat 
this  procedure  several  times.     The  changes  in  the  oil  produced  by 
allowing  the  lipase  to  act  for  progessively  longer  and  longer  periods 
are  seen.     Note  carefully  changes  in  behavior  of  the  oil  on  passing 
along  the  series  of  watch-glasses. 

D.     PROTEIN-STLITTIXG  OR  PROTEOLYTIC  FERMENTS.     PROTEASES. 

87.  Pepsin.     Enzyme  active  in  acid  solution.     Use  an  artificial 
gastric  juice  made  by  extracting  commercial  scale  pepsin  with  warm 

Q.2%   HC1. 

Take  six  clean  test-tubes  labeled  A  to  F,  and  place  in  each  a  few 
shreds  of  fibrin,  or  strips  of  hard  boiled  egg-white.  To  the  tubes 
add  the  following: 

a.  0.2%  HC1. 

b.  Artificial  gastric  juice. 

c.  The  same,  but  previously  boiled. 

d.  Gastric  juice  carefully  neutralized    (using  as  indicator  very 
weak  neutral  red  solution)   with  o.2(/f   KOH  added  from  a  pipette 
drop  by  drop. 

e.  Gastric  juice  neutralized  as  in  d  and  then  rendered  alkaline 
with  an  equal  volume  of  ic/r  Na,CO,.. 

f.  Distilled  water. 

Warm  and  place  in  a  water  bath  at  40°.  At  intervals  note  any 
changes  occurring  in  the  digests,  especially  in  b.  After  forty  min- 
utes test  each  solution  for  protein  by  the  biuret  test,  after  removing 


21 


the  undigc-ted  lihriu.  C  <  unpare  the  color  of  the  hinrei  tost  given  hy 
tuhc-  li  t"  e.  Kc-ult?  I'onclu-ion-  as  in  condition-  of  activity 
..fpepsin?  Does  0.29!  I  U'l  alone  effect  hydrolysis  of  proteid?  Xote 

cially   difference  in   appearance  oi    a  and   1. 

l:.vtimiiiatiini      »f     pcpsin-HCl     tlit/cst.       Allow     a      solution 
gg  white   to   digest    overnight    with    pcpsin-IK'1.       Then 
:inine  tin-  digc-l  as  foll"\\  -  :     A.   Xcutrali/e;  i-  there  any  precipi- 
\\hat    i-    tin-    precipitate  ?      I'..    filter;    to   a    part    of    filtrate 
appl\   ferrocyanide  or  picric  acid  tc-t ;  re-ult?  Conclusion?   C.  Satu- 
rate   the    remainder   of    the    filtrate    from    A    with    (XII,),S<>,    and 
filter;  apph   the  hiuret  U  st  to  a  portion  of  the  filtrate;  re-ult?     Con- 
clusion?     Add    hr   mine    water    to    the    remainder    of    the    filtrate. 
Am    coloration? 

I'oc-  (/(•/</  alone  digest  proleid?  lloil  a  few  thin  -fip-  of  egg- 
white  for  s,>nie  time  (at  lea-t  jo  minute-)  in  \a' ,  lll'l.  Coo]  and 
apply  the  hiuret  test;  re-ult?  1  )oe-  pepsin  digest  proteins  in  the 
ah-encr  of  free  acid  ? 

'/>Y/\\-;;; — an  enx\me  occuring  in  pancreatic  juice  and  active 
in    alkaline    solnti    n.       Use    a    -olution    of    tryp-in    in     \' ,     XaA'<'. 
Taki    5  lr-t-tnhr-  and  to  each  add  a  -trip  of  fihrin.      I  hen  add: 
a.      5  or   ID  cc.   l   '  ,    Xa_.l  '<  i  . 
1).      $  OT   i< '  CC.  t  r\  ji-in  -i  >lutii  m. 

The  -ame.  e\actl\  neutralized  with  \' ,  IIC'l,  using  i  drop 
of  neutral  red  a-  an  indicator. 

d.  The  -ame  a-  c  -(-  ecjiial  x'olur.ie  of  o._>',    11(1. 

e.  5  or  KI  cc.  trvpsin  solution  previoiish'  hojled. 

I'lace  in  the  water  hath  at    }»     I  '..  and  oh-er\'e  at   intervals  to  -ee 
it  dige-tion  occurs.      After    |o  min.  remo\e  the  undigested  lihrin  and 
•    tor  peptone   with   hiuret    reagent.      Xote  especially    the   color  of 
the  hinret   n-ai'tion  in  each  case.      In  uhich  i-  peptone   formed? 

niiniliini    /if   tryptu'    <//</<•>/.      Let    5    cc.    egg-while   digest 
"\'Tiiight    witli    ]iancrea-    extract.       Lxaminc   as    follows: 

Lilh  r  the  digest.  Saturate  a  ]iorti..n  of  the  fillrate  with 
ammonium  sulphate;  filter  and  te-l  the  fillrate  for  peptone. 

To  another  porti,  >n  of  the  filtered  di-e-t  add  gradually  hroniinc 
Xote  tin    ensuing  coloration.     ( 'oinpare   with  gastric  digest. 
(  V'-v/1/. •/•//(///,•  i.  -t.  i 

tration   of   amino  acids    i  Icucin   and    tyrosin).       1;.\  aporale 

the  remainder  .  .l"  the  original   filtrate  to  the  consistency  of  a   -yrup. 

al'ohol  gradually  t<>  tin-  s\  rnp\    solution   until   no   further 

te     form-.    >tirrin-    co;itinuall\     \\ith    a    gla-s    md.       \\'hat 


substances  are  precipitated?  Gather  the  precipitate  together  with 
a  glass  rod  so  far  as  possible  and  filter  the  remainder  of  the 
mixture  through  a  dry  filter.  This  filtrate  contains  the  ainino 
acids.  Concentrate  to  a  syrupy  consistency,  transfer  to  a  flask  and 
allow  to  stand  until  next  day  for  crystallization.  Crystals  of  leucin 
and  ty rosin  separate  out.  Examine  under  microscope,  identify 
and  draw. 

91.  Examine  the  preparations  of  amino-acids ;  glycocoll,  tyrosin, 
leucin.     Test  water-solubility,  coagulation  by  heat  and  response  to 
protein    color    reactions    ( xanthoproteic,    Millon's,    biuret).      Note 
carefully  differences  from  and  resemblances  with  protein. 

92.  Vegetable     protease.       Broiiielin.      Cut    a    pineapple    into 
small  portions  and  express  the  juice  into  a  mortar.     Filter.     Test 
the  reaction  of  filtered  juice.     Place  strips  of  fibrin  in   four  test- 
tubes  and  add  as  follows:  to  a:  filtered  juice  (unaltered)  ;  to  1) :  the 
same,  exactly  neutralized  with  o.2c/f    KOH  ;  to  c:  the  same  made 
alkaline   with   an   equal  volume  of    \','<    Na^CO, ;   to   d:   pineapple 
juice   previously   boiled.      Place    at   40°    and    examine   at    intervals 
as  with'other  ferments.     Note  carefully  any  differences  from  pepsin 
or  trypsin.    In  which  tube  is  digestion  most  rapid?    After  40  minutes 
remove  the  undigested  fibrin  from  each  tube  and  test  for  peptone 
by  the  biuret  reaction. 

II.  COAGULATING  ENZYMES. 

93.  Reniiin.      Place    10   cc.    of    milk    in    each    three    test-tubes. 
To  a  add  3  cc.  rennin  solution ;  to  b  the  same,  previously  boiled ; 
to  c  add  a  few  cc.  dilute  NH4  oxalate  solution  and  then  3  cc.  rennin 
solution.     Place  at  40°  for  15  minutes,  examining  at  intervals.     Re- 
sult?   After  20  minutes  add  a  few  drops  CaCL  solution  to  c.   Result? 
Explain. 

III.  OXIDIZING  AND  REDUCING  ENZYMES. 

94.  Catalase.    To  5  cc.  neutralized  hydrogen  peroxide  (H,O.,)  in 
a  test-tube  add  about  3  cc.  crushed  liver  suspension.     Note  result. 
\Yhat   is  the   reaction?     Repeat   using  liver  which   has   previously 
been  boiled.     Result?     Also   (a)   using  a  mixture  of  5  cc.  of  liver 
suspension  and  5  drops  of  ioc/c  HC1,  (b)   5  cc.  of  liver  suspension 
and  i  cc.  of  i(/r-  Na,CO,.  (c)  liver  suspension  plus  equal  vol.  n/io 
KCN    solution,    (d)    liver    suspension    plus    equal    vol.    saturated 
HgCl,   solution.      In   which   of   the   above   is   an   evolution   of   gas 


ino-t   acti\c:     What   d<>  you  conclude   from   rc-ult-   of   experiments 
a.  1>.  c.  and  «1 : 

'  >.\-iiliist-s.  <  ixidixing  enxyme-  existing  in  the  cell-  of  organ- 
i-m-  are  readily  -olnblc  in  water.  \  eatable  cell-  yield  extract- 
of  -pccial  o\idativc  activity.  The  initially  colorle-s  surface  of  a 
pared  p..t;itn  i-  turned  l»n>\vn  by  the  oxidation  of  paraoxy-phenyl 
'.nets  -uch  a-  tyro-iu  in  the  potato-cells.  The  brown  pigment 
produced  i-  a  melanin  and  the  enxyme  is  lyrosina-c.  Tyro-ina-e 
ha-  the  po\\er  of  oxidixing  oxidixable  com]),  mud-,  like  phenol, 
added  to  the  juice  a-  well  a-  the  t\  ro-in  already  pre-ent  in  the  juice. 
Into  each  of  eight  test-tubes  place  5  cc.  of  filtered  potato  juice, 
made  by  -craping  potatoe-  on  a  grater,  expressing  and  filtering  the 
re-ult  through  clue-e  cloth;  the  -larch  is  allowed  to  -etile  and  tin 
-upernatent  fluid  removed,  filtered  and  used  in  the  tests.  I  Place  the 
March  in  the  \e--el  dc-tined  for  it.  to  be  later  purified.  i  Treat  the 
portion-  a-  foll<>\\  -  : 

a.  'ntato   |;.\tract   -|-  5  drop-  (,f  toluol. 

b.  -".xtract  boiled,  then  cooled  and  5  drops  toluol  added. 
v.       -".x tract  —  5  drop-  m',    lk'1. 

d.  Extract  +  5  drop-,,, f   10',    |\<  ill. 

e.  -'.x tract  -j-  5  drops  phenol   i  i',    i. 

l".       -"xtract   boiled,  then  cooled,  and  5  drop-    \< ,    phenol  added. 
•".\tract  —  5  drop-  guaiac  -olution. 

h.      ;.x tract  +  5  drops  a-napthol  +  5  drop-  para-phenylene 
tliamine  -<  ilutii  •!!. 

i.     To  another  lube  add   water   —   5   drops  u-napthol   -j-   5   drops 
para-phenylene  diaminc  -olution. 

Thoroughly  -hake  each  mixture.  Xotice  any  change-  in  15  or 
,;o  minutes  \\here  i-  the  color  mo-t  marked?  \\"hy  :  Set  aside 
until  the  next  laboratory  |ieriod  and  record  change-  in  each.  The 
r  liio]  i-  ad«U-d  a-  a  preservative.  It  \\oiild  kill  any  cell-  pre-ent 
but  doe-  not  de-lro\  the  o\ida-e.  I-  the  phenol  a  preservative: 
b.  i.  and  i  are  control  experiment-  for  e  and  h  re-pectively.  Do 
ou  see  why:  .Vote  that  potato  juice  accelerate-  a  proces-  which 
take-  place  -louly  in  the  ab-encc  of  potato  juice,  a-  in  i. 

'»'•.  (  utiiltts,-  diitl  ,i.vi<lii.\-t-  in  tininitil  tissues.  a.  Tea-e  finch 
\\ith  a  pair  of  needle-  p.irti«m-  of  the  s^l.'cn,  H:\-r.  linii/,  kidncv, 
and  inn.",!,-  oi  tlu'  t'rog.  I'lace  portion.  ,,f  the-e  ti--ne-  in  a  seric- 
of  \\atch  gla--e-  and  add  to  each  dilute  IL<  >,.  \\'hich  tissue  gives 
the  nio-t  ].ronounced  i-atalytic  action: 

lo    a    -imilar    series    of    \\atch-gla--r-    containing    the    -ame 

-4 


freshly  teased  tissues  add  an  alkaline  mixture  of  u-naphthol  and 
p-diamino  benzene  solutions  in  0.7' ','<  NaCl.  Note  the  results.  Ar- 
range the  tissues  in  the  order  of  their  actiritv  in  accelerating  this 
oxidation. 

97.  Localization  of  iinloplicnol  formation  in  blood  corpuscles  of 
the  fro(j.     Prepare  4  cc.  of  a  mixture  of  equal  parts  saturated  solu- 
tion of  alpha  naphthol  in  an  alkaline  NaCl  solution  (m/8  NaCl  -)- 
m/ioo  NaL,CO:;).  and  i%  di-methyl  para-diamino  benzene  in  0.7% 
NaCl.     Add  this  mixture  to  a  few  drops  of  a  suspension  of  frog's 
blood  corpuscles  in  a  solution  of  m/8  NaCl  +  m/ioo  K.,C,O4  in  a 
watch-glass.    Mount  two  or  three  drops  of  this  mixture  en  a  slide  so 
as  to  have  several  air  bubbles  under  the  cover.     In  the  course  of  a 
few  minutes  the  indophenol  will  appear  in  the  cells. 

In  which  cells  does  it  appear  first?  Where  is  it  chiefly  localized 
in  the  cell  ?  Is  there  any  relation  between  the  position  of  the  air  bub- 
bles and  the  rate  of  formation  of  the  oxidation  product? 

98.  O.vidation  test  for  blood  with  (juaiac  tincture.     Apply  a  little 
guaiac  suspension  containing  hydrogen  peroxide  to  a  blood  stain. 
Note  the  result.     Other  dried  animal  tissues  give  a  similar  reaction. 

IV.     SPECIAL  CHARACTERISTICS  OF  ENZYME  ACTION. 

99.  Specificity  of  enzyme  action.    Collect  saliva,  dilute  with  four 
volumes  of  water  and  filter.     Place  about  3  cc.  in  each  of  five  test- 
tubes.     Then  add : 

a.  5  cc.  of  starch  paste. 

b.  5  cc.  of  5^-  cane  sugar  solution. 

c.  One  drop  of  olive  oil  -(-  5  cc.  I  '/(   Na.CO.,. 

d.  A  shred  of  fibrin  -f-  5  cc.  of  0.2%  HO. 

e.  5  cc.  milk. 

Set  tubes  a  to  e  ( inclusive )  in  water  bath  at  40°  for  i  hour,  and  then 
test  the  tubes  as  f  ollo\vs : 

Tube  a,  for  starch  and  glucose. 

Tube  b,  for  glucose. 

Tube  c,  for  soap  by  emulsification. 

Tube  d,  for  peptone. 

Tube  e,  note  if  coagulation  occurs. 
What  do  you  conclude  as  to  the  action  of  ptyalin  in  the  saliva? 

100.  Reversibility  of  enzyme  action.    Many  enzymes  are  reversi- 
ble in  their  action,  i.  e.  they  can  accelerate  either  decompositions  or 
syntheses  of  the  substances  on  which  they  act,  according  to  the  con- 


tratioiis  <>f  the-c  -nit-lances  in  the  -olmion.      I.ipa-c  is  the  mo-t 
fa\.  .ral.le   en/yme    for   tin.-   <K monstrati>  >n    of    this   property.      Take 

a  iv  »••  u-e  tlu-  exact  amount-  called   f<  >r  in  the  directions, 
a.      //V(/r«'/v/».  .  •  r  -plating,  action  of  lipa-e.     I  .ahel  _'  -mall  tla-ks 
•  I ul. es  a  am!  h.      In  a  place    i<>  CC.  of   neutral   pancreatic  ex- 

I  etlnl  butyrate  u'l !  <  I  I  t  I  I..O  H  U'JL). 

•k  tiidith   and  keep  at    )"     for  4"  minute-.     Kemove  to  ice  \\ater 

until  quite  cold.    ('..].. r  with  litmu-.     \\liat  is  reaction?     Xow  empty 

the  content-  into  a  heaker  ami  titrate  iniinciinitcly  with  n    jo  \a(  >l  1. 

i.e.   ad-1   the  alkali   carefully    from   the   hurette   until    the   color   ju-t 

change-  to  blue.      N'ote  a-  exactly  a-   |>o--iMe   the  amount   of  alkali 

lla-k    h    a«M    the    -ame    (|uantitie-    of    ethyl    hutyrate 

and  |>ancreatic  juice  f'rcriottsly  />«;7r</  and  proceed  in  the  -ame  man- 

(  -..npare  the  re-ult-  of  a  and  h.      Write  the  equation    for  the 

-plittini,'  of  ethyl  hutyrate:  of  glycerol  hutyrate  ihuttcr  fat). 

h.     Synthetic  nction  <>)  Lipusi-.     Mix  _'5  cc.  of  m   _'O  hutyric  acid 

and   10  CC.  -  ''  ,    ethxl  alcohol.      I'lace  _'()  cc.  in  each  of  two  flasks,  a 

and    h  ;    to   a    add    5    cc.    pancreatic    extract;    to    h    5    cc.    pancreatic 

i  xtracl    previously   h<>iled.      ( '<>rk   hoth   tla-k-   tightly   and   place  in   a 

water  hath   at   .\»      for    J<>  minutes.     'Then   compare   the   »d«r    from 

the    two   tla-k-.      1-    the    ethyl    hutyrate    to    he    detected?       In    which 

I  low   forim-d?     \\rite  the  e<|uali'.n.      l;.ni|>tv  the  contents  ot 

li   tla-k   into  a  heaker  and   titrate   with   n    Jo   K()ll.  usiiij^  litmti- 

aii    indicat'ir.      Mow   much    i-   required    for  each   tla-k?      1  )o  your 

•'.It-  indicate  that  -ome  acid  ha-  disappeared  through  -ynthe-i-  to 

!'•  inn    the   ester? 

i'   i.      Influence    .•/    temperature    an    enzyme    action.       (  (Jimntita- 

tleteniiiiiiitioii.  i     I'ollect   -aliva,  dilute  with  ~  volumes  of  water 

and  t'dter.       \d<l   ;  cc.  <lilutc-  starch  paste  to  each  of   four  test-mite-. 

"lie  t< -t  tnhe  in  the  \\ater  hath  at  40     » '..  and  keep  another  at 

in  temperature  i  ahont  ji  t     ('.  ).      Have  two  series  of  iodine  drops 

"n  a  porcelain  plate.      When  the  te-t   tuhe-  have  attained  the 

j-p.per   temperature-  add   an   equal   volume  of   dilute   saliva   to   each 

tnhe.    mix    thoroughly,    and    keep   at    their    respective    temperature  -. 

At   inteixal-  iif  niie  half  minute  remove  a  drop  ,>f  the  mixture  and 

h.      <  'ne    -indent    attend    to    the    le-l-    from   4C      -ali\a 

in:  the  other  to  the  20    mixture.     Determine  the  time  required 

convert    the    March    into  pruduct-    L;ivint,r   no   colur    reaction    \\ilh 

ich  temperaf 

.ictl\     the    -ame    experiment    n-iiii;    the    Siiinc    diluted 

\-"'  hut  I  .....       it  o°  C  and  compare  it  with  another 


at  20°  C.  Record  your  results  in  the  form  of  a  table.  How  near 
in  agreement  are  the  two  determinations  at  20°  C.  ?  \Yhat  is  the 
increase  in  rate  of  enzyme  action  for  a  rise  of  20°  C.? 

IC2.  Rate  of  decomposition  of  H.2O.2  by  catalase  at  different' 
temperatures.  Place  25  cc.  of  dilute  filtrate  from  crushed  liver 
suspension  in  a  bottle  provided  with  a  perforated  cork  and  bent 
or  flexible  tube  ;  place  carefully  in  the  bottle,  witJiout  spilling,  a  small 
vial  with  5  cc.  H.,(  ).,  solution.  Insert  the  end  of  the  bent  tube  below 
the  mouth  of  a  eudiometer  or  inverted  graduate  filled  with  water 
and  placed  mouth  downward  in  a  beaker  of  water.  Then  shake 
the  bottle  so  as  to  spill  the  vial  and  mix  its  contents  with  the  liver 
extract.  Measure  the  volume  of  O.2  evolved  from  minute  to 
minute  and  record  the  results.  Try  the  experiment  with  ( a  )  cold 
mixture  (liver-extract  ami  HoO,  cooled  just  before  mixing  to 
about  o°  C. ),  (b)  mixture  at  room  temperature  (20  ),  and  (c)  at 
40°.  Estimate  the  relative  rates  of  action  at  the  different  tem- 
peratures. 

103.  Effect  of  concentration  of  enzyme  on  enzyme  action. 
(Quantitative  determination. ) 

Collect  saliva  and  filter.  Add  5  cc.  dilute  starch  paste  to  each  of 
two  test  tubes. 

Have  a  series  of  iodine  drops  ready  on  a  porcelain  plate  as  in  exp. 
101.  Add  as  follows  to  the  two  test-tubes: 

a.  5  cc.  of  r  pt.  saliva  to  3  pts.  water. 

b.  5  cc.  of  i  pt.  saliva  to  /  pts.  water. 

Keep  at  20°  C  and  determine  by  trials  at  one-minute  intervals  when 
the  iodine  gives  no  color  reaction  with  the  mixture. 

After  making  the  first  series  of  experiments  perform  the  same 
experiments  using  the  same  saliva  diluted  as  follows : 

c.  5  cc.  starch  paste  -|-  5  cc.  of  i  pt.  saliva  to  15  pts.  H.2O. 

d.  5  cc.  starch  paste  +  5  cc-  ot~  l  pt-  saliva  to  31  pts.  H2O. 
Keep  at  20°  C.  and  determine  by  trials  at  two-minute  intervals  the 

time  required  to  convert  the  starch  to  a  substance  giving  no  color 
with  iodine. 

E.     ENZYMES  AND  METABOLISM   IN  CELLS. 

In  living  cells  syntheses,  hydrolyses,  and  oxidations,  all  of  which 
are  under  the  influence  of  enzymes,  interact  in  the  metabolic  pro- 
cesses. The  interdependence  of  syntheses  and  oxidations  is  well 
shown  in  Pasteur's  experiment.  The  metabolism  of  the  cell  varies 
according  to  the  quantity  of  oxygen  available. 

27 


/\'e!ntii'ii  <>f  u.viildtu'iis  ti>  synthetic  processes  in  yeast 
tells.  l\istenr's  experiment,  a.  'lake  I  K>  oi  a  \ea>t  cake  and 
mix  thoroughly  in  a  mortar  with  _'<  >  CC.  water.  Add  10  cc.  of  thi> 
mixture  t»  i  "f  nutrient  tluid.  Mix  tin  >n  Highly,  and  while  the 

i-t  i-  uniformly  suspended  divide  int<>  two  equal  part-,  a  and  b. 
Mace  a  in  a  buttle  pr  \  ided  with  a  oirk  and  exit-tube  to  carry 
1  ;  tin-  tube  -hoiild  end  bel<  >w  the  >urt';n'e  of  water  in  a 
beaker.  iWtimi  b  >hnuld  be  poured  int<>  one  <>r  more  wide  jars  or 
di-he-  t"  fcirin  a  thin  layer  well  exposed  t  air;  cover  loo>ely  with 
^la-- plaie^.  Place  a  and  b  aside  tor  2  days  or  more.  Then  examine 
both  carefully,  comparing  tlu-  a]i]>earance.  relative  turbidity,  odor, 
and  d<  •!"  ett'erve-cence.  I  ran-ter  b  to  a  bottle.  Shake  each 

|»«rti"ii  <a  and  b)  ;  take  equal  (|uantitie-  of  each.     Centrifugalize  and 
eumpare  the  am   unt-  of  the  >ediment.     l\e>ult>?     Conclu>i<>n>? 

b.  .llciilinl  formation  in  presence  and  absence  of  <>.\'\'</cn.  Ar- 
range a  -imple  >till  b\  allo\\in^  a  lR.n;  tu])C.  from  a  rla>k  to  run  to  the 
Inittoin  (,f  a  narrow  te-t-tube  immersed  in  cold  water.  I)i-till  ot'f 
from  both  a  and  b  (  usini,r  a  moderate  tlame )  a  few  cc.  of  the 
di-tillate.  idol  the  di-tillate-.  ( 'oinpare  their  >mell  and  ta-te. 
\\arm  each  \\itb  lx.(  r.''-  and  1I..S'),.  Kestilt  ?  d  nclusion  ? 

1  -Meet  i  CC.  of  j^a-  from  a  fermenting  yeast  culture  in  an 
inverted  te-t-tube.  Te-t  the  .^as  by  introducing  in'.  K<)11  solution, 
h\  mean-  of  a  bent  pipette,  into  the  tube  in  contact  with  the  <,ra-. 
Ah-orptiiin  indicate^  (  "i  i  .  \\'hy?  \\rite  the  eiiuati'  n. 


PART  II 
PHYSICAL  CHEMISTRY  OF  CELLS 

A.     SOLUTION  AND  DIFFUSION 
A.     GENERAL  PHENOMENA. 

The  purpose  of  the  following  experiments  and  simple  observa- 
tions is  to  enable  the  student  to  form  a  concrete  idea  of  the  reality 
and  character  of  diffusion  and  solubility,  and  to  work  out  some  of 
their  general  laws  for  application  later  to  organic  processes. 

105.  Fill  a  test-tube,  supported  on  a  clamp,  to  within  an  inch 
from  the  top  with  distilled  water.     Place  in  a  position  where  it  can 
remain  entirely  undisturbed.     Then  with  a  long  pipette  drawn  to  a 
slender -point,  place  in  the  bottom  of  the  test-tube  2  cc.  of  potassium 
permanganate  solution,  in  such  a  way  as  not  to  disturb  the  water 
and  to  leave  all  the  purple  solution  at  the  bottom.     On  the  outside 
of  the  tube  mark  the  level  of  the  potassium  permanganate  solution. 
Observe  that  it  very  slowly  rises,  by  diffusion.     Mark  the  position 
reached  by  the  end  of  the  laboratory  period,  and  leave  the  experi- 
ment in  progress  until  the  next  period.     Mark  the  point  to  which 
the  fluid  has  then  risen.    What  is  the  approximate  rate  of  diffusion  ? 

106.  Seal  off  a  glass  tube  at  one  end  and   fill   with  hot    1.570 
agar-agar,  colored  red  with  neutral  red  solution.     Set  in  an  upright 
position  to   solidify.     When   solidified,  place   in  a  bottle   of  0.2', 
KOH  open  end  down.     Measure  by  the  change  in  color  of  neutral 
red,   the  amount  of   diffusion   during  the   laboratory   period.      Set 
aside   for  measurement  next  day.     What   is  the   rate  of   diffusion 
of  KOH  as  thus  measured  ? 

107.  Fill  a  petri  dish  with  warm  agar-agar  solution   (1.5%  )  and 
allow  to  cool.     When  solidified  place  one  drop  of  each  of  the  fol- 
lowing   solutions    on    the    agar-agar :      a.     Saturated    CuSO4 ;    b. 
KoCr..O7 ;     c.     KMnO4 ;     d.     aqueous     neutral     red ;     e.     aqueous 
methylene   blue ;    f .    haemoglobin  ;    g.    chlorophyll   solution.       Com- 
pare the  rates  of  diffusion.     Is  there  any  difference?     Is  it  related 
to  the  molecular  weights  of  the  substances?     Cover  and  leave  until 
the  next  day  when  the  diffusion  may  be  again  noted. 


-9 


-onie  alcoholic  solution  of  mcthylene  blue,  a  few 
dn>p-  at  a  time,  "ii  tile  -urfacc  of  \\ater  in  a  wutch-^las-.  1>  CS 
mixture  taki  place  -lowly  or  rapidly:  (In  this  case  \\  e  liave  an 
additional  factor  inv- .lve<l. — change-  in  surface  ten-ion.)  Try  in 
tlu-  -ami-  wa\  an  a«|Ui-ou-  -olmion  of  niethvlene  blue.  Are  ibe  results 
•it  ?  \\  li\  : 

lla\r    a    Uaker    full    "f    water    wbieh    i-    kept    undisturbed. 
and   oh-er\e   from   the   side.      I 'laee  on   the   -urface   -onie   powdered 
metluleiie  blue:  ob-ervc  the  solution  and  diffusion.      Describe  accu- 
!y  what  you  -• 

i  i  i.  a.  I'lace  a  lilanient  "f  Spiro^yru  in  a  dilute  solution  I  I  drop 
tn  a  wateh-.yla--  of  \\ateri  of  neutral  red  in  pond  water.  Is  the  dye 
taken  up:  hoe-  it  beeome  more  eoneentrated  than  in  the  external 
-nluiioii;-  U  thi-  eonirary  t"  phy-ieal  law-?  l;.\ainine  the  cells 
under  the  inien>-eope  and  determine  the  condition  in  which  the  dye  is 
held  within  the  cell.  Ik-scribe. 

b.  Place  in  another  dilute  solution  of  neutral  red  some  Spirojjyra 
tilanuiits  that  have  hreii  killed:  (a)  by  immersion  for  one  minute 
in  chloroform-saturated  water,  ibi  by  immersion  for  one  minute 
in  boiling  \\ater.  How  do  tlii'  re-ult-  coni])are  \\ith  tho-e  on  living 
ci-11  \plain. 

ill.  Kepeat  t-xperinient  lio  with  l-'.lodea  leaves.  How  i-  the 
d\e  accnmulatid  in  these  cells?  U  this  in  opposition  to  ]>hysieal 
law-  More  concentrated  dve  may  be  nece-sarv  here.  1  low  does 
I'.iiamecium  take  U|>  neutral  red.  Try  a  very  dilute  dye  -  >lntii>n. 

SEMIPERMEABLE   MEMBRANES  AND  OSMOSIS. 

iij.      \\hat    i-   a   setni-pcrmcable   membrane.'     Such   membrane- 

ma\   be  made  a-  follows;      iai    Introduce  by  a  tine  mouthed  pijiette 

<  u><  i,  lielo\\   ilu-  -urface  of  -'' -     K4Fe(CN  i,,  in  a  watch-.^la--. 

\\'hat    i-   the  membrane    formed?      Give   the   reaction.      (  >h-ervc   the 

Doe-  it  change  in  thickness?      Do  the  drops  of 
(  u><  i,   ilnis    formed  change   in    size?      If   SO,   why? 

1  b.  i  Tannate  of  gelatin  membrane-:  a  -olnlion  of  _"  ,    tannic  acid 

tainin^'  su^ar  \(>  ,-<;.  m    _'  conci-ntration  i-  allo\\ed  to  llow  a-  abo\i- 

i  the  ~urface.it"  a  noil  -elatini/in^  m',    i^.-latin  -olntion.     Study 

tin   (  bara.  ter  "f  the  membrane,  form,  and  changes  of  si/e  as  lu-fore. 

\\  hat  part  doe-  the  -n^'ar  plav  in  this  e\pi-rinient  ? 

1l  t!ii-,>n;/li  til>s»r^ti<'ii  ,'f  zvatcr.     Suspend  by  a  thread 

m  a  3^5    (   nS<  i,   -olution  and  place  where 

be    undi-turbed.      Observe    the    general    characteri-tic-    and 

30 


growth  of  the  resulting  formation.     Uo  you  note  any  movements? 
What  are  they  due  to?    Draw. 

114.  Sea-vueed-like  formations  tliroiu/h  t/ro^'tli  of  precipitation 
membranes.     Place  a  lump  of  fused  CaCL,  in  the  bottom  of  a  tall  jar 
filled  with  concentrated  XaL,CO,  solution.     Set  aside  for  several  days 
and  watch  the  development  of  a  plant-like  growth. 

115.  Membranes  formed   throiii/Ji   surface   action,     a.    Shake  a 
few  drops  of  olive  oil  in  a  test-tube  with  the  following  fluids    ( a  ) 
water,  and  aqueous  solutions  of   (b)    XaCl   (0.65^  ),   (c)   peptone, 
(cl)  soap,   (e)  haemoglobin,  (f)  albumin  (g)  sugar,  (h)  starch   (i) 
lecithin,  and  ( j )  gelatin  (  2('/(  ).    In  which  does  a  permanent  emulsion 
occur  ?     \Yhy  ? 

1).     Perform  the  above  experiment  using  chloroform  instead  of 
olive  oil.     Results  ? 

1 16.  Formation  of  artificial  cells  surrounded  by  a  film  of  modified 
protein.      Shake    chloroform    with    an    albumin    solution.      Can    the 
film  be  removed  b}-  repeated  washing  in  water?     Pour  some  of  the 
chloroform    globules    into    a    watch-glass    of    water    and    examine 
under  the  microscope.    Can  you  see  the  film?    Does  the  chloroform 
slowly  evaporate  ? 

If  lecithin  is  dissolved  in  the  chloroform,  it  will  absorb  water  as 
the  chloroform  evaporates,  and  a  watery  solution  of  lecithin  can  be 
obtained  surrounded  by  a  protein  film.  Such  a  cell  resembles  in 
many  of  its  properties  a  sea  urchin  egg.  Prepare  some  of  these  cells 
by  shaking  a  lew  drops  of  a  chloroform  solution  of  lecithin 
(m/8o)  with  albumin  solution,  washing  with  water  and  pouring 
the  globules  into  a  watch-glass  of  water.  X>cte  from  time  to  time 
during  the  course  of  the  hour  their  appearance  under  the  microscope 
and  draw  the  stages  observed.  "When  chloroform  has  been  com- 
pletely replaced  by  water  add  a  drop  of  neutral  red  to  the  water  in 
the  watch-glass.  Is  it  accumulated  by  the  cells?  Try  pricking  the 
cell  with  a  fine  needle  to  determine  the  consistency  of  the  contents 
and  also  of  the  membrane. 

C.     FORCE  OF  DIFFUSION— OSMOTIC  PRESSURE. 

The  energy  of  diffusion,  or  the  tendency  which  two  substances 
have  to  mix,  may  be  measured  by  separating  them  by  a  membrane 
through  which  one  of  them  can  pass  while  the  other  can  not.  The 
latter  then  produces  pressure  on  the  membrane  and  this  pressure  can 
be  measured.  This  is  partly  accomplished  by  the  following 
experiment. 

31 


i  17.  I 'our  out  a  thin  layer  »f  collodion  -olution  mi  a  mercury  sur- 
face in  a  petri  di-h  and  allow  it  to  harden  -omewhat.  \\'hile  still  flexi- 
ble remove  tin-  lilni  fnun  the  mercury  >urface  and  tie  tightly  on  the 
end  of  a  thi-tle  tnhe.  Prepare  two  of  thc-c  "osmometers".  Fill 
.  iiu  I  \\ith  in  J  -ugar-formol  solution  to  a  p  int  on  the  stem 
•  me  inch  aho\e  the  hull).  Fill  the  other  (  />  )  with  water  to  a  point 
\\nhin  "lie  inch  of  the  top  of  the  tuhe.  Place  i/  in  water  and  /'  in 
m  _•  -ugar-formol  -olution  in  a  heaker.  .Vote  any  change  in  the 
level  .  >f  the  nieiii-cn-  of  ,;  and  /'.  I  low  high  doe-  the  level  of  sugar 
in  (/  become?  Does  it  then  remain  stationary?  Kxplain  all  the 
plieii'-mena  noted  in  connection  with  the  experiment,  leaving  the 
.  Milometers  -et  up  for  -cveral  day-. 

I  |S.  Prepare-  two  test-tnhe  collodion  hag-  a-  follows:  The  te-t- 
tuhe  mu-t  he  cleaned  with  alcohol  and  dried  thoroughly.  Pour 
ollodion  -olutioii  into  a  dry  clean  test-tuhe  and  then  pour  it  out 
-lowly  into  the  collodion  hottle.  revolving  the  test-tube  so  a-  to  coat 
the  gla--  evenly  \\ith  a  thin  film  of  collodion.  Allow  this  to  harden 
-onieuhat.  then  gently  pry  off  the  edge  and  allow  -oine  water  to 
run  down  hetween  the  tilm  and  the  te-t-tuhe.  P.y  carefully  ]>rying 
and  pulling  the  film  may  he  entirely  -eparated  from  the  glass.  Fill 
with  water  to  make  -ure  there  are  no  leaks.  If  the  collodion  hags 
are  pu-fcet  till  one  (a)  with  m  4  salt  solution,  and  tie  with  a 
piece  i  if  -tring  ahont  the  middle  50  a-  to  ohtain  a  firm  -alt  water 
tilled  hag.  Fill  the  other  (  />  )  half  full  of  alhumin  solution  and  tie 
off  so  a-  to  lea\e  -onie  air  enclosed.  Note  that  it  is  not  firm.  Then 
place  <;  in  Jm  -all  -olutioii  ami  /'  in  water.  Xote  any  change-  in 
rigidity  of  the  hag-?  F.xplaiiation  ? 

i  i1;.  Place  a  drop  of  frog'-  him  id  corpu-cles  in  water  in  a  watch- 
gla-s  and  examine  <//nV/.-/v  under  the  micro-cope. 

Place  a  drop  of  -n-pended  frog'-  lilood  corpuscles  in  m  XaCl  solu- 
tion and  examine  under  micro-cope. 

What  happen-  in  each  case?  Kxplain.  Xote  -imilarity  to  the 
preceding  experiment. 

i  jo  Osmotic  prcssitri'  in  li-iii,/  c^lls.  f'litsni<>lvsis.  Place 
>pirog\i-.i  lilament-  in  cane--ngar  -ohnion  of  concentrations:  m  J, 
in  i,.  m  4.  m  5.  m  '',  m  -.  m  S.  and  in  I\X<  >.  -olution-  of  the  -ame 
.-..infiltration-,  in  \\atch-gla-  X'ot,'  carefully  under  the  micro- 

pe    am    changi--    of    volume    of    the    prot.,pla-m    within    the    cell 
walls.      What    solution   jn-t    fail-   to  pla-mo]y-e   in   the  ca-e  of   hoth 

[ar  and    l\     '  '         Take  the  avera-e  of   tin-   filament-   in   the   di-h. 
.hat  i-  the  o-niotic  pr,  x-,,re  of  the  -ap  of  Spin.gyra  cell-  a-  deter- 


mined  by  sugar  plasmolysis  ?  Calculate  the  isotonic  coefficient  (i) 
for  KNO8  at  the  isotonic  concentration.  Then  calculate  the  degree 
of  dissociation  (a)  of  the  KNO,,  using  the  formula: 

i  1  -)-  (K  l)a,  where  K  =  number  of  ions  from  one 
molecule,  and  a  the  proportion  of  molecules  split  into  ions. 

121.  Perform   the    same    experiment   as    above,    and    make    the 
same   calculations,   using   instead   of    Spirogyra   the   leaves   of    the 
water  plant,  Elodea,  only  two  cell  layers  in  thickness :  a  layer  of 
large  cells  above  and  of  smaller  cells  below. 

122.  Place  blood  corpuscles  of  the  frog  in  m/2,  111/4,  m/6,  111/7, 
m  8,  ni/io,  and  m/i6  NaCl  solutions.     In  which  solution  do  the 
corpuscles  retain  their  volume  unchanged?     What  is  the  osmotic 
pressure  of  this  solution  if  the  isotonic  coefficient  is  1.83? 

123.  Try  plasmolysing  the  cells  of  a  marine  plant  (Ulva)  using 
2m,  i.^ni  and  /;/  sugar  solutions.     In  which  solution  does  plasmolysis 
take  place?     Why  are  stronger  solutions  needed? 

B.     CELL  PERMEABILITY 

Dead  membranes  such  as  parchment,  or  collodion  or  cellulose  are 
readily  permeable  to  crystalloidal  substances  in  general,  but  not  to 
many  colloids.  The  membrane  surrounding  living  cells  is  permeable 
to  some  crystalloids  but  not  to  others,  thus  exhibiting  a  ''selective 
permeability",  which  is  characteristic  of  both  animal  and  plant  cells. 
Some  of  the  permeability  relations  of  living  cells  are  brought  out 
in  the  following  experiments. 

124.  Permeability   to   non-electrolytes.     Place  Elodea  leaves   in 
the  following  solutions  of  both  111/2  and  111/3  concentrations  in  watch- 
glasses  :     Cane  sugar,  grape  sugar,  urea,  glycerine,  alcohol.     Which 
of  these  solutions  have  the   same  osmotic  pressure?     Do  they  all 
plasmolyse?     Note  carefully  whether  plasmolysis  is  permanent  (till 
the  end  of  the  laboratory  period)  when  it  occurs,  or  only  temporary. 
How   do   you   explain   any   differences   noted?     Which    substances 
penetrate  the  cells  most  rapidly  ? 

125.  Pcnncabilit\  to  salts.     Place  Elodea  leaves  in  111/2  and  111/3 
solutions  of  common  salt  and  KNO3.    Have  these  solutions  the  same 
osmotic  pressures?     Have  they  the  some  osmotic  pressures  as  the 
above  mentioned  solutions?     Is  plasmolysis  permanent  in  both  salt 
and  KNO3?    Leave  in  the  solutions  for  i  hour  or  longer.    What  does 
this  indicate?  ^i 

126.  Permeability  to  alkalies.     Elodea  leaves  stained  in  neutral 
red  are  to  be  used.    Note  if  they  show  protoplasmic  rotation.     If  the 

33 


alkali  enters,  the  neutral  red  will  he  -tained  yellow.  I 'lace  in  11.40 
Xa<  ill.  Ba(<  '11  '  .  ami  Xll,<  >ll.  and  reo.nl  the  time  required  for 
the  change  of  r»l«ir  to  o^cur  in  each  ca-e.  As  SOOn  as  the  leat  i> 
\\h- 'lly  \ello\\  place  in  pond  water.  hoe-  the  red  o>lor  return? 
\\  Inch  leave-  have  heen  killed?  1  >oe-  recovery  of  pmt.  ipla>niic  n>ta- 
ur  ? 

ij-.  liiiperiiieahility  to  \a<  'II  may  he  -h  wn  in  a  -inking  man- 
ner h\  the  following  experiment:  I 'lace  an  Klodea  leaf  stained 
in  neutral  red  in  n  40  Xll,<  ill  till  yellow;  then  remove  it  to  n  40 
Xa<  >11.  h.  ics  it  hcci>me  red  ? 

Stain    1'aramecia  in  a   watch-gla--  of   pond  water  to   which 
only  a  /><;<v  of  neutral   red  ha-  heen  added.      Place  a  drop  dcn-cly 

•  ude.l  with  red  -tained  I'aramecia  in  n  500  Xa')ll  and  11/500 
(XII,i<>H  in  pond  water.  XoU-  the  time  of  color  change  and 
length  of  life  in  each  ca-e.  Are  the  I'aramoecia  killed  in  Xa()II 
hefiire  the  alkali  enter-:  How  ahont  Xll,()ll?  Which  region 
of  the  cell  HUM  the  Xa<  >11  attack? 

U'(.  c7i(/;;</r  /;/  pcnnc(ih\l\t\  nil  death.  Place  an  Klodea  leaf 
-tained  in  neutral  red  in  n  40  Xa<  )||  and  another,  previously  killed 
h\  J  minute-  immer-ion  in  saturated  chloroform  water,  in  the 
-ame  -ohition.  \\'hich  i-  more  permeable? 

1^0.  Place  -mall  cuhe-.  cut  from  a  heet  and  washed  in  running 
\\ater,  in  a  te-t-tuhe  containing  water.  Place  similar  cuhe-  washed 
and  then  heated  to  hoiling  for  a  moment  in  another  test-tuhe  of 
water.  Xote  from  time  to  time  the  amount  of  diffusion  in  each 

C,  of  the  red  pigment   from  the  heet  cuhe>.     Significance? 

i  _}  i .  The  rapid  penetration  of  Xll,<  HI  into  cells  is  probably  to 
he  correlated  with  it-  solnhility  in  fat  solvents  or  Hpoid  substances. 
Suluhiliu  of  XII,' )!I  in  fat  solvents  max  he  demonstrated  as 
follow-:  Shake  a  \\lol  -olmion  of  lecithin  with  egg  alhumin  solution 
coiiiaining  a  fi-w  <lrop-  of  neutral  red.  Xote  that  the  glohule-  of 
lieii/ol  taki-  up  the  neutral  red.  X'o\\-  place  -ome  of  the  latter  in 
n  I«H)  Xll,()||  and  n  too  Xa<  >l  I  in  watch-gla--i--.  Which  alkali 
penetrate-  and  \\liy? 

132.       Sll/ill    and    Xa<>ll    diffuse    through    dead    memhranes    at 

the  -ame  rate,      hem   n-trale  thi-  hy  o>\-ering  the  end-  of  two  tuhe- 

uith   a   e. -llodion    film    (made  on   mercury    -urface   and    tied   tightly 

the  tuhe-i.  then  tilling  with  agar-agar  colored  \\Jth  neutral  red 

and  immersing  the  covered  end  of  the  tuhes  in  n    loo   Xll,<)ll   and 

v,"a<»ll    re-].e<  ii\el\.      Tlu-   diffu-ion    rate-    of    Xa<>||    and    Xll,(>ll 

in  pure  \\ater  are  ].racticall\    the  -ame. 


133-  Salts  change  cell  permeability.  Often  a  pure  salt  affects 
the  permeability  of  the  plasma  membrane  so  that  it  cannot  be  used 
to  determine  the  osmotic  pressure  of  the  cell.  A  mixture  of  certain 
salts  (especially  salts  of  Na,  K,  and  Ca )  maintains  the  cell  surface 
in  a  normal  condition. 

Place  Spirogyra  cells  in  the   following  solutions : 

1.  Cane  sugar:  m  2,  m  4,  m/6,  m/8. 

2.  XaCl :  m/2,  111/4,  ni   ().  ni/8. 

3.  XaCl  (95  vols. )  -+-  CaCl,  (5  vols. )  :  m/2,  m/4,  m/6,  m/8. 
Determine   the   plasmolytic    limiting   concentration    in    each    case. 

The  cane  sugar  will  giye  the  true  osmotic  pressure.  Note  carefully 
if  at  first  plasmolysis  begins,  then  disappears  and  then  appears  again 
(false  plasmolysis)  in  any  of  the  XaCl  solutions.  Compare  with 
the  mixtures  of  Xa  and  Ca.  Explanation? 

134.  Salts  affect  the  penetration  of  NaOH.     Place  Spirogyra  or 
Elodea,  stained  in  neutral  red,  in  the  following  mixtures. 

1.  111/40  XaOH. 

2.  111/40  NaOH  +  m/8  XaCl. 

3.  m/40  XaOH  -f  m/8  (95  vols.  XaCl  +  5  vols  CaCl,). 
Determine  the  time  required   for  entrance  of  alkali  in  each  case. 
As  a  control  kill  the  cells   in  chloroform  water  and  place  in  the 
above  solutions.    Conclusion  ? 

C.       SURFACE  TENSION  AND  RELATED  PHENOMENA 

Note  in  all  the  following  observations  and  experiments  that  the 
surface  behaves  in  many  ways  as  if  it  were  a  stretched  elastic  mem- 
brane. 

A.     THE  SURFACE  FILM. 

Experiments  to  give  a  concrete  realization  of  the  existence  of 
surface  tension. 

135.  Drop    a   needle    carefully   on    the    surface    of    still    water, 
so  that  the  entire  side  of  the  needle  strikes  the  surface  at  once. 
\\~hy  does  it  float  ?     Notice  the  depression  on  the  surface  film  close 
to  the  needle. 

136.  Drop  water  slowly  in   drops   from  a  pipette.     Notice  the 
form  of  the  drops,  and  how  they  become  stretched  or  elongated 
just  before  they  drop.     \Yhy  do  they  take  the  form  they  do? 

137.  Form  a  film  on  a  circular  wire  frame  with  soap  solution. 
(This  is  really  a  double  film.)      Does  the  film  pull?     Test  this  by 
forming  a  film  on  a  circular  wire  frame,  laying  a  loop  of  thread  on 

35 


the  film,  and  then  breaking  the  film  within  the  loop.     What  happen-  ? 

i^S.  hip  a  camel'-  hair  hrn-h  in  water;  notice  how  it  spreads 
•  nit  mi  tile  water  hut  clo-e-  U|>  when  taken  out.  What  pulls  the 
l»ri-tle-  together? 

1^0.  Cses  made  <>f  surface  film  l>\  ori/anisms.  (  >bscrve  any  of 
the  following  for  which  there  is  an  opportunity:  Spiders, 
"whirligig-".  «•!•  other  animal-  moving  on  the  upper  surface  of  the 
water;  tlatwnn-  or  -nail-  creeping  on  the  under  -urface  of  the 
film. 

n>K.MS     OF      FLUDS      I'RoDL'CKI)      I'.V     SURFACE 
PENSK  'X. 

1411.  f'latean's  experiment.  Half  till  a  gla.-s  tumhler  with 
alcohol.  With  a  pipette  drop  a  few  drop-  .  f  olive  oil  into 
tin- ;  notice  that  they  -ink.  (  If  they  do  not.  add  a  -mall  quantity  of 
!  alc»hol.  i  Xo\\-  add  a  certain  amount  of  50' <  alcohol  -^'ithont 
stirriin/.  The  drop-  will  lie  found  not  to  -ink  to  the  bottom.  If 
nece--arv  add  more  50' ,  alcohol  :  or,  if  the  drops  do  not  sink  at  all, 
add  -ome  70'.  alcohol.  When  a  mixture  has  been  obtained  where 
the  drop-  -ink  part  way  but  not  to  the  bottom,  it  is  ready  for  obser- 
vation. What  form  do  the  drops  take?  Add  a  considerable 
quantity  of  olive  oil  with  the  pipette,  until  a  sphere  an  inch  or  more 
in  diameter  i-  produced.  Why  does  it  retain  the  spherical  firm? 
Try  changing  the  -hape  with  a  glass  rod;  doe-  it  return  to  the 
-pherical  form?  Why  doe-  such  a  ma--  not  keep  the  -pherical 
lorm  when  placed  on  a  Hat  -urface?  Place  a  drop  of  olive  oil  on 
water  and  note  it-  -hape. 

I'KINVII'LK  <>l;  LEAST  <  >K   MINIMAL  SURFACES. 

<  >wing  to  the  pulling  .  f  the  surface  film  in  all  direction-  the 
surface  "f  <>  fluid  tends  to  hccome  as  small  as  possible  under  the 
liinitin,/  conditions. 

i-Ji.  I  lie  -pherical  form  of  the  oil  drop-  or  a  -oap  bubble  ex- 
emplifies tin'-.  Why  do  they  take  the  -pherical  form? 

i  J-'.  In  a  di-k  of  paraffin  oil  floating  <.n  70',  alcohol,  increa-e 
the  -urfare  by  pulling  out  |irojections  with  a  gla-s  rod.  \\bat 
happen-  to  the-. 

i  (.v      I- \amine  a   film  of  -oa|>  -olution  on  a  circular  wire   frame. 

Xou-  that    it   i-   Hal.      Why?      Now   bend   the    frame  in   the   form  of 

•op   curved   at    right    angle-    to   the    radii   of   the   loop. 

.hat  i-  the  f.  inn  of  the  film  on  -uch  a  loop?     Kxplain  in  term-  of 

the  •    principle. 

• 


D.  INTERNAL  PRESSURE  DUE  TO  CURVED  FILMS. 

144.  Blow  a  soap  bubble  on  a  glass  tube;  why  is  it  spherical? 
note  decrease  in  size  when  left  to  itself,  clue  to  expression  of  air 
(let  it  blow  against  a  flame).     Why  must  a  curved  film  press  in- 
ward?    Does  this  internally  directed  pressure  vary  with  the  degree 
of  curvature?     How?     Determine  this  by  blowing  and  balancing 
two  unequally  sized  bubbles  on  opposite  ends  of  a  Y-tube.     Which 
presses  air  into  the  other?     Explain.     Internally  directed  pressure 
in   inversely   proportional   to   the    radius   of   the   spherical   drop   or 
bubble. 

145.  Try  deforming  with  a  glass  rod  olive  oil  drops  of  different 
sizes  in  the  alcohol.     (Experiment  140.)     Which  show  most  resis- 
tance to  deformation,  the  large  or  the  small  ones?      \\hich  most 
quickly  regain  their  shapes  ?    Explain. 

146.  Is   the    cylinder    a    stable    form    when    the    principle    of 
minimal  surface  is  considered?     The  following  experiment  shows 
how  stability  is  attained  when  the  form  of  a  cylinder  is  imposed 
upon  a  liquid.     Make  a  cylinder  of  70^    alcohol  in  paraffin  oil,  in 
the  following  way :     Have  a  layer  of  alcohol  in  the  bottom  of  the 
tumbler  under  the   oil.      Take   a   glass   tube   about    l/j    to    !<    inch 
in  diameter,  and  holding  the  finger  over  one  end,  put  the  other  end 
down  to  the  bottom  of  the  vessel,  then  remove  the  finger  and  allow 
the  alcohol  to   rise  in  the  tube.     Now  rather  gently  lift  the  tube 
straight  out  of  the  oil,  when  a  cylinder  of  alcohol  will  be  left  in  the 
oil.      Observe   how    it    instantly    breaks    up    into    spheres.      ( Why 
does  fluid  from  a  spout  usually  break  up  into  drops  ? ) 

E.  CHANGES  OF  SURFACE  TENSION  IN  FLUIDS. 

147.  Different  fluids  have  different  surface  tensions.     Try  drop- 
ping   slowly    water    and    toluol    from   a    pipette.      Which    has    the 
greater  surface  tension?    How  indicated  in  this  experiment? 

148.  The    surface    tension   of    a    fluid    may   be    altered    by    the 
presence  of  another  substance.     Float  a  clean  thin  rubber  band  on 
the  surface  of  a  dish  of  clean  water.     Now  touch  the  end  cf  a  glass 
rod  to  the  surface  within  the  band,  the  rod  having  been  previously 
dipped   in   oil.     Result?     Explanation?      Now   touch   similarly   the 
water  outside  the  band.     Explain  the  result.     Test  the  influence  of 
alcohol  and  chloroform  on  the  surface  tension  of  water. 

149.  Why  does  a  drop  of  alcohol  break  a  soap  bubble?     Drop 
a  drop  of  alcohol  on  a  thin  layer  of  water  on  a  clean  glass  plate. 
Result?     Explain. 

37 


i  ;o.  I 'lace  a  small  piece  of  camphor  on  the  surface  of  clean 
water.  Result?  Kxplain  movements.  What  determines  the  direc- 
tion of  the  movements?  Touch  the  surface  with  a  rod  containing 
a  trace  of  «>il.  Kxplain  what  happens. 

151.  ('//(/ m/t\v   of  form    due   to    chemically    induced   chaiii/es    of 

surface  tension.      Place  a  g 1   -i/ed  drop  of  mercury  in  a  watch- 

gla--;    n    te    its    form    and    size;   now   cover   with    2'  <     IIN<>.;    any 
change?     I'lace  near  the  1  Ig  a  crystal  of  K,A'r._.<  >T  ;  result?     De-cribe 
the  phenomena  carefully;  draw  the  drop-  at   intervals  showing  the 
nature  of   the   form   changes.      Xote   the    resemblance    to   amoeboid 
movement,    ingestion    of    bichromate    crystals,    movement    ot     drop 
toward  crystal   (  anal  gy  to  chemotaxis). 

152.  Movements  and  formation   of  projections,  due  to  changes 
in   surface   tension    in    a   drop    of   olire   oil.     a.    <  hi    a   slide    fasten 
some  glass  reds  i  mm.  in  diameter,  a  sufficient  distance  apart  so  that 
they   will   support  a   rectangular  cover  glass  near  its  ends.      Make 
a  mixture  of  two  parts  glycerine  and  one  part  70'  '<   alcohol — place  on 
the  slide,  and  cover  it.     Then  with  a  fine  capillary  pipette  introduce 
:i  -mall  dm])  of  clove  oil  beneath  the  cover.     Observe  that  the  drop 
changes  its  shape  and  moves  about.     \\  by  J.      (  If  the  drop  does  not 
move    or    moves    too   violently,    vary    the    experiment    by    changing 
the  proportion  of  glycerine  and  alcohol   in  the  fluid.) 

b.  Into  Mich  a  drop  of  clove  oil  prepared  a-  above  introduce  with 
a   tine  capillary  a  very  little  70'  <    alcohol.  closC  to  the  edge  of  the 
drop.      Notice  the   formation  of  a  projection  and  movement  toward 
this  edge.      \Vliy? 

c.  In   the  same  preparation,  or  a   similar  one,   touch   the  upper 
-iirface  "f  the  cover  gla>s  near  the  drop  of  clove  oil  with  a  hot  wire. 
What  happens?     \Yhy? 

153.  I'.lectricallv   conditioned  chain/es  of  surface   tension.     The 
phenomena  of  K\p.  151   are  at  bottom  due  to  electrical  change-.     I  he 
influence  of  the  current  may  be  shown  thu-:      Dip  wires   tr  in  two 
OF  three  dry  cell-  on  the  opposite  -ide-  of  a  watch-glas-  containing 
a   drop  of   mercury   in   dilute  acid.      Result  ?      Note   the   direction   .>t 
movement   of  the  drop  relatively   to  the  poles,  and  also  ot    particles 
on  the  surface  of  the  mercury.     Make  a  diagram  -bowing  this. 

F.  SURFACE  TENSION   I'.KTWKKN   SEVERAL  SUB- 
STANCES. 

When  a   fluid  i-   in   contact   with   another   -ub-iance    i -olid,   liquid 
or  ga-  )  the  degree-  "l"  the  -urface  ten-inn  depend-  on  both  -ub-tances. 

38 


(Perhaps  it  may  be  said  that  it  depends  on  the  dearer  nf  attrac- 
tion  or  repulsion  between  the  particles  of  the  two  substances. ) 

154.  In  the  case  of  a  solid,  if  there  is  a  strong  attraction  between 
the  fluid  and  the  solid,  the  fluid  wets  the  solid.      Will   water  wet 
clean    glass?      Will    it    wet    paraffin?     Will    mercury    wet    glass? 
Try  this  by  dipping  a  piece  of  the  solid  into  the  fluid. 

155.  \Vhere  there  is  an  attraction  the  fluid  may  be  lifted  against 
gravity.     Try  this  by  dipping  a  plate  of  glass  into  the  water.     Does 
the  water  rise  at  the  sides  of  the  plate?     Try  with  a  glass  tube. 
Does  the  water  rise  in  the  tube?     What  is  the  form  of  the  water 
surface  in  the  tube?     Try  water  in  a  paraffined  glass  tube. 

156.  This  attraction  may  likewise  pull  the  solid  into  the  fluid. 
Place  a  drop  of  water  on,  the  edge  of  a  glass  plate.     Then  take  a 
very  small  splinter  of  wood,  and  with  forceps  bring  one  end  of  it 
into  the  drop.     Notice  how  it  is  pulled  in.     Observe  that  the  water 
rises  along  the  splinter  so  that  the  spherical  surface  of  the  drop  is 
altered.      What    pulls    the    splinter    into    the    drop?      Show    by    a 
diagram. 

157.  Place  a  drop  of  chloroform  on  the  bottom  of  a  watch-glass 
full   of   water.      Bring  against   it   a   piece  of   hard   shellac.     What 
happens  ?    \Vhy ? 

158.  "Choice"  in  a  drop  of  fluid  owing  to  varying  surface  tension 
in  contact  with  different  substances.     With  drops  of  chloroform  as 
in  experiment  157,  try  bringing  other  substances  into  contact  with 
it.     Are  they  accepted  or  rejected?    The  following  should  be  tried: 
shellac,  glass,  paraffin,  gum  arabic,  chlorate  of  potash,  resin,  potas- 
sium iodide.    Is  there  any  relation  to  the  solubility  of  the  substances 
in  chloroform? 

159.  "Artificial  Difflngia  Shells."      Grind  up   some  glass   finely 
with  chloroform   in  a  mortar.     Inject  drops  of   this   with  a   fine- 
pointed  pipette  into  a  watch-glass  of  water.     Notice  how  the  glasj 
grains  come  to  the  surface  and  arrange  themselves  in  a  layer,— as  in 
a  Difiiugia  shell. 

The  same  experiment  may  be  performed  with  linseed  oil  in  place 
of  chloroform  and  jo(/c  alcohol  in  place  of  water. 

G.     FORMATION  OF  FILMS  UNDER  THE  INFLUENCE  (  )F 
SURFACE  TENSION. 

1 60.  Laws   of   Gibbs.      Note    in    a   warmed   glass   of    milk    the 
gradual  formation  of  a  film  at  the  surface.     Prove  that  film  forma- 
tion is  net  due  to  evaporation.     Substances  that  lower  the  solution 

39 


U-n-iou  of  the  Advent  tend  [<  >  accumulate  at  the  surface  in  higher 
concentration  than  in  the  interim'  and  may  there  form  coherent 
membranes  or  films.  The  formation  of  cell  membranes  and  similar 
structures  in  organism.-  has  been  thus  e\])lained. 

II.     Fl  \RCE  (  )!•'  FA  AI'oKATK  )X. 

i  iii.  Fill  a  porous  cup  with  water  and  place  in  a  beaker  <>t 
water  for  in  or  15  minute-.  Then  insert  a  rubber  -topper  through 
which  pa-ses  a  gla-s  tube  into  the  porous  cup,  and  till  the  whole 
apparalu-  with  water.  Then  place  the  free  end  of  the  water-tilled 
tube  under  mercury  in  a  glas-  vc-sel  and  clam])  in  an  upright  position. 
lias  the  mercury  begun  to  rise  in  the  tube  by  the  end  of  the 
laboratory  period?  Why?  Leave  till  next  da}'. 

I.     AM<  >FIU  )ll)   M(  >YK.MKXT. 

\><2.  The  preceding  e.\])eriments  have  brought  out  various  princi- 
ples bearing  on  surface  teiisii  n.  The  student  should  now  ende:i\  or 
to  apply  them  to  the  study  of  movement  in  the  living  organism, 
amoeba,  whose  mode  of  locomotion  is  commonly  attributed  to 
change-  in  it-  surface  tension.  The  experiments  and  observations 
-hould  be  devi.-ed  by  the  student  and  conducted  in  the  spirit  of  re- 
-earch.  in  an  endeavor  to  prove  or  disprove  the  surface-tensi  n 
theory  of  movement.  First  make  sure  of  the  facts  by  a  careful 
study  of  the  Amoeba  from  above.  Determine  which  species  of 
Amoeba  you  are  observing  from  I 'late  I  of  I'onn's  "Protozoa". 

The  following  are  suggested  as  points  worthy  of  notice: 

Can  a  pseudopodium  be  thrust  out  freely  into  the  water,  or  mu.-i 
it  be  in  contact  with  the  substratum? 

(  'bserve  a-  exactlv  a-  possible  the  current-  ot  protoplasm  in  the 
amoeba  and  the  currents  in  the  water  about  the  amoeba.  The 
latter,  if  any.  may  be  oh-erved  by  india  ink  granule-  in  the  water. 

I  )o  particles  of  soot  or  debri-  clinging  to  the  surface  <  t  amoeba 
move  completely  around  the  animal  as  if  it  were  a  bag  rolling 
ab<  int  <  '11  the  slide  ? 

I  )oes  the  amoeba  move  forward  in   jerks  or  gradually? 

\\hat  i-  the  character  of  the  movement  when  viewed  I  mm  the 
side.'  I  -e  the  -peciallv  prepared  -lide  for  this  purpose.  Study  care- 
lnll\  ;  manv  of  the  phenomena  connected  with  the  movement  become 
clear  when  examined  from  the  -ide.  I  )oe-  the  amoeba  adhere  to  the 
substratum?  *  an  it  move  up  a  vertical  -urface? 

40 


After  a  careful  study  of  amoeba,  compare  with  what  you  find  in 
the  two  kinds  of  movement  described  below. 

163.  The  movements  of  some  species  of  amoeba  may  be  imitated 
by  causing  a  drop  of  water  or  glycerine  to  adhere  to  the  substratum 
more  strongly  on  one  side  than  the  other.     This  can  be  done  a-  fol- 
lows:    Place  a  piece  of  smooth  cardboard  in  the  bottom  of  a  flat 
dish,  and  on  a  certain  spot  on  the  paper  place  a  drop  of  water.    Then 
cover  the  whole  with  oil,  which  soaks  into  the  paper  everywhere 
except  in  the  spot  covered  by  the  drop  of  water.     After  it  is  well 
soaked   remove  the  drop   of   water   and  oil   and   proceed   with   the 
experiments.    Mix  some  soot  with  a  drop  of  water,  or  better,  glycer- 
ine, and  place  this  on  the  surface  of  the  cardboard  near  the  spot 
that  was  protected.    Allow  one  side  of  the  drop  to  come  against  the 
protected  spot.    What  happens?    How  does  the  movement  resemble 
that  of  amoeba  and  how  does  it  differ?     Study  the  movement  of 
particles    on    the    surface    of    such    a    drop.      Lycopodium    powder 
sprinkled  on  the  drop  will  make  clear  the  surface  movements. 

Or  does  amoeba  move  in  the  manner  of  the  drop  indicated  below? 

164.  Make  a  mixture  of  equal  parts  glycerine  and  water,  add 
some  bone-black,  and  place  two  cr  three  drops  on  a  clean  mercury 
surface.    Mercury  is  not  wet  by  the  mixture.    Powder  lightly  the  sur- 
face of  the  drop  with  lycopodium.     Now  place  a  very  small  drop 
of  95  c/'t  alcohol  at  one  side  of  the  glycerine  drop.     \Yhich  way  does 
the  drop  as  a  whole  move?    Make  a  diagram  of  the  currents  within 
(as  shown  by  the  lamp-black)  and  the  currents  on  the  surface  (as 
shown   by   the   lycopodium ).     Do   they  agree   with   those   observed 
in  amoeba  ? 

165.  Perform    exactly    the    same    experiment    but    use    a    clean 
glass  surface  instead  of  mercury.     The  glass  surface  is  wet  by  the 
glycerine  water  mixture.     Do  the  results  obtained  here  agree  with 
those  observed  in  amoeba  ? 

What  are  your  conclusions  in  regard  to  the  cause  of  movement 
in  amoeba  ? 

D.     COLLOIDAL  SOLUTIONS 

In  colloidal  solutions  the  particles  of  solute  are  large  and  consist 
of  many  molecules  (distinction  from  solutions  of  crystalloids,  e.g., 
sugar).  Hence  these  solutions  resemble  suspensions  in  many  of  their 
properties.  Usually  the  colloidal  particles  are  electrically  charged. 
The  charge  keeps  the  particles  permanently  suspended  by  preventing 
their  union  to  form  larger  particles ;  it  also  determines  many  of 

41 


the  propertie-  of  tlu-  -olution.  In  -onie  o>ll.  idal  solutions  the 
particle-  an.-  positively.  in  other-  nei/ativcly  charged.  The  chemical 
behavior  <>f  the  colloid  is  largely  determined  by  the  sign  of  the  charge 

•  'ii  ihe  particle-. 

A.     SUSPENSK  >X  C(  >LL<  >II>S  (SUSPENSE  >IDS). 

MX..  The  colloid-  to  be  -tudied  are  ferric  hydroxide  (positive) 
and  arsenious  sulphide  i  negative).  I  'roceed  as  to]lo\vs: 

a.  Xote  the  phvsical  properties  (optical  properties,  vi-cosity,  difiu- 
-ion  in  ]»ure  solvent,  filterability)  ;  diffusion  through  collodion  mem- 
liraiu  s. 

I',.  Action  of  non-electrolytes.  To  3  cc.  of  each  solution  add  its 
volume  of  m-sugar  -olution.  l\e-ult? 

C.  Action  of  electrolytes.  Ion  action.  l/sing  3  cc.  of  ferric 
hyilroxidc  hydmsol  in  each  experiment,  add  six  drops  of  each 
of  ihe  following  -olution-.  Shake  gently  and  note  the  result  care- 
fully in  each  case. 

a.  n    jo    I  If!,    n    20    !!><>,.    n   2O    H3PO4    or    n   JO    H8C6H5O7 
(  citric  acid  ). 

b.  n,  20  XI  l,i)  1  1.  ,,  20  KOH  or  NaOH,  n  20  Ba(OH)2. 

c.  m   _'o  Xa('l.  in   jo  Xa._.S<  >,,  m  Jo  Xa  ..  citrate. 

d.  m   jo  Xai'l.ni  2O  CaQ2,  rn   20  A1C1  , 

M>-.  Repeat  this  series  with  arsenious  sulphide  hydrosol.  \\iiat 
(litTerence^  do  you  (hid  in  the  action  of  the  acids,  ba-cs  and  -alts  upon 
the  tw  hydrosols?  What  relation-  do  yon  find  between  ^rcci^itnt- 
Inij  /i(Ti'(T  and  :•<//('//(•(•  of  the  ion-  of  the  electrolyte?  Which  ion- 
precipitate  the  positive  and  which  the  iu-</dti:'c  colloid?  Explain. 

||<S.  Mix  e<|iial  \-olume-  of  the  two  hydro-ol-.  Any  result? 
Explain. 


' 


EMULSION    COLLOIDS    UK    HYDROPHILOUS    COL- 
l.(  )II)S   i  EMULSl  )IDSi. 

In  tin-  cla-s  of  colloidal  -oluti  ns  the  union  with  the  -oKrnt 
(water)  i-  more  intimate.  The-e  colloid-  incorporate  or  combine. 
water  spontaneously  ;  the  colloidal  particle-  are  thus  probably  water- 
-\\olK-n  or  liydrati'd.  and  hence  not  SO  -har|>ly  separated  I  nun  the 
medium  a-  in  the  -u-pen-oid  hy<lro-ol-.  Their  -ohition-  are  more 
like  cry-talloid  solution-,  and  the  condition-  of  precipitation  from 
-ohition  are  different  from  tho-r  -lio\\n  1>\  -u-pen-  'ids. 


-J-' 


C.  SWMLL1XG  PROCESSES 

169.  Arrange  a  series  of  test-tubes  of  uniform  diameter  and 
place  in  each  tube  i  gram  of  granulated  gelatin.  Determine  the  rela- 
tive degrees  of  swelling  undergone  by  the  gelatine  in  the  following 
solutions.  The  gelatine  is  mixed  with  10  cc.  of  the  solution  and 
allowed  to  stand  until  the  height  of  the  swollen  gelatine  in  the  tube 
is  constant. 

a.  distilled  water. 

b.  n/5,  n/io,  11/20,  11/40  HC1. 

c.  11/5,  n/io,  11/20,  11/40  NaOH. 

d.  n/5  NaCl,  11/5  Na2SO4. 

e.  n/20  HC1  +  n/io  NaCl;  11/20  NaOH  -f  n/io  NaCl. 

Note  the  influence  (a)  acid,  salt,  and  alkali  acting  alone;  (b)  opti- 
mum concentration  for  swelling;  (c)  action  of  acid  and  alkali  in 
presence  of  neutral  salt.  Antagonisms  of  this  latter  kind  are  of  great 
physiological  importance. 

D.  OSMOTIC  PRESSURE  OF  COLLOIDS. 

Direct  determinations  of  osmotic  pressure  are  difficult  to  make  with 
crystalloid  substances  because  of  the  difficulty  of  preparing  satis- 
factory semi-permeable  membranes.  On  the  other  hand,  semi- 
permeable  membranes  for  colloids  are  easily  prepared,  so  that, 
although  the  osmotic  pressure  is  low,  there  is  no  difficulty  in  measur- 
ing it  directly.  The  osmotic  pressure  of  colloids  varies  with  their 
"state  of  aggregation",  and  this  varies  with  the  concentration  of  the 
electrolytes  present  in  solution  along  with  the  colloid,  and  also  with 
several  other  conditions:  as  (i)  rate  of  admixture  of  electrolyte; 
(2)  degree  of  mechanical  agitation  to  which  the  solution  has  pre- 
viously been  exposed;  (3)  the  temperature;  and  (4)  in  general,  the 
lapse  of  time  and  the  nature  of  the  previous  history  of  the  colloid 
(method  of  preparation,  etc. ). 

i/o.  A  simple  and  efficient  osmometer  is  made  as  follows:  make 
a  collodion  membrane  of  the  shape  and  capacity  of  a  50  cc.  round- 
bottomed  flask:  this  is  done  as  follows:  Pour  a  moderate  quantity 
of  the  10%  collodion  solution  (in  equal  parts  alcohol  and  ether)  into 
a  50  cc.  flask  ;  invert  the  flask  and  turn  till  an  even  layer  of  solution 
is  formed  on  its  wails;  pour  back  the  surplus  solution  into  the  bottle; 
blow  a  current  of  air  into  the  flask  through  a  glass  tube;  then  add 
some  warm  water  and  change  this  two  or  three  times.  The  mem- 
brane is  then  ready  to  remove  from  the  flask  ;  removal  is  facilitated 

4.3 


bv  first  running  a  Mream  of  water  between  the  membrane  and  the 
glas-  wall.     Prepare  three  membranes. 
Prepare  the  following  solution-: 

a.  50  cc.   2' ,    egg  albnniin   ]>lns    10  cc.   distilled   water. 

b.  50  cc.  2' ,    egg  albumin  ])lus   10  cc.  m   S  XaL'l. 

c.  50  cc.   _'' ,    egg  albumin   ])lus    10  cc.   m  S   XaL'l.,. 
In  the  outer  vessel  of  each  osinometer  add  respectively: 

a.  distilled  water. 

b.  m   4*  XaL'l. 

c.  in  4S  L'aL'L. 

t'se  the  same  volume  of  outer  fluid,  e.g.,  420  cc.  in  each  osniometer. 
I- ill  each  membrane  with  its  corresponding  solution  :  insert  the  rubber 
cork  and  manometer  tube  into  the  neck  of  the  membrane  (excluding 
air-bubbles)  and  bind  in  position  \\-ith  a  rubber  band.  Then  place 
the  membrane  in  position  in  its  corresponding  outer  fluid  and  clamp 
the  manometer  tube  in  a  vertical  position. 

Note  the  rise  ot  the  fluid  in  the  manometer  tubes  and  the  different 
rates  of  rise.  What  is  the  maximum  pressure  in  each  solution: 
\\hat  do  you  conclude  as  to  the  influence  of  salts  on  the  osmotic 
pressure  of  colloids  ? 

171.  The  prc-cnce  of  acid  and  alkali  Increases  the  osmotic  pres- 
-ure  of  certain  proteins,  e.  g..  gelatine.  This  action  is  prevented  by 
the  presence  of  neutral  salts  in  appropriate  concentrations.  Deter- 
mine the  osmotic  pressure  of  the  following  solutions: 

a.  I' '  f  gelatine. 

b.  \'e    gelatine  containing   IU'1  to  n  300  concentration. 

c.  I',    gelatine  containing   Xa<>ll   or   K<>ll   [>  11/300  concentra- 
tion. 

d.  and   e.      Same  as   h  and   c  but   containing  also    XaC'l   to   m   4S 
concentration. 

Keir.unber  that  the  outer  fluid  contains  the  same  electrolvtes 
in  the  some  concent  ration  as  in  the  colloidal  solution. 

<  ompare  the  action  of  electrolytes  on  osmotic  pressure  with  their 
action  on  the  s^'elliiit/  process. 

!•:.     EFFECT     <>K     SALTS     ON     COLLOIDS     IX     LIVING 
TISSUES. 

S;nre  the   solid   |>ortions  (,f  living  tissue-   are  cnlliiids.   it    i-   to   be 
expected    that    electrolytes    \\j]l    have    a    marked    influence   on    vital 
activities.    The     following    experiments     show    the    importance    of 
lvtcs  for  the  aclivitv  of  ciliated  cells. 


-14 


1/2.  Separate  carefully  with  a  pair  of  needles  a  number  of 
filaments  from  the  gills  of  an  oyster  or  clam.  The  filaments  will 
remain  living  in  sea-water.  Prepare  the  following  solutions.  In 
each  experiment  transfer  several  of  these  filaments  with  forceps 
to  a  clean  dry  watch-glass;  then  add  several  cc.  of  the  solution 
whose  action  is  to  be  tested.  Examine  the  filaments  in  the  solutions 
at  frequent  intervals  and  determine  as  accurately  as  possible  the 
action  of  each  solution,  as  follows: 

(a)  The  character  and  duration  of  the  ciliary  movement.     If  the 
cilia  are  still  active  at  the  end  of  the  period  cover  the  watch-glass 
and  examine  again  next  day. 

(b)  Are  there  any  visible  structural  changes  as  a  result  of  its 
action   ( swelling  of  cells,  breakdown  of  cilia,  etc. )  ? 

a.  Pure  isotonic  solution  of   the  chief   chlorides  of   sea-\vater : 
m/2  NaCl,  m/2  KC1,  m/2  MgCL,  m/2  CaCL. 

b.  Combinations  of  two  chlorides    (to  show  antitoxic  action  of 
salts). 

(a)   25  vols,  m/2  NaCl  -(-  i  vol.  m/2  KC1. 
(b>  25  vols.  m/2  NaCl  -|-  I  vol.  m/2  CaCl,. 

( c )  25  vols.  m/2  NaCl  -f-  i  vol.  m/2  MgCL. 

c.  Combinations  of  three  or  four  chlorides. 

(a)  25  vols.  m/2  NaCl  -+-  i  vol.  m/2  KC1  -f  T  v°l-  m/2  CaCl,. 

(b)  25  vols.  m/2  NaCl  -(-  i  vol.  m/2  KC1  -f  i  vol.  m/2  MgCL. 

( c )  25  vols.  m/2  NaCl  -+-  i  vol.  m/2  CaCL  +  i  vol.  m/2  MgCL,. 

(d)  25  vols.  m/2  NaCl  +  T  v°l-  m/2  CaCl,  -+-  i  vol.  m/2  KC1 
-+-  i  vol.  m/2  MgCL. 

Note  especially  the  difference  between  the  pure  solution  of  NaCl 
and  the  mi.rtnrcs.  \Yhich  solutions  are  the  most  favorable?  Note 
the  differences  betwen  KC1  and  CaCl,  or  MgCL  as  antitoxic  salt 
(with  NaCl  as  the  toxic  salt).  The  valence  of  the  cation  is  impor- 
tant in  antitoxic  action. 


PART   111 

I  'll  YHoUiCY   OK    .MoYK. \1K.\T 

/.     MUSCLE  rHYSIOLOGY 

In  the  following  experiments  the  catalo-ue  of  the  Iltirvard 
Apparatus  Company  is  to  he  used  as  an  apparatus  reference  hook. 
.Moi\-  detailed  explanations  of  the  apparatus  than  can  he  given  in 
the  laboratory  direction  sheets,  will  he  found  there.  From  now  on 
dissecting  instruments  will  always  be  needed. 

STRIATED  MUSCLE 
\.     MKTIK  )1)S  (  n-'  STIMULATK  >X. 

The  apparatus  used  in  electrical  stimulation  .should  he  carefuly 
studicd  in  all  details  before  the  preparation  of  a  frog's  muscle  for 
experiment. 

173.  Batteries.      Observe   the   dry   cell.      Carbon    (-f-)    and    zinc 
(--)   plates  are  immersed  in  a  ma--  of  porous  clay  permeated  with 
-trong  XI I  ,C1  solution.     Attach  wires  to  the  binding  po-t-  of  the  bat- 
tery and  apply  the  ends  of  the  wires  to  neutral  litmus  paper  moistened 
with     XaCl     solution.       (  L'se     insulated     platinum-tipped     wire-,  i 
Xote    (a)    reaction   at    -f~   ail(l       '  poles ;    (b)    rapidity   with    which 
color  change   a]t])ears   with   poles    i  i  )    close   together   and    (2)    one 
cm.  apart.  (  3  )  several  cm.  apart.     Kxplain  this.     What  is  (  )hm's  law? 

174.  Ap|)ly  the  ends  of  the  wires  as  in  the   former  experiment  to 
filter  paper  moistened  with  starch  solution  containing  KI.      Result? 
At   which  pole  doe-  a   reaction  appear?      Kxplain        Repeat,  varying 
the    di-tance    between    the    two    electrodes     |  anode     (positive)     and 
cat  hi  >dc    I  negative  I  | . 

175.  I  )ip  the  end-  of  the  platinum-tipped  wires  into  weak  acid  so- 
lution.    What  gase-  are  evolved  and  at  which  poles.'     Try  Xal  1  -olu- 
li    M.  distilled  \\ater  and  -ugar  s,  .hitii  HIS.      Results?     1;n>m  the  above 
e.\]>eriments   formulate  a  rule   for  distinguishing  anode  and  cathode 
in  an  unknown  circuit. 

17''.  Connect  the  cell  in  circuit  with  a  simple  key.  Xow  without 
closing  the  circuit  place  the  platinum  electrodes  on  the  tip  of  the 

t" 


tongue  about  i  cm.  apart.  Then  close  the  circuit.  Xote  the  effect. 
Is  there  any  perceptible  difference  between  anode  and  cathode?  Ex- 
plain. 

1/7.  The  rheocord.  A  device  for  introducing  resistance  into  a 
circuit  or  for  obtaining  fractions  of  the  electromotive  force  of  a 
cell.  Lead  wires  from  the  dry  cell  through  a  key  to  posts  o  and  I 
pnd  then  from  post  o  and  the  slider  to  platinum  electrodes.  Place 
the  electrodes  in  salt  or  acid  solution  and  determine  the  relative 
amount  of  electrolysis  when  the  slider  is  moved  toward  the  o  or  i 
post.  Draw  a  diagram  showing  the  course  of  the  current. 

178.  Induction  coil.  Induced  currents  are  usually  employed  for 
stimulation.  These  are  momentary  currents  which  appear  in  any 
circuit  when  a  current  in  an  adjoining  circuit  is  made  or  broken, 
or  its  intensity  altered.  In  the  instrument  the  wires  of  the  two 
circuits  are  arranged  in  two  parallel  coils — primary  circuit  ( induc- 
ing circuit)  and  secondary  (induced)  circuit — to  intensify  the 
effects.  An  automatic  interrupter  is  inserted  in  the  primary  circuit. 
Study  the  instrument  and  make  a  diagram  showing  its  essential  con- 
struction. Place  the  primary  coil  in  circuit  with  a  simple  key 
and  dry  cell. 

a.  Direction  of  induced  currents.    Attach  wires  to  the  secondary 
coil  and  apply   the  ends   to  starch   iodide  paper.     Attach   primary 
circuit  wires  for  single  shocks.     Close  and  open  the  key  in  primary 
circuit  a  number  of  times.     Effect?     Note  the  cross  circuiting  key 
at  the  poles  of  the  secondary  coil.     Its  purpose?     Apply  the  elec- 
trodes to  test  paper  as  before  and  close  the  key  in  the  primary  circuit 
several  times  in  succession  but  cross  circuiting  the  secondary  coil 
each  time  before  opening.     Result?     Repeat,  cross  circuiting  before 
making  so  as  to  allow  only  the  break  induced  current  to  pass  through 
the  electrodes.     Result?     \Yhat  conclusions  do  you  draw  regarding 
the   direction   of   the  induced   current  on  making  and   on   breaking 
respectively  ? 

b.  Separate  the  primary  and  secondary  coils  to  some  distance. 
Close  the  primary  circuit  and   then  place  the  secondary  electrodes 
(platinum-tipped  wires  attached  to  secondary  coil)  on  the  tip  of  the 
tongue.    Any  result?    Now  make  and  break  several  times.     Result? 
Slide  secondary  nearer  primary,  testing  as  before.     Note  the  effect 
on  the  intensity  of  the  shock.     \Yhich  is  stronger,  make  or  break 
shock?    Explain  the  difference  in  intensity  of  shock.     Place  the  coils 
at  right  angles  to  each  other.    Are  shocks  perceptible?    Change  the 
angle  between  the  coils,  and  test  strength  of  shocks.    Give  a  generali- 


-.7 


nation  as  to  the  relation  between  the  angle  of  crossing  of  the  coil- 
a\e-  and  the  strength  of  the  shocks. 

\~n.  '1  he  re:-ersiiii/  (or  roekiny)  key  or  pule  eliaiit/cr.  Xote  the 
inechani-m  of  the  reversing  key  and  draw  diagrams  showing  the 
connections  to  he  made  in  order  to  n-e  it  (l)  in  reversing  the  direc- 
tion of  the  current  ;  (2)  as  a  double  key  without  changing  the  wire- ; 
I  3  )  a-  a  single  key. 

i  Si.  Kxamine  the  non-polarizable  electrodes.  They  are  -oaked  in 
phy-ioln^ieal  salt  solution,  then  tilled  with  ZnS<  )4  solution  in 
which  is  immersed  a  Zn  rod.  Take  great  care  not  to  spill  ZnS<  ), 
on  the  outside  of  the  boots.  Zinc  is  dissolved  at  the  anode  (  -(-  pole  ). 
and  i-  deposited  on  the  7n  rod  at  the  cathode  (- -  pole).  The  Xa 
and  (.'1  ions  carry  the  current  through  the  tissue. 

Set  up  the  non-polarizable  electrodes,  place  on  litmus  paper  mois- 
tened with  physiological  -alt  >olmion,  and  determine  the  effect  of 
pas.-ing  a  current  through  them.  Does  electrolysis  take  place?  Do 
you  see  why  they  are  used  in  phy-iol  gy  ? 

Immediately  after  using,  wash  out  the  ZnS(  ),  very  carefully  and 
place  the  electrodes  in  physiological  salt  solution  to  soak.  Wipe  off 
the  Zn  rods  so  that  they  will  be  ready  for  another  experiment. 

I!.     PHENOMENA    OF    CONTRACTILITY    AXD    IRRITA- 
BILITY. 

Muscular  Contractility.     Muscle  cells  are  typically  stimulated  to 
contraction    by    impulses    conveyed    through    tracts    of    conducting 
ti--ue  called  nerves.      A  muscle  with   its  attached   nerve   represent 
the  chief  motor  organ  of  higher  animals. 

i  .Si.  \crrc-mnsclc  preparation.  A  muscle  with  nerve  attached 
(gastrocnemius-sciatic)  can  be  isolated  a-  foil  ws :  Destroy  the 
brain  and  -pinal  cord  of  a  frog  bv  pithing,  as  demonstrated.  All 
-pontaueons  movement  -honld  cease.  Do  you  know  \\liv:  l\e- 
move  the  --kin  from  the  whole  body  of  the  frog  except  the  head,  as 
secretions  of  the  -kin  injure  the  muscle.  The  object  now  IS  to 
remove  tin-  gastrocnemius  muscle  (.-till  attached  to  the  femur),  and 
the  whole  -ciatic  nerve  (-till  attached  to  the  gastrocnemius  muscle) 
from  it-  origin  in  the  -pinal  cord.  Xote  on  the  dorsal  side  of  the 
thijji  a  longitudinal  depression  between  the  va-tu-  externus  and 
?emimembranosns  mu-cle-  ( -ee  l-.cker'-  l'r«g.  \^.  <)$).  The  -ciatic 

ner\c  lie'-  in  thi-  groo\-».'  along  with  the  hi 1   \e--els.      Lift   up  the 

m-rve    vi-r\-    genth     with    a    gla-s    -eeker   and    caretullv    i-olate    it    as 
far  a-  the  knee  in   a  downward   direction.      'I  hen   -eparate   well    the 

48 


thigh  muscles  with  forceps  and  isolate  the  nerve  upward,  taking 
care  not  to  injure  it  where  it  passes  over  the  dorsal  side  of  the 
pelvic  bones,  and  thence  forward  ventrally  to  arise  from  the  cord 
by  several  roots,  clearly  visible  when  the  intestine  and  kidneys  have 
been  removed. 

Cut  the  roots  as  near  the  spinal  cord  as  possible.  What  happens? 
Cut  through  the  femur  in  the  middle  and  remove  the  thigh  muscles 
without  injuring  the  nerve,  cut  the  tendon  of  Achilles  below  the 
ankle,  separate  the  gastrocnemius  muscle  from  the  other  muscles 
of  the  calf  and  cut  the  calf  just  below  the  knee.  You  now  have 
a  gastrcenemius-sciatic  preparation.  Keep  moist  with  physiological 
salt  solution  ( why  ? )  and  avoid  touching  muscle  or  nerve  tissue  with 
forceps 

Fix  the  cut  end  of  the  femur  in  a  femur  clamp  and  lay  the  nerve 
on  a  glass  slide  supported  by  another  clamp.  Attach  with  thread 
a  lo-gram  lead  weight  to  the  tendon  of  Achilles. 

182.  Mechanical  stimuli.      Pinch   the   end  of  the  nerve,  or  tap 
with  a  glass  red.     Result  ?     Try  tapping  a  muscle  directly,  using 
some  other  muscle  of  thigh.     Result? 

183.  Thermal  stimuli.     Touch  the  nerve  with  a  warm  glass  rod. 
Also  a  muscle  as  in  the  preceding  experiment. 

184.  Chemical  stimuli.      Place   a   few   drops  n/io   HC1  on   the 
extremity  of  the  nerve.     Result? 

185.  Electrical  stimuli.     Galvani's  experiment.     Prepare  a  nerve- 
muscle  preparation  and  lay  the  nerve  across  a  pi>ece  of  filter  paper, 
soaked  in  physiological  salt  solution,  on  the  china  plate. 

a.  Gently  touch  the  nerve  with  a  copper  wire  and  the  muscle 
with  an  iron  wire.    Any  result?    Now  touch  the  ends  of  the  Cu  and 
Fe  wires  together.     What  happens?    Can  you  explain  this?     If  the 
\\ires  are  corroded,  file  a  clean   surface  at  the  points  of  contact. 
Try  the  same  'experiment  with  two  wires  of  of  the  same  metal. 

b.  Touch   the   muscle   alone   and  the   nerve   alone   with   the   Cu 
and  Fe  wires  in  contact  at  the  opposite  ends.     Result? 

c.  Touch   the   filter   paper   near   the   nerve    (but   do   not   touch 
the  nerve  itself )  with  Cu  and  Fe  wires  about  i  cm.  apart  with  their 
line  of  junction  parallel  with  the  nerve.     Xow  bring  into  contact 
the  opposite  ends  of  the  wires. 

d.  Try  the  same  experiment,  but  place  the  wires  on  each  side  of 
the  nerve,  but  not  touching  it. 

>e.  Try  the  same  experiment  with  the  muscle.  Do  you  obtain 
the  same  result?  Explain.  If  the  gastrocnemius  muscle  does  not 


49 


respond,  try  tin.-  sartorius  or  some  other  muscle  of  th«i  leg.     (  ICcker, 
The   Frog,  p.  <>S.  i 

1.  Osmotic  stimuli,  (a)  I 'lace  a  few  -alt  crystals  on  the  nerve 
or  dip  in  J1  ^  m.  Xal'l.  Result  ?  Wash  off  the  salt  with  ph\  -i<  (logical 
salt  solution.  Result? 

(hi  Allow  the  nerve  t«  dry.  What  is  the  effect  i  n  the  musele? 
I  >oes  the  nerve  lose  its  irritability?  \\  a>h  with  salt  solution  to 
see  if  the  power  of  functioning'  returns? 

(ci  Kern  ve  a  sartorius  muscle  and  suspend  it  half  immersed  in 
distilled  water.  Note  carefully  any  movements  or  changes  in  length 
« >r  color? 

i  Si  i.  Independent  irritability  of  muscle.  A  muscle  is  stimulated 
by  the  electric  current,  but  we  cannot  be  certain  that  nerves  in  the 
muscle  are  not  also  stimulated.  These  nerve  endings  can  be  para- 
lyzed by  curare.  Proceed  as  follows:  Ktherize  a  fr  g  lit/htly  with 
ether  soaked  in  cotton  under  a  glass  jar.  Kxpose  the  sciatic  nerve 
in  the  thigh  by  a  small  slit  in  the  skin  over  the  course  of  the  nerve; 
be-  especially  careful  not  to  injure  the  femoral  artery  which  runs 
close  to  the  nerve.  Carefully  separate  the  nerve  for  a  length  of  half 
an  inch;  pass  a  strong  thread  under  the  nerve,  and  tightly  ligature 
the  \vhcle  leg  except  the  nerve.  The  circulation  is  thus  interrupted 
below  the  ligature  without  injury  to  the  nerve.  Xow  inject  into  the 
dorsal  lymph  sac  a  few  drops  of  a  \' ',  solution  of  curare.  When 
paralysis  is  complete  (15-30  min.i,  expose  both  sciatic  nerves  and 
stimulate  with  tetanizing  currents.  Xote  the  difference  between  the 
two  legs,  and  explain.  Is  the  nerve  trunk  affected  by  the  curare? 
Where  i-  the  point  of  actim  ?  Is  the  muscle  itself  affected?  Stimu- 
late the  curarized  muscles  directly.  Do  they  contract?  What  do 
you  conclude  a-  to  the  independent  irritability  of  muscle?  Place  the 
non-poisoned  muscle  with  its  attached  nerve,  in  a  watch-glass  with 
curare  solution.  At  intervals  test  its  irritability  through  the  nerve". 
U  it-  direct  irritability  affected?  Test  the  direct  irritability  of  a 
curarized  muscle  to  the  make  and  break  of  the  constant  (or  gal- 
vanic) current.  Using  non-polarizable  electrodes  applied  at  opposite 
end-  of  the  muscle.  Trv  similarlv  make  and  break  singly  induction 
(or  faradic)  shocks,  and  tetanizing  shocks  (with  interrupter).  Is 
there  anv  difference  from  indirect  stimulation  with  regard  to  the  rela- 
tive readiness  or  response  to  the  different  tornis  o)  electrical 
stimulation  ? 

|S~.  I'olar  stimulation  of  muscle.  a.  Stimulation  at  cathode 
and  anode.  Slit  a  curari/ed  sartorius  from  its  lower  end  about 


5" 


two  thirds  of  its  length.  Then  apply  to  each  of  the  two  halves  t!m> 
separated  a  non-polarizable  electrode.  Use  a  galvanic  current. 
Make,  and  note  which  limb  contracts.  After  an  interval  break. 
\Yhere  does  contraction  start  at  make  and  break,  respectively? 

b.  Cool  a  curarized  muscle  by  placing  on  ice  covered  with  paraffin 
paper   (to  protect  the  muscle).     \Yhen  thoroughly  cool  place  in   a 
Gaskell  clamp  and  bathe  with   ice  cold  salt  solution.     Bring  non- 
polarizable  electrodes  against  the  opposite  ends  of  the  clamped  mu>- 
cle  and  stimulate  as  before.    Results  ? 

c.  Remove  the  rectus  abdominis  muscle  from  a  frog,  lay  on  a 
dry  glass  or  porcelain  plate  and  apply  non-polarizable   electrodes 
to  either  end.     Stimulate  with  the  galvanic  current  and  note  what 
occurs    (close  observation   is   required  here)    in   the   region   of   the 
tendinous  bands  which  divide  the  muscle  into  segments?     Is  polar 
stimulation  indicated?    The  effect  is  most  distinct  with  a  cold  muscle. 

188.  Does  muscle  change  volume  in  contraction/     Remove  the 
skin  from  the  hind  limb  of  a  frog  and  place  the  limb  in  the  volume 
tube.     Hook  electrodes  into  the  muscle  at  opposite  ends  of  the  limb. 
Fill  the  tube  quite  full  of  isotonic  NaCl  solution,  and  replace  stopper 
in  such  a  way  that  air  is  absolutely  excluded  and  fluid  is   forced 
part  way  up  the  capillary  tube.     Ajust  the  position  of  the  meniscus 
by  the  glass  rod.     Stimulate  the  muscle  by  an  interrupted  induction 
current.      Note    movements,    if    any,    of    the    meniscus    and    draw 
conclusions  as  to  the  nature  and  extent  of  the  change  of  volume 
during  contraction. 

C.  GRAPHIC  RECORD  OF  CONTRACTIONS. 

189.  The  graphic  method  of  recording  muscular  contractions  and 
other  physiological  processes.    The  muscle  is  so  arranged  that  its  own 
contraction  describes  en  a  uniformly  moving  surface  a  curve  from 
which    the   extent,   character   and   time-relations   of    the   movement 
can  be  seen.    Usually  smoked  paper  is  used  wrapped  around  a  drum 
on  a  vertical  axis  moved  by  clock  work.      Such   an   instrument  is 
a  kymograph.     Examine  thoroughly.     Learn  how  to  wind  it,  regu- 
late  speed,   etc.,    from   the   description   in   the   Harvard   Apparatus 
Company  catalogue.     Learn  how  to  cover  the  drum  with  paper  and 
smoke  it. 

Examine  also  the  following  pieces  of  apparatus  and  learn  their 

use :    Light  muscle  lever,  writing  lever,  scale  pan  and  signal  magnet. 

To  prevent  drying  during  experimentation,  the  muscle  is  often  kept 


ill  a  moist  cliamhcr.  Kxamine  the  moi-t  chamber;  note  the  muscle 
rlanip  in  which  the  I'ennir  may  In.-  placed,  ami  the  binding  posts. 

Make  diagram-  of  all  the  pieces  <>f  apparatus 

Adjust  the  muscle  lever  and  moist  chamher  on  the  support  and 
arrange  \oiir  wh»le  a]t])aratus  in  a  convenient  po-Hion  for  taking  a 
record  of  contraction.  (See  demonstration.)  Diagram. 

MM.  ('iirrcs  «f  siin/le  contraction,  summation  of  twitches,  and 
t claims.  Kach  student  is  t  preserve  one  set  of  record-,  so  two 
rec»rd-  should  he  made  by  a  pair  of  students  working  t  gether. 
When  the  paper  mi  the  drum  is  covered  it  should  lie  removed, 
clearly  labelled,  shellacked,  and  hung  up  to  dry;  then  the  records 
-hould  be  cut  out  and  pasted  in  the  laboratory  book,  with  the  descrip- 
tion of  the  experiment.  Always  draw  a  base  line  under  the  muscle 
curve  before  shellacking.  The  student's  name  and  data  of  the  experi- 
ment -hould  also  be  written  on  the  record. 

a.  Simjlc  contraction.  Connect  the  inductorium  with  two  cells  only 
and  push  the  coils  near  enough  together  to  give  a  good  single  shock. 
Set  the  drum  on  high   speed  and  allow  it  to  revolve  after  pre-sing 
the  writing  lever  and  signal  magnet  lever  lightly  against  the  drum. 
Take  a   record  of  a  single  twitch  on  the  make  of  the  current,  and 
another  :  n  the  break.     Which  is  greater?     Why? 

b.  Summation    of  stimuli.      Send    in    two    shocks    in    rapid    -uc- 
ce--i<>n   by   making  and   quickly   breaking,   so   as   to  obtain    a   curve 
Allowing  what  happens  when  a  muscle  is  stimulated  at  the  height  of 
contraction.      If  you   fail  in  obtaining  the  correct   time   interval   be- 
tween shock-  the  first  time,  try  again. 

c.  Incomplete  tetanus.     Repeat,  making  and  breaking  by  hand  in 
rapid  -ucccssion.     Summation  of  -everal  -hock-  -hould  be  obtained, 
giving  an  incomplete  tetanus. 

I  letter  record-  of  incomplete  tetanus  can  be  obtained  by  mean-  of 
the  spring  interrupter.  Study  the  instrument  and  make  a  diagram 
"f  it.  Connect  with  an  inductorium  whose  coils  are  so  far  separated 
that  only  break  shocks  stimulate,  and  obtain  record-  -bowing  dif- 
ferent degree-  of  incomplete  tetanus. 

d.  ('omplete    tetanus.       I  )e-cribe    a    curve    of    contraction    with 
letani/ing  current,  i.e.,  with  interrupted   laradic  or  induced  current. 
!><>  not   -timulate  f   r  longer  than  ,}  -econds. 

e.  l-'atii/ne  cnn-e.      With   very   slow   -peed   of   drum,    fatigue  the 
inu-cle  by  prolonged  tetanu-.      Note  the  gradual   relaxation   in   -pile 
of  0'iitimied  -limulation.     When  completely   fatigued,  allow  to  rest. 
Wa-h  with  -alt   -olntion.     Then  take  curve-  of   single  twitehe-  with 


maximal  break  shocks.     Compare  with   the  curves   from  the   fre^h 
muscle. 

191.  The  preceding  experiments  will  give  practice  in  handling  the 
apparatus.     The  student  should  now  make  a  neat  record  of  a  single 
muscle  twitch,  as  before,  but  introducing  also  a  tuning  fork  which 
will  make  a  time  curve  on  the  drum  so  that  the  duration  of  the 
phases  of  a  contraction  may  be  recorded.     Turn  the  drum  a  siiu/le 
revolution   fairly  rapidly  by  hand  instead  of  clockwork  and   while 
revolving  stimulate  the  muscle  with  a  single  induced  shock  or  break 
shock.     The  writing  levers  and  vibrating  tuning  fork  with  writing 
point  attached  should  be  pressed  against  the  drum  before  turning. 
The  writing  points  should  all  be  in  a  vertical  line.    After  the  record 
has  been  obtained  place  the  lever  point  of  the  signal  magnet  over 
the  point  of  stimulation  as  indicated  on  the  record  and  with  the  drum 
stationary,   stimulate  the   muscle   to   contract.     This   will  give  the 
exact  latent  period  in  case  the  writing  points  are  not  exactly  over 
each  other.     Practice  may  be  necessary  to  obtain  a  good  record. 

D.     EFFECT  OF  VARIOUS   FACTORS   IX   Ml'SCLE  CON- 
TRACTION. 

192.  Influence  of  repeated  stimuli — Treppe.     Fasten  the  muscle 
in  a  moist  chamber  and  arrange  the  apparatus  for  recording  contrac- 
tions on  a  drum.     Connect  the  muscle  through  a  key  with  the  binding 
posts  on  the  desk.    A  current  will  be  made  and  broken  by  a  revolving 
key  which  automatically  excludes  the  break  shocks  from  the  induc- 
torium.     Thus  the  muscle  will  be  stimulated  at  a  certain   rate  by 
make  induced  shocks  alone.     Record  the  contractions  on  a  slowly 
moving  drum  until  nearly  fatigued.     Allow  to  rest  several  minutes 
and  again  record  the  contractions  till  fatigued.     Mark  the  rate  of 
stimulation  on  the  record.     One  record  for  a  pair  of  students  will 
be  sufficient. 

193.  Repeated  stimulation  at  tlie  moment  of  relaxation.    Records 
can  be  obtained  by  means  of  a  special  muscle  lever.     Consult  the 
instructor  for  directions. 

194.  Influence  of  strength  of  stimulus  on  lieif/lit  of  contraction. 
Arrange  the  muscle  as  in  experiment  190  for  direct  stimulation  with 
single  induction  shocks.     Separate  the  coils  (  connected  with  one  dry 
cell)   to  a  distance  at  which  both  make  and  break  shocks  are  inef- 
fective.   Then  slowly  increase  the  strength  of  stimulus  by  moving  the 
coils  nearer  until  the  break  shock  just  begins  to  be  effective.     Now 


the  height  «>f  make  and  break  contracti>  n  on  a  stationary 
drum  with  gradually  increasing  stimuli — moving  the  coils  <  cm. 
closer  at  each  trial,  until  both  make  and  break  shocks  give  maximal 
-timuli.  Kotate  the  drum  by  hand  about  5  mm.  betuccn  each  con- 
traction record.  Take  also  a  tetanic  contraction.  Write  on  the  drum 
bel'U  each  contraction  line  the  distance  between  the  coils  in  centi- 
meter-. Xote  carefully  any  relation  between  height  of  contraction  and 
intensity  of  stimulus.  What  is  the  general  law  describing  this  rela- 
tion :  Compare  the  height  of  a  single  contraction  with  that  of  a 
tetanus.  l:rom  the  height  of  the  curves  ami  the  relative  lem/tlis  of 
the  two  anus  of  the  lever,  estimate  the  actual  distance  throiii/h  \chich 
the  muscle  contracts,  both  In  siiit/le  twitch  and  tetanus.  II  hat  pro- 
portion of  its  o-^'ii  Icin/th  does  the  muscle  contract  in  both  forms  of 
contraction.' 

i'^.  Influence  of  load  on  licit/Jit  of  contraction.  Same  arrange- 
ment as  in  the  preceding  experiment.  I'sc  a  fresh  unexhausted 
muscle.  L'se  the  same  stimulus  (maximal  break  shock)  through- 
out. Attach  the  large  weight  pan  to  the  lever  and  take  contractions 
with  the  following  loads:  I  i  )  unloaded,  lever  alone;  (  _' I  lever  and 
scale  pan;  13  and  foil,  wing  (  same  +  10,  20,  30,  40,  etc.,  grams 
up  to  100  gms. ;  then  increase  by  _><>  gius.  each  until  the  limit  ot 
contraction  is  reached.  Describe  the  relation  between  load  and  height 
of  contraction.  Kstimate  the  work  performed  by  the  muscle  in  each 
contraction  in  gram-centimeters.  At  which  load  is  the  work  done 
maximal?  What  is  the  absolute  lifting  power  ("absolute  force") 
of  the  muscle  stimulated  by  a  single  twitch?  Is  the  lifting  power 
increased  in  tetanus? 

i</>.  Influence  of  temperature  on  contraction.  The  muscle,  is 
fastened  in  the  "muscle  warmer"  by  binding  the  femur  to  the  metal 
rod  with  tine  wire.  The  tendon  of  Achilles  i<  connected  by  a  bent 
pin  and  tine  wire  with  the  short  arm  of  a  special  light  muscle  lever. 
I 'lace  a  signal  magnet  in  the  primary  circuit,  and  stimulate  with 
-ingle  induced  make'  or  break  shocks.  I'lace  a  thermometer  lon-ely 
in  the  "musi-le  warmer"  so  that  the  fluid  may  be  stirred  occasionally 
and  the  temperature  maintained  even.  Sec1  that  the  muscle  is  irritable 
and  then  lower  the  temperature  to  0—1  C'.  but  do  nut  frC( 
lake  a  record  of  contraction  on  a  rapidlv  moving  drum.  I  lien 
raise  the  temperature  slmvly  and  take  records  at  5  .  l<>  .  15  .  2O°, 
_>5  ,  30  and  35  .  Xote  carefully  the  differences  in  licit/lit,  dnrti'ion 
and  i/eneral  form  of  co'itracti<  n  curve  at  the  different  temperatures. 
\"te  al-o  tin-  variation  in  the  latent  period  with  temperature. 


197-  Heat  ric/or.  Using  the  muscle  of  the  la>t  experiment,  dis- 
connect the  inductcrium  and  attach  to  the  signal  magnet  wires  from 
the  desk  binding  posts  which  will  give  time  intervals  of  15  seconds. 
Revolve  the  drum  very  slowlv  and  at  the  same  time  raise  the 
temperature  of  the  "muscle  warmer"  about  one  degree  in  2  minutes. 
Mark  on  the  rigor  curve  thus  obtained  the  temperatures.  Note 
especially  the  temperature  at  which  marked  shortening  or  heat 
rigor  of  the  muscle  begins. 

198.  Action  of  salt-solutions  on  muscle.  a.  Use  curarized 
muscles.  Remove  small  muscles  ( sartorius,  biceps,  tibialis,  etc.) 
from  the  leg  of  the  frog,  with  as  little  injury  as  possible,  and  place 
in  the  following  solutions,  which  should  be  cJuuujcd  two  or  three 
tunes  to  remove  all  foreign  substances. 

a.  m/ 4  sugar  solution  (non-electrolyte). 

b.  Mixture  of  4  vols.  111/4  sugar  -)-  i  vol.  m/8  NaCl. 

c.  m/8  NaCl  pure. 

d.  m/8  NaBr  pure. 

e.  m/8  NaXO4    (calcium  precipitant). 

f.  .Mixture  of  24  vols.  m/8  NaCl  +  I  vol.  m/8  CaCl,. 

g.  m/8  KC1  pure, 
h.     m/8  CaCl.,  pure. 

Note  the  following  points  :  ( i  )  Any  immediate  change  on  placing 
the  muscle  in  the  solution;  (2)  behavior  of  the  muscle  after  it  has 
been  in  the  solution  for  several  minutes;  (3)  changes  in  the  irrita- 
bility of  the  muscle :  test  with  single  induction  shocks  at  ten-minute 
intervals. 

(a)  Which    solutions   cause   the   muscle   to    contract    or   shorten 
permanently  ? 

(b)  Which  produce  rhythmical  contractions  or  twitches?    Which 
do  not?     Which  solutions  have  the  greatest  effect  of  this  kind? 

(c)  Which    solutions    deprive    the    muscle    of    irritability    most 
rapidly  ?    Compare  especially  solutions  a  and  b. 

b.  After  irritability  has  disappeared  try  the  effect  of  transferring 
the  muscle  to  normal  saline  solution  (  NaCl  in  tap  water).     Does  the 
irritability  return? 

c.  Compare  the  behavior  of  the  muscle  in  m/8  NaCl,  m/8  Nal'.r, 
m/8  Na,SO4.     Any  difference?     Then  try  the  effect  of  adding  to 
each  solution  a  few  drops  of  m/8  CaCl.,    What  is  the  effect?    What 
do   you   conclude    from    this    experiment   and    from    the    action    of 
solution  f  above,  as  to  the  influence  of  Ca  on  the  spontaneous  activ- 
ity of  skeletal  muscle?     Try  returning  the  muscle  after  an  interval 
to  the  pure  m/8  NaCl,  etc.     Does  the  former  behavior  return? 

55 


(1.  Try  tin-  effect  . .f  adding  to  the  pure  m/8  XaCl  or  m  S  XaBr 
a  little  m  S  KC1  solution:  e.  g.,  <)  vols.  m  S  XaCl  -)-  I  vol.  m/8 
KC1.  How  d.>cs  the  KC1  influence  the  behavior  of  the  muscle? 

MO.  Muscular  t:^itcliiii</  in  salt  solutions.  Sensiti.::int/  and  de- 
sensiti.'iiii/  actiini  of  'I'driotts  salt  solutions,  d  ret  pine  record.  The 
muscle  (gastrocnemius)  is  attached  to  the  extremity  of  a  bent  glass 
rod  by  a  wire  encircling'  bone  and  rod.  and  is  arranged  to  pull 
vertically  downward  on  the  short  arm  of  the  light  muscle  lever. 
Connection  is  made  with  the  lever  by  a  silk  thread  attached  to 
an  S-shaped  honk  passing  through  the  tendon.  The  glass  rod 
with  the  attached  muscle  is  immersed  in  the  solution  contained  in  a 
beaker,  which  stands  on  a  block  s< >  that  the  solution  can  be  readily 
withdrawn  without  disturbing  the  muscle  (see  demonstration). 
Test  the  action  of  the  following  solutions.  The  muscle  is  arranged 
for  the  lever  to  write  on  a  slowly  moving  drum.  Take  a  base  line 
with  the  muscle  in  Ringer's  solution. 

a.  Transfer  the  muscle   from  Ringer's  solution  to  a  mixture  of 
7  vols.  m/8  XaCl  -{-  i   vol.  m/8  KC1  by  substituting  a  beaker  with 
j;o  cc.  of  this  solution  for  that  containing  the  Ringer.     Make  the 
change  quickly,  but  be  careful  to  avoid  jarring.     Xote  the  behavior 
of  the  muscle   and   the  characteristics  of   the  contraction-curve   in 
this    solution,   tone-change,    etc.      At    the   end    of    _'   minutes   return 
tn  Ringer.     Xote  the  effect. 

b.  After  the  muscle  has  been  in   Ringer  2  or  ^  minutes  transfer 
to  pure  m    S  Xal'.r;  leave  in  this  solution  exactly  4  minutes.     Then, 
while    the    drum    is    moving    transfer    to    the    XaCl-KCl    mixture. 
Xote  the  difference  in  the  resulting  activity.     After  _'  minutes  return 
to   Ringer  as  before.     Result? 

Repeat  this  experiment  using  Xa.,S<  ),  instead  of  m  8  Xal'.r. 
Xote  the  difference  in  action.  Xote  carefully  the  behavior  ot  the 
muscle  both  /;/  the  solution  and  at  the  moment  ot  exposure  to  the 
air  when  the  transfer  is  made.  (Contact-Irritability,  shown  espe- 
cially after  treatment  with  solutions  ()f  salts  whose  anions  precipi- 
tate calcium;  as  XaK.  X  a, .('._,<  >,.  etc.)  Xote  also  carefully  the  be- 
havior in  the  XaCl-KCl  solution.  Leave  here  _'  minutes  and  then 
transfer  to  Ringer. 

e.      l)cscnsiti:::in</    salts.       Repeat     using    m   S    CaCI,     instead    of 
m   S   Xal'.r.      Xote  the  difference  in  effect.      U  this  action  of   Cat  | 
reversible?      If  there  is  time  try  also  m  S  MgCl... 

2OO.      Influence  of  muscle  poisons — Yeratrin.      Inject   one  or  (wo 
drops  of  a  saturated  solution  of  vcratrin  into  the  dorsal   Ivmph  sac 


of   a    frog.      Xote    from   time   to   time    the   condition    of    the    frog. 
What  is  the  effect  en  reflexes  and  on  general  activity? 

\\  hen  the  animal  is  "veratrinized"  remove  the  gastrocnemius 
muscle  plus  sciatic  ncn'c  and  mount  in  a  moist  chamber.  To  stimu- 
late the  nerve  it  is  laid  across  "needle  electrodes"  in  the  moist 
chamber;  to  stimulate  the  muscle,  connections  are  made  as  usual 
through  the  muscle. 

a.  Record   contractions   when   the   muscle   is   stimulated   by   the 
make  or  break  of  the  induced  current  and  a  short  tetanizing  current. 

b.  Record    contractions    when    the    ncrrc    is    stimulated   by   the 
make  or  break  of  the  induced  current. 

Note  character  of  curve.  What  part  of  the  neuro-muscular 
mechanism  is  affected  by  veratrin? 

201.  Unipolar    method    of    stimulation.      To    stimulate    human 
nerves  a  large  "indifferent"  electrode  is  placed  over  the  skin  where 
there  are  no  large  nerves  and  a  smaller  "stimulating  electrode"  over 
the  nerve  to  be  stimulated.     Place  the  indifferent  electrode,  covered 
with  cotton  soaked  in  physiological  salt  solution,  over  the  biceps 
muscle  and  connect  with  the  indtictorium  for  single  induced  shocks. 
Explore    the   inner    surface   of    the    forearm    with    the    stimulating 
electrode,  also  covered  with  salt-soaked  cotton,  and  note  the  con- 
tractions of  the  muscles  of  the  fingers  when  their  nerves  are  stimu- 
lated.    (See  fig.  in  Howell,  p.  93.)     Draw  an  outline  of  the  arm  and 
mark  the  "motor  points"  of  four  or  five  muscles  which  you  have 
been  able  to  find  on  your  arm. 

202.  Record   of  human    contractions — Tlie   er</o(/raph.     Tie   all 
the  fingers  of  the  right  hand  except  the  index  finger  in  the  wooden 
block  of  the  ergograph  and  adjust  the   rod  between   index  finger 
and  ergograph  lever  so  as  to  record   isotonic  contractions  of  the 
abductor  indicis  muscle.     A  celluloid  writing  point  should  be  at- 
tached by  wax  to  the  lever,  or  an  aluminium  point  if  the  movement  is 
very  slight.     Take  the  following  records : 

a.  Unipolar  single  make  or  break  induced  shocks.     The  indif- 
ferent electrode  should  be  placed  in  the  palm  of  the  hand  and  the 
nerve  stimulated  at  the  angle  between  first  and  second  metacarpals. 
Try  also  tetanizing  shocks  and  record  the  contraction. 

b.  Voluntary  contraction,  of  a  very  short  duration.     Voluntary 
contraction  of  several  seconds  duration. 

Then  shift  the  rod  toward  the  cast  iron  support  of  the  spring 
and  take  an  isometric  record  of: 

c.  Voluntary   contraction   of   very   short   duration. 


57 


(1.      Continued   contraction   until   marked    fatigue   results. 

Xdte  difference  l)et\veen  voluntary  tetanu-  and  tetanus  due  to 
electric  simulation. 

jo^.  I'rmiiiction  of  (/(•/</  /'/;  muscle,  a.  On  contraction.  1'ith 
a  frog  and  remove  the  skin  from  the  hind  legs.  Stain  these  in 
ph\ -iolo^-ical  salt  solution  containing  neutral  red.  Then  stimulate 
iinc  leg  with  the  tetanic  current  for  _'  or  3  minutes.  Any  change  in 
color:  Xo\v  place  both  legs  in  n  2<)(:  XI  I, <  )|  I  in  physiological  salt 
solution.  Any  difference  between  stimulated  and  unstimulated  legs? 

1).  ()n  lictit  ri</<»'.  Heat  the  unstimulated  leg  in  the  Xll,<  Ml  -alt 
solution  gradually  till  heat  rigor  sets  in.  Xote  any  change  in  color 
and  also  in  positi  n  of  the  leg  as  a  \vhole.  What  do  you  conclude 
a-  to  the  relative  -trength  of  the  flexor-  and  extensor-  of  the  leg? 

SMOOTH  MUSCLE 

As  material  for  experiment,  rings  about  3  mm.  broad  cut  from 
the  -tomach  of  a  frog  or  strips  from  a  cat's  bladder  may  be  n-ed. 

204.  Attach  by  hue  copper  wire  one  end  to  an  L-shapcd  gla-s 
rod.  the  other  to  the  >hort  arm  of  a  light  muscle  lever  as  in  experi- 
ment KJ'I.  Immerse  in  Ringer's  -olution  and  record  any  change-  in 
length  on  a  very  slow  moving  drum.  1  );  >  you  obtain  rhythmical 
contractions  i  r  tone  change-?  Try  raising  the  temperature  of  the 
Ringer's  solution  to  about  30°  C.  Ke-ult ': 

-'05.  .Make  appropriate  electrical  connections  and  study  the 
response  of  smooth  muscle  as  under  striated  mu-cle.  i  Kxps. 
[90-191.  I 

HEART  MUSCLE 

The  properties  of  heart  muscle  will  be  examined  in  studying  the 
physiology  of  the  heart  (p.  8l). 

//.     NERVE  PHYSIOLOGY 
\.     XKKYK  HI'.KUS. 

Stimulation  of  a  muscle,  -u-pendcd  in  a  nioi-t  chamber,  through 
it-  nerve  will  give  exactly  the  -ame  type  of  contraction  and  mu-cle 
curve  a-  direct  stimulation  of  the  mu-cle  fibers  thcm-clve-. 

Many  of  the  fundamental  phenomena  of  stimulation,  however, 
can  be  demonstrated  to  greater  advantage  on  nerve.  Muscle  shows 
irritability,  conductivity  and  contractilit \ .  Nerve  -how-  nly  irrita- 
bili;\  and  c  uductivity  ;  these  \\\  >  properties  are  interconnected  and 

58 


only  partly  separable.  ( ireat  care  must  be  taken  not  to  injure  the 
nerve  in  removing  it  from  the  frog.  Never  pinch  a  nerve  with 
forceps. 

206.  Effect   of  alcohol  and  CO.^  on   nerve,     a.   Carbon  dioxide. 
Arrange    the    inductorium    for    single    induced    currents.      Connect 
the  secondary  coil  with  the  main  posts  of  the  pole  changer  (used  in 
this  experiment  as  a  double  key  ).  Connect  the  two  other  pairs  of  posts 
with  the  usual  stimulating  electrodes  and  the  electrodes  of  the  small 
gas   chamber.     Join   the   inflow   tube  of  the  gas  chamber   with   the 
outflow  tube  of  the  O  ).,  bottle.     The  gas  chamber  should  be  clamped 
in  position  on  a  glass  plate.     Make  a  nerve-muscle  preparation,  prc- 
scrviinj  the  full  length  of  the  sciatic  nerve  up  to  the  vertebral  column. 
Pass  the  nerve  through  the  holes  of  the  gas  chamber  so  that  it  lies  on 
the  electrodes.    The  nerve  should  be  drawn  through  until  the  muscle 
is  close  to  the  gas  chamber.     Stop  the  holes  through  which  the  nerve 
passes  with  normal  saline  clay.     Bring  the  outer  pair  of  electrodes 
against  the  central  (i.e.,  towards  spinal  cord)  end  of  the  nerve  near 
its  exit   from  the  gas   chamber.      Determine  which  position   of   the 
double  key  corresponds  to  each  pair  of  electrodes.     Stimulate  the 
nerve  first  within  the  chamber,  and  then  on  the  central  end  of  the 
nerve,  using  a  current  just  sufficient  to  cause  tetanus.     What  is  the 
result?      Xow    pour    ioc/r    HC1    on    the    marble    in    the    generator 
and   pass   the  gas   through   water   and   then   through   the   chamber. 
After  a   few  minutes   stimulate  as  before.     Result?     What  is  the 
explanation  ? 

b.  Alcohol.  Disconnect  the  rubber  tube  from  the  gas  generator, 
and  blow  through  the  gas  chamber  until  the  CO.,  is  driven  out. 
Does  the  nerve  recover  its  irritability  ?  Determine  this  by  stimulating 
from  time  to  time.  When  the  nerve  has  recovered,  drop  a  little 
alcohol  through  the  long  glass  tube  of  the  gas  chamber,  hein</  very 
careful  that  only  the  vapor  of  the  alcohol  comes  into  contact  t^'itli 
the  nerve.  Stimulate  both  within  and  without  the  chamber.  What 
results  do  you  now  obtain  ?  Which  property  of  nerve  does  the 
alcohol  affect?  To  obtain  good  results,  the  electrodes  within  the 
gas  chamber  should  not  be  too  far  from  the  opening  through  which 
the  nerve  passes  to  the  muscle. 

207.  Threshold  value  of  Stimulation.     Prepare  a  gastrocnemius 
muscle  with  the  sciatic  nerve  from  its  point  of  origin  in  the  spinal 
cord  down,  and  place  in  the  moist  chamber.     Hang  the  nerve  over 
the  needle  electrodes.     Determine  the  single  break  induced  stimulus 
which  just  causes  contraction.     This  is  the  threshold  value.     Xow 


59 


apply  the  -ame  needle  electrode-  t< .  the  mu-cle  directly.  I-  the 
threshold  value  for  muscle  (  or  the  nerve  fibers  in  muscle  \  the  same? 
I  'eterniine  the  thrc-hold  value  f  r  different  points  along  the  nerve. 
I-  there  any  dift'ereiice  ?  I  Alii  >\\  ance  mu-t  he  made  in  this  experi- 
ment l"«r  differences  in  the  electrical  resistance  of  the  two  tissue-. 
or  of  the  different  regions  of  the  nerve.) 

_'uS.  Summation  of  snhminimal  stimuli.  t'-ing  the  la-t  uerve- 
mu-cle  preparation,  place  the  coils  of  the  inductorium  just  far 
enough  a]>art  to  prevent  contraction  on  stimulation  of  the  muscle 
with  needle  elect n>de-.  Allow  the  muscle  to  rest  a  few  minute-. 
Xow  stimulate.  If  in  contraction  results  keep  stimulating  about 
twice  a  -eomd.  Doe-  the  muscle  eventually  contract?  Does  it  con- 
tract with  a  tetanizing  current?  Does  your  result  indicate  that  the 
excitation>  outlast  the  stimulus  and  reinforce  subsequent  stimuli? 

209.  The  excitation  i^\rce  remains  in  the  muscle  or  nen-e 
fiber  in  'leliich  it  starts.  In  order  to  limit  the  stimulus  to  one  or  two 
filters,  the  method  of  unipolar  stimulation  may  lie  adopted.  Fasten 
in  one  |  ii  ist  lit  the  sec<  ndary  coil  of  the  inductorium  arranged  for 
tetanizing  currents  a  wire  soldered  to  a  blunt  needle.  F.xpose  the 
sacral  plexus  in  a  brainless  and  spineless  frog  in  which  the  skin 
has  been  removed  from  the  hind  limbs.  Connect  the  preparation  by 
means  of  a  copper  wire  to  the  earth  through  the  gas  or  water  pipes 
by  connecting  with  the  desk  binding  p<  sts.  Touch  the  sacral  nerves 
here  and  there  with  the  needle  electrode,  watching  meanwhile  the 
sart<irius  muscle.  Do  all  fillers  contract?  Stimulate  the  sartoriu- 
directly.  Do  only  the  libers  touched  by  the  needle  contract? 

_'io.  The  same  ner:-e  fiber  ma\  conduct  impulses  both  ccntri- 
petally  anil  ccntrif in/ally,  a.  The  nerve  of  the  sartorius  divide-  at 
the  muscle,  part  going  to  each  half  of  the  muscle.  Microscopical 
c ••  animation  sh  \\--,  that  the  division  is  not  -imply  a  parting  of 
individual  nerve  filters,  but  that  each  axis  cylinder  tork-.  one  limb 
going  upward-,  the  other  downward-.  If  the  mu-cle  i-  -evered 
between  the  fork-,  no  impul-e  started  in  one  halt  -  t  the  mu-cle 
could  reach  the  other  half,  except  by  going  up  one  branch  to  the 
original  axis  cylinder  and  down  the  remaining  branch;  for  it  has 
been  -howii  that  the  nerve  impulse  does  not  escape  transversely 
in -m  "lie  axis  cylinder  to  other  neighboring  ones. 

Keinovc  a  -artoHu-  mu-cle  with  great  care.  Split  the  mu-cle 
in  the  middle  line  for  one-third  of  its  length,  beginning  at  the 
bn-.ad  end.  Stimulate  the  right  -egment  by  snipping  it  with  a  pair 
of  scissors.  Xote  can-fully  if  the  liber-  of  the  left  -egment  contract. 


b.  The  gracilis  muscle  of  the  frog  is  divided  by  a  fascia  into  an 
upper  shorter  part  and  a  lower  longer  part.  Remove  carefully  the 
muscle  with  its  attached  nerve  and  note  that  the  nerve  and  blood 
vessels  divide  so  as  to  go  on  each  side  of  the  tendon.  Cut  the  muscle 
in  half  at  the  tendon  without  injuring  the  nerves.  Then  stimulate 
one  half.  Does  the  other  half  contract? 

211.  Elcctrotonits.     a.  Effect  of  constant  current  on   irritability. 
Two  currents  are  to  be  sent  through  the  nerves,  a  galvanic  polarizing 
current  \vhose  effect  on  irritability  is  to  be  studied  and  a  stimulating 
current  of  single  induced  shocks  for  testing  the  irritability.     Set  up 
the  nerve  muscle  preparation  in   the  moist  chamber   for  recording 
en  a  drum,  placing  the  nerve  over  non-polarizable  electrodes  about 
1-2  cm.  apart,  connected  through  the  rheocord  and  a  reversing  key 
with  the  desk  binding  posts  which  will  give  a  strong  galvanic  current. 
Place  the  stimulating  needle  electrodes  (see  that  they  are  clean )  on 
the  muscle  side  of  the  boot  electrode  nearest  the  muscle  and  connect 
with  the  secondary  of  an  inductorium.     Stimulate  the  nerve  with  a 
minimal    break    induced    shock    and    record    the    contraction    on    a 
stationary  drum.     Xow  send  a  weak  constant  current  through  the 
nerve  and,  disregarding  the  contraction  on  making  the  galvanic  cur- 
rent,   stimulate   again.      Is   the   height   of   contraction   increased   or 
diminished?    Indicating  what?    Break  the  galvanic  current  and  in  a 
minute  or  so  stimulate  again.    Result  ?    The  record  should  show  the 
result  of  stimulation  in  all  phases   of  the  experiment.     At   which 
pole  is  irritability  increased? 

Repeat  the  above  but  reverse  the  direction  of  the  constant  current 
through  the  nerve.  Result  and  conclusions? 

The  best  results  are  obtained  with  a  certain  strength  of  polariz- 
ing current  which  must  be  determined  by  experiment.  Do  you  see 
now  why  the  make  galvanic  stimulus  is  greater  than  the  break  ? 

b.  Effect  of  constant  current  on  conductivity.  Apparatus  the 
same  as  in  the  preceding  experiment  except  that  the  stimulating 
electrodes  are  placed  midway  between  the  boot  electrodes  and  a 
piece  of  muscle  is  introduced  in  the  stimulating  circuit  to  increase 
resistance.  Do  you  see  why?  Xo  record  need  be  taken.  Cse 
minimal  stimuli  as  before.  Determine  if  a  weak  galvanic  current 
can  block  the  passage  of  a  nerve  impulse  and  the  pole  at  which  the 
block  occurs.  Increase  the  strength  of  the  polarizing  current  and 
determine  its  effect  on  the  blocking  of  the  impulse. 

212.  Speed  of  the  nerve  impulse.     Adjust  the  drum  for  turning 
by  hand.     Place  two  pairs  of  needle  electrodes  in  the  moist  chamber 


61 


connected  through  a  double  key  with  the  >ec<  >ndarv  of  an  induc- 
lorium.  Make  a  nerve  muscle  preparation,  preserving  the  full 
lent/tit  nf  the  sciatic  iier:-e,  and  place  in  the  in  i-t  chamber  with  the 
electrodes  under  the  nerve  and  as  far  apart  a-  possible.  With  a 
tuning  fork  and  signal  magnet  pressed  against  the  drum.  >timulate 
with  a  maximal  make  shock  and  record  the  latent  period  and  con- 
traction (as  in  exp.  \<)\  ).  fii'ft.  with  the  electrode  far  trom  the 
muscle  in  circuit,  and  then  with  the  near  electrode  in  circuit. 
The  time  differences  in  the  latent  periods  inn-i  be  the  time  required 
for  a  nerve  impulse  to  pass  along  a  space  equal  to  that  between  the 
tuo  cath:  des  (why?).  Measure  this  distance  and  calculate  the 
rate  of  the  nerve  impul-c  per  second. 

213.  The  salt  <>f  fatii/nc.  Pith  the  brain  of  a  frog  (but  not 
the  spinal  cord  )  and  plug  the  cavity  with  cotton  to  prevent  bleeding. 
Kxpose  the  sciatic  nerves  of  both  sides  and  pass  a  thread  under- 
neath so  that  they  may  be  lifted  readily  for  stimulation.  Stimulate 
the  right  sciatic  (with  a  current  strong  enough  to  cause  contraction 
of  the  left  leg)  and  note  the  time  required  for  the  muscles  of  the 
left  leg  to  relax,  completely  fatigued.  Have  the  right  leg  muscles 
also  relaxed?  Call  this  time  A.  Then  stimulate  the  left  sciatic 
(do  not  fatigue  it)  to  see  if  the  muscles  still  contract.  Result? 
\Yhcre  has  fatigue  occurred?  (Juickly  remove  the  skin  from  the 
right  leg.  tie  the  thread  about  the  sciatic  and  cut  centrally  to  the 
ligature,  remove  the  muscles  fn  in  the  thigh,  cut  the  femur  and 
fasU-n  in  a  clam]).  Stimulate  the  sciatic  with  a  weak  stimulus 
i  tciani/.ing  )  until  fatigue  occurs,  recording  the  time  required.  Call 
this  time  \\.  Xow  stimulate  the  muscle  directly  until  fatigued.  Call 
this  time  C.  What  ha-  been  fatigued  here? 

To  prove  that  the  sciatic  nerve  has  not  been  fatigued  at  the 
point  of  stimulation  proceed  as  tollow-:  Remove  the  left  leu;. 
rcttiiiiiiH/  the  <\7/<'A'  leiit/tli  of  the  sciatic  ncn'c.  and  place  in  a  lemur 
clamp  witli  the  nerve  across  non-polarizable  electrode-  near  the 
muscle.  A  galvanic  current  (  from  desk  electrodes)  is  to  be  passed 
through  the  nerve  in  order  to  block  the  nerve  impulse,  and  the  far  end 
of  the  nerve  i-  stimulated  with  a  weak  tetanixing  current  \<  r  a 
length  of  linn/  equal  to  A  -J-  B  -f-  C.  If  the  muscle  contracts  when 
the  polarixing  current  i-  turned  off.  but  the  tctani/ing  stimuli  arc 
-till  given,  we  can  >afelv  assume  that  no  tatigue  has  occurred  at  the 
part  of  stimulation  during  the  time  of  the  experiment.  Which  are 
fatigued  in  order  in  this  experiment — nerves,  nerve  endings,  nerve 
cells,  muscle  liber? 


<  _• 


B.     XERYE  CELLS.     (PHYSIOLOGY  OF  CENTRAL   XLR- 
VOUS  SYSTEM  1.) 

A.     REFLEXES. 

214.  In  a  normal  frog  observe  the  following:     Movements  of  the 
head  when  the  animal  is  revolved  in  (  i  )  a  vertical  plane  parallel  to 
the  axis  of  the  body,   (2)  in  a  vertical  plane  perpendicular  to  axL 
of   body,    (3)    in   a    horizontal    plane.      Make   a    general    statement 
denning  movements  in  the  above  cases. 

215.  Pith   a    frog    (brain   only)    and    stimulate   by   pinching  or 
touching   the    following    regions :    a    toe    of    right    foot :    a    toe    of 
left    foot ;    a   finger ;    an    eye ;   the    skin   of   the   abdomen.      Record 
the  movements  resulting  in  each  case. 

216.  Record  what  happens  in  the  following  reflexes  in  yourself 
or  partner:     Pupil  rcflc.rcs.     (i)   Light  reflex.     Close  one  eye  for 
several  seconds,  then  open  it  quickly.     Xote  any  change  in  pupil. 
(2)  Consensual  reflex.    Close  one  eye  as  before,  but  watch  the  pupil 
of  the  other  eye  when  the  first  is  opened  again.     (  3)  Accommodation 
reflexes.     Look,  alternately  at  a  near  and  a  far  object.     Xote  any 
change  in  pupil.    This  experiment  cannot  be  performed  on  yourself. 
(4)    If  you  are  not  already  familiar  with  the  "knee  jerk",  demon- 
strate   this. 

217.  Purposiveness  of  rcflc.rcs.     Suspend  from  a  hook  a   frog 
with  its  brain  pithed.    Dip  in  acetic  acid  a  piece  of  filter  paper  about 
a  quarter  cm.  square.     Shake  oft"  the  excess  of  acid,  then  apply  the 
paper  to  the  front  of  the  frog's  body,     \Yhat  movements  result? 
Remove  the  paper,  dip  the  frog  in  water  to  remove  the  acid  from  his 
skin,   and   again   suspend   the   animal    from   the   hook.      After   five 
minutes  repeat  the  experiment,  but  apply  the  acidulated  paper  to 
the  inside  of  the  thigh.      If  only  one  foot  is  drawn  up  hold   that 
foot.      Does    the    other    foot    now    move?      After   washing   off   the 
acid  and  waiting  again  for  five  minutes,  apply  the  acidulated  paper 
to  the  back  near  the  tip  of  the  urostyle.     To  what  region  is  the  re- 
sponse now  directed?     Are  the  directions  of  the  reflex  movements 
sufficiently  different  in  these  three  instances,  and  pointed  toward  a 
definite  end  with  sufficient  clearness,  to  indicate  purposive  action  ? 

Are  the  reflexes  in  sections  214,  215,  and  216  obtained  when  the 
spinal  cord  is  also  destroyed  J. 

218.  Summation.     Suspend  by  a  hook  a  frog  with  brain  pithed. 
Tie  two  fine  copper  wires   i   cm.  apart  around  the  left   foot,  near 
the  toes,  and  attach  the  wires  to  a  secondary  coil  of  the  inductorium. 


i '"iiiKct  the  primary  c«>il  through  a  sjmple  kev  in  a  dry  cell.  I  )n 
single  make  and  break  shocks  evoke  a  reflex  response?  Me  ver\ 
careful  in  this  ca-e  t"  di-tin^uish  bet  \\cen  a  direct  stimulation  of 
llie  muscle  liy  the  eleciric  current  and  a  reflex  stimulation  from  the 
central  nervous  system.  Stiinulale  with  regularly  repeated  iiva// 
make  and  break  -hnck^,  and  test  \vhether  under  these  circumstance^ 
retlex  action  can  result  fr  m  the  summation  of  afferent  impulses. 
I  i"  the  -^timuli  are  repeated  more  rapidly,  does  the  reflex  occur 
sooner?  \\'hat  is  the  effect  of  increasing  the  strength  of  the  stimuli 
and  maintaining  the  -ame  rate  of  stimulation? 

2\().  Inhibition.  Use  the  fmg  and  a]>])aratus  as  described  in 
the  foregoing  experiment,  hut  arrange  the  inductorium  to  deliver 
a  tetanixing  current. 

I'n  vide  a  vessel  of  \vater.  Immerse  the  toes  of  the  right  leg 
nf  the  fmg  in  0.5',  stil])huric  acid  and  note  the  time  required 
before  retlex  action  occur-.  Without  any  delay  wash  off  the  acid 
in  water.  After  an  interval  of  3  minutes,  stimulate  the  left  foot 
with  a  weak  tetanixing  current  a>  the  right  is  again  immersed  in 
the  acid.  If  the  foot  is  not  withdrawn  from  the  acid  after  20 
seconds,  st  p  the  tetanizing  currents.  What  has  been  the  effect  of 
the  afferent  impulses  frnm  the  left  foot  on  the  efferent  imputes  to 
the  right  leg?  After  again  washing  the  leg  in  water,  prove  that 
the  sensory  endings  in  the  skin  are  still  irritable  to  the  acid. 

^2(i.  Irradiation.  Use  a  tetanizing  current  and  arrange  as  in 
experiment  Ji<).  Start  with  a  subminimal  stimulus  and  then  gradu- 
ually  increase  its  strength,  determining  the  effect  on  the  retlex 
movement  produced.  I  )oes  the  reflex  become  "crossed"?  H  es  it 
extend  to  anterior  regions?  Kecnrd  the  order  nf  spread  with 
increasing  strengths  of  stimulus. 

_'_'!.  Augmentation.  Determine  the  ease  with  which  the  "knee 
jerk"  is  -iven,  Using  your  partner  a-  a  subject.  Let  him  then  pull 
upon  his  clasped  hands  in  a  maximum  muscular  eft'nrt  and  again 
determine  the  activity  of  the  "knee  jerk".  I>  there  any  difference? 

222.  Mmiifictitiiin  nf  rr//<M-  response  hy  altcriin/  condition  of 
//.  rrr-i -m//m/.v.  I'rnductioii  of  hypersensitiveness  of  cntane  >us 
nerve-endings  can  be  induced  by  s  dium  citrate.  Suspend  the  frog 
that  the  feel  dip  in  in  S  Xa  citrate  solution.  After  mie  to  two 
minutes  withdraw  the  feet  from  the  citrate  solution  and  dip  in 
ordinary  tap  water.  Note  the  effect.  Kcplace  in  citrate  solution. 
Note  the  effect. 

\fter  producing  the   -eiisjiivc  condition   as  before,  dip   the    Frog's 


feet  into  m-cane-sugar-solution.  Note  the  effect.  What  is  the 
general  physiological  effect  of  such  a  solution  ?  Then  dip  the  feet 
into  the  water  as  before.  Is  there  any  response? 

Can  the  hypersensitive  condition  he  restored  by  the  citrate  solu- 
tion? i.  e.,  are  these  changes  of  sensitivity  reversible? 

223.  Modification   of  rcflc.r  by  altering  condition   of  cord.     Ef- 
fect of  strychnine  on  rcflc.v  action.    Inject  with  a  fine-pointed  pipette 
a  few  drops  of  0.5^   solution  of  strychnine  sulphate  into  the  dorsal 
lymph  sac  of  a  frog  whose  brain  has  been  destroyed.     After  a  few 
minutes  test  the  reflex  excitability  of  the  animal  by  touching  the 
foot  with  a  needle.     Note  carefully  the  character  of  the  response 
and  how   it   differs   from  that  of   an  unstrychninized   frog.     Note 
evidence    that    as    the    influence    of    the    strychnine   becomes   more 
marked  the  afferent  impulses  spread  more  and  more  readily  through- 
cut  the  entire  cord.    Then  destroy  the  spinal  cord  and  stimulate  the 
animal  as  before.     What  is  the  essential  nature  of  the  change  pro- 
duced in  the  cord  by  strychnine? 

224.  Production      of     hyper-irritability      of     the      ucrrc-trnnk. 
Under  some  conditions  the  nerve-trunks  become  abnormally  sensi- 
tive, and  a  reflex  response  may  be  modified  by  this  cause.     Hyper- 
sensitiveness  to  contact  may  be  induced  as  follows :     Immerse  the 
nerve  of  a  nerve-muscle  preparation  in  m/8  Na  citrate  solution  for 
about  5  minutes.     (Let  the  muscle  rest  on  moist  filter  paper  on  a 
glass  plate  from  the  edge  of  which  the  nerve  hangs  down  into  the 
beaker  containing  the  solution.)      Is  there  any  effect  on  the  muscle 
while  the  nerve  remains  in  the  solution  ?     Then  remove  the  nerve 
from  the  solution  and  let  it  hang  in  the  air.     Any  effect?     Touch 
it  with  a  glass  rod  or  the  handle  of  a  scalpel.     What  is  the  result? 
Dip  the  nerve  in  Ringer's  solution  for  a  short  time  and  again  test  its 
contact  reaction.     Can  the  hypersensitiveness  be  produced  a  second 
time?     Muscle  may  be  similarly  rendered  hypersensitive. 

225.  Reaction  time.     Place  a  signal  magnet  in  circuit  with  two 
simple  keys  and  the  primary  of  an  inductorium  arranged  for  single 
shocks.     The  signal  magnet  is  arranged  to  write  on  a  drum  (turned 
by  hand  )  just  above  a  vibrating  tuning  fork.    One  student  is  to  place 
the  stimulating  electrodes  from  the  secondary  terminals  of  the  induc- 
torium on  his  tongue  and  his  right  hand  on  one  of  the  keys  which 
must  be  closed.     He  should  close  his  eyes  and  concentrate  attention 
on    the    stimulation    of    his    tongue.      When    stimulated    he    should 
instantly  open  the  key  in  his  right  hand.     The  other  student  must 
start  the  tuning  fork,   rotate  the   drum   and   close   the   second   key 


65 


which  give-  the  -timulu-.     Take  at  least  two  records  and  determine 
the  average  time  required  to  react  to  a  -limnlus. 

226.  f\'ciiction  time  ;*.'ith  choice.  Apparatus  as  in  the  preceding 
experiment  except  that  three  ke\  -  are  placed  in  the  circuit  and  the 
stimulating  electrode-  are  luld  mi  the  tongue  by  the  lips.  Right  and 
left  hands  are  placed  on  two  of  the  key>  (  hoth  closed)  and  if  a 
5tr  ng  -timulus  is  received  the  left  hand  opens  a  key;  if  a  weak 
stimuli!-  the  right  hand.  Strong  and  weak  stimuli  must  he  deter- 
mined beforehand  in  terms  of  coil  distances  and  of  course  the 
•ubject  must  not  know  which  he  is  to  receive.  Take  two  nv  inl- 
and compare  with  the  preceding  experiment. 

i  227.  AY//r.r  tone  of  muscles.  I'ith  the  brain  Miily  of  a  frog 
and  suspend  from  a  hook  hy  the  lower  jaw.  Xote  especially  the 
position  of  the  leg-,  \o\\-  make  a  small  -lit  in  the  abdomen  and 
cut  the  roots  nl  all  the  nerves  going  to  the  right  leg  where  they 
leave  the  -phial  cord.  Again  -u-pend  the  frig  fnun  the  honk  and 
note  the  position  of  the  legs.  \\  hat  does  thi-  experiment  indicate.' 

I!.     Till'.   IJkAi.v 

In  the  following  operations  proceed  very  -lowly  and  cautioti-ly. 
as  the  frigs  are  to  he  kept  alive  for  as  long  a  time  as  pn--ihle  t" 
recover  from  "cerebral  shock",  due  to  the  operation.  I'se  well 
sharpened  instruments.  At  intervals  during  the  operation  wash  the 
skin  with  antiseptic  salt  solution  (  I  IgVL  I  :  JCOG). 

228.  I\'emo:'al  of  hemispheres.  Select  a  male  hog.  characterized 
by  a  thickened  pad  on  the  innermost  digit  of  the  front  limb. 
Anacstheti/c  by  placing  him  under  a  battery  jar  with  -  me  absorbent 
cotton  \\et  with  ether.  If  during  the  following  operation  the  effect 
of  the  anaesthetic  diminishes,  place  under  the  jar  again. 

The  cerebral  hemispheres  of  the  trog  extend  back  to  a  line  con- 
necting the  front  margins  of  the  two  tympanic  membranes.  (  ni  the 
skin  along  this  line  over  the  top  .  I  the  -kull.  Lrom  this  cross  cut 
make  a  median  incision  forward  nearly  to  the  iio-tn]-.  Lav  back 
the  Hap-.  With  -cis-or  point-  separated  to  either  side  o|  the  top 
of  the  skull,  immediately  in  front  of  the  transverse  skin  incision, 
cautiously  bring  ihe  points  together,  cutting  hareh  through  the  bone. 
In-ert  the  -bar]»  blade  forward  and  at  one  side  under  the  bony 
covering  of  the  cerebrum,  and  snip  the  bone.  Kcpeat  the  operation 
on  the  other  sjdi.'.  l\ai-e  the  bone  with  -mall  lorccps  and  carefully 
cut  forward,  alternate!)  on  one  sjdc  and  the  Other,  until  the  cere- 
brum i-  entirely  eNpn-ed.  Sever  the  Connections  between  the  optic 


lubes  and  the  cerebrum  and  remove  it.    With  silk  thread  MW  together 
the  flaps  of  the  skin. 

Xote  the  posture  of  the  animal  immediately  after  the  operation. 
To  what  factors  may  this  be  due?  While  the  animal  is  recovering 
perform  other  experiments.  During  the  interval,  however,  keep 
the  frog's  skin  moist,  for  he  breathes  in  large  part  through  his 
moist  skin.  In  about  an  hour  test  the  capabilities  of  the  frog  as 
follows :  ( a  )  Posture.  Record  the  difference  between  the  decerebrate 
frog  and  a  spinal  frog  as  to  posture.  (b)  Locomotion.  Simi- 
larly record  the  differences  in  leaping  and  swimming.  (  c  )  Respira- 
tion. Is  there  a  difference  in  respiratory  activity?  (d)  Vision. 
Compare  the  eyelids.  Place  an  opaque  object  between  the  decere- 
brate frog  and  a  source  of  light.  With  the  animal  facing  the  object, 
which  should  be  only  6  or  7  cm.  distant,  stimulate  him  to  jump. 
Does  the  frog  jump  against  the  object,  or  avoid  it?  (e)  Equilibra- 
tion. Turn  the  decerebrate  frog  on  his  back.  Compare  his  reaction 
to  that  of  a  spinal  frog.  Place  him  on  the  palm  of  the  hand  or  on 
the  frog  board.  Slowly  tilt  his  support.  What  happens  as  his 
equilibrium  is  disturbed?  See  if  the  frog  can  be  made  to  crawl 
to  the  other  side  of  the  hand  or  frog  board  as  the  support  is  fur- 
ther turned.  (  f  )  Croak  reflex.  Hold  the  decerebrate  frog  gently 
between  the  thumb  and  first  finger,  placed  immediately  behind  the 
front  limbs.  Apply  slight  pressure  for  a  moment.  The  frog  should 
croak  in  response  to  each  application  of  stimulus.  Stroke  with  the 
moistened  finger  the  skin  of  the  back  or  flanks,  and  note  if  this 
also  evokes  a  reflex. 

(g)  If  the  operation  is  successful  the  animals  live  for  several  days. 
They  will  be  kept  in  the  laboratory,  and  if  possible  the  student  ought 
to  examine  their  general  reactions  on  the  clay  after  the  operation. 

229.  Influence  of  optic  lobes  on  refle.res.  Endeavor  to  find  some 
marked  difference  between  a  decerebrate  and  a  normal  frog.  a. 
Expose  the  brain  according  to  the  directions  already  given.  Imme- 
diately posterior  to  the  hemispheres  lie  the  optic  lobes,  two  gray 
spherical  bodies.  Separate  the  cerebral  hemispheres  from  the  optic 
lobes  by  a  transverse  incision,  and  carefully  remove  the  hemispheres. 
Wait  until  the  shock  of  the  operation  has  passed.  Xo\v  suspend  the 
frog  without  injury  so  that  the  tips  of  the  toes  hang  above  a  shallow 
dish  containing  0.5 r;  sulphuric  acid.  Determine  the  reflex  time. 
Wash  off  the  acid  and,  after  a  moment's  rest,  sprinkle  a  very 
little  finely  powdered  common  salt  on  the  cut  surface  of  the 
optic  lobes.  Again  determine  the  reflex  time.  Is  it  markedly 
changed  by  the  stimulation  of  the  optic  lobes? 

67 


1).  Remove  (.-are fully  the  optic  l<il>es.  wash  oft  any  exec--  -alt 
with  physiological  salt  -olntion.  and  again  determine  the  reaction 
tinu.  Any  change:  X«»\v  sew  carefully  together  the  tla]>-  of  -kin 
over  the  brain  cavity.  C  •  mpare  it-  reactions  with  tho>e  of  the 
decerebrate  frog  of  experiment  jjS.  Place  the  frog  in  the  box 
to  be  kipt  t"r  observation  next  day. 

///.     BIOELECTRIC  CURRENTS 

j^o.  (.  \i  pillar  \  elect r  i nneter.  The  inertia  of  the  coil  of  the 
ordinary  d'. \r-on\al  gal\  anometcr  is  so  great  that  it  is  nnsuited  to 
rec  I'd  rapid  changes  in  potential  such  as  are  produced  by  actively 
functioning  plant  and  animal  ti--ues.  For  this  purpose  the  capil- 
lary electrometer  is  used.  The  wire-  are  attached  to  two  snrtace-  of 
mercury,  one  large  and  one  small  I  in  a  capillary  tube),  -eparated 
from  each  other  by  _>o' ,  -ulphuric  acid.  When  a  current  passes,  the 
mercury  in  the  capillary  moves  in  the  direction  of  the  current. 
M  "\cimnt  is  proportional  to  the  strength  of  the  current  and  de- 
pend- on  a  change  in  the  surface  tcn-i;  n  of  the  mercury.  It- 
-urface  tension  i-  greatest  when  the  potential  difference  across  the 
surface  i-  least.  Draw  a  diagram  -bowing  the  construction  ot  the 
capillary  electrometer.  A  detailed  description  of  the  instrument 
will  be  found  in  I  b  >well,  p.  o,S. 

X  te  the  >hort  circuiting  key  on  the  instrument.  Non-polarizable 
electrode-  nm-t  alwa\  -  be  u-ed  in  leading  oil  the  wires  trom  the 
ti--ue  to  the  galvanometer. 

Fill  the  tubes  of  the  capillary  electrometer  with  mercury  and  Jo', 
sulphuric  acid  and  -et  up  on  the  stage  of  a  microscope  a-  demon- 
strated. I  landle  the  parts  rrry  carefully,  as  the  in-trumcnt  is  expen- 
sive and  easily  broken.  Wax  cement  may  be  used  to  hold  the  tube- 
firmly  in  the  block  support.  The  prc--urc  tube  on  the  right  will 
n  t  lie  u-ed  for  these  experiments. 

2$\.  The  action  current  of  the  heart.  Non-polarizable  electrode- 
are  applied  to  ventricle  and  auricle  of  the  frog'-  bear!  while  in  th 
body  and  connected  to  the  capillary  electrometer  on  the  micro-cope. 
Heart  mu-cle.  like  every  other  muscle,  becomes  during  contraction 
electrically  negative  relatively  to  inactive  portions  ot  the  tissue.  A 
wave  of  contraction  accompanied  by  a  wave  ''I  negative  potential 
pa  —  is  o\er  the  heart  and  is  recorded  b\  the  electrometer.  I  >o  the 
excursions  of  tin-  mercury  correspond  to  the  heart  heat-: 

j^j.  "/'//(•  current  of  injury  (current  of  rest  or  demarcation  cur- 
rent) of  muscle.  A  -artorius  muscle  i-  carefully  prepared  and  one 

•  3 


•end  cut  off.  Non-polarizable  electrodes  are  placed  one  on  the 
////injured  surface,  near  one  end,  and  the  other  on  the  ////injured  sur- 
face, near  the  other  end,  and  led  off  to  the  capillary  electrometer. 
A  deflection  may  he  noted  on  opening  the  short  circuiting  key,  indi- 
cating differences  in  the  electrodes.  Note  its  direction  and  amount. 
Now  one  cf  the  non-polarizable  electrodes  is  placed  on  the  injured 
end,  the  other  is  left  near  the  uninjured  end  of  the  muscle.  Again 
note  the  deflection  of  the  galvanometer.  Is  it  greater  than  before? 
In  which  direction  does  it  indicate  that  a  current  is  flowing? 

233.  Measurement  of  current  of  injury.     Connect  injured  and 
uninjured  surfaces  of  a  cut  sartorius,  with  an  electrometer  in  cir- 
cuit, to  the  slider  and  O-post  of  a  rheocord.     Connect  a  dry  cell 
through  a  key  to  the  O  and  lo-meter  post  of  the  rheocord.     Either 
both  negative  or  both  positive  poles  of  muscle  and  cell  must  be  con- 
nected to  the  O-post.      (See  fig.  in  Harvard  Apparatus  Company 
Catalogue,  p.  22. )     Move  the  slider  to  a  position  where  no  current 
flows  through  the  galvanometer  when  the  cross  circuiting  key   is 
opened.     The  fractional  voltage  of  the  dry  cell  (1.4  volts)  can  then 
be  read  directly  from  the  rheocord  and  will  just  balance  the  voltage 
of  the  muscle.     Result  ? 

234.  Current  of  action.     Stimulate  the  muscle  by  pinching  while 
a  current  of  injury  is  flowing.     How  is  the  electrometer  affected? 
Do  you  see  now  why  the  action  current  was  called  the  "negative 
variation''  of  the  current  of  rest? 

235.  The    bio-electric    currents  are   stront/   enouyh    to   stimulate 
nerre.     The  "Rheoscopic  Fro;/  Preparation". 

a.  Make  two  nerve-muscle  preparations,  A  and  B.  Lay  the 
nerve  cf  A  lengthwise  over  the  muscle  of  B.  Stimulate  B  through 
its  nerve.  Does  the  muscle  of  A  contract  as  well  as  that  of  B  ? 

1).  Cut  the  B  muscle  near  its  tendon  end.  Lay  nerve  of  A  care- 
fully on  muscle  of  B,  touching  injured  and  uninjured  surfaces.  Is 
there  any  contraction?  Nowr  stimulate  the  nerve  of  B.  Does  the 
A  muscle  also  contract?  How  do  you  interpret  each  of  the  results 
obtained  in  the  above  experiments? 

c.  Lay  the  nerve  of  A  lengthwise  across  the  beating  heart  of  the 
frog.  The  heart  is  left  in  the  body  of  the  frog  but  exposed  by 
cutting  away  the  pectoral  girdle  and  pericardium.  \Yhat  happens  ? 
Explain.  This  experiment  succeeds  best  if  the  frog  furnishing  the 
nerve  muscle  preparation  is  previously  kept  on  ice  for  some  time. 

236.  Polarization    current.      Connect   two    keys    (A    and    15)    in 
circuit  with  two  dry  cells.     Connect  a  frog's  muscle,  by  means  of 


69 


non-polarizable  electrode-  about  one  cm.  apart,  with  the  binding 
posts  .if  one  <il"  tlu-  ke\s  i  A  ).  Close  the  circuit  through  the  muscle 
for  -cvcral  minutes  hy  means  of  key  I'.,  leaving  key  A  open.  \"«>w 
<>pen  key  \\  and  immediately  make  and  lireak  key  A  several  time-. 
l>  es  the  mu-cle  contract?  \\'hcrc  is  the  -ource  of  the  current  in 
tliis  experiment:  The  muscle  act-  as  a  delicate  gal\  am  mirter. 
In  what  other  way  may  a  polarization  current  IK  obtained? 

IV.     CILIARY   MOVEMENT 

J  V '-  I'ith  a  frog,  destroying  hoth  hrain  and  spinal  cord,  and  pin 
it  on  it-  hack  on  the  frog  hoard.  Cut  away  the  ventral  body  wall 
and  remove  all  of  the  viscera  except  the  oesophagus  and  stomach. 
With  the  scissor-  cut  through  the  lower  jaw  in  the  middle  line  and 
continue  the  cut  hack  to  the  stomach.  I  )raw  hack  the  flaps  of  the 
lower  jaw.  and  pin  out  the  oesophagus  to  form  a  flat  surface  on  a 
level  with  the  roof  of  the  mouth.  Keep  the  oc-ophagus  and  the 
mucous  memhrane  of  the  roof  of  the  mouth  moist  with  normal  saline. 

a.  Lay  one  of  the  -mall  pieces  of  cork  on  the  exposed  mucous 
memhrane.     In  which  direction  does  the  cork  move?     Lay  a  weight 
on  the  cork  block  and   repeat   the  observation.      Repeat   again  after 
tilting  the  frog-hoard  so  that  the  weight  must  be  carried  up  an  incline. 

b.  Remove    the    weight,   and    determine   the   time    in    seconds    in 
which  the  cork  moves  one  inch.     Make  a  second  determination  after 
warming  the  preparation  with  saline  solution  at   t}<>     C. 

c.  Saturate  a  piece  of  filter  paper  with  ether  and  blow  the  funie- 
d   wn    upon    the   preparation.      After   a    few    seconds    make   a    third 
determinate  >n. 

(1.  Similarly  test  the  effect  of  vapor  of  ammonia,  hut  in  this  case 
it  will  he  sufficient  to  blow  across  the  open  mouth  of  the  bottle. 

Record  the  re-ults  of  the   four  determinations. 

-'^7.  (  )pen  a  clam,  mussel  or  oyster  shell  and  catch  the  contained 
fluid  in  a  beaker.  Cut  a  small  piece  of  tissue  from  the  mantle,  tease 
it  well  with  needles  and  mount  under  cover-glass.  India  ink  may 
be  added  to  ascertain  the  direction  of  current-.  Study  and  draw 
under  the  high  power  various  phases  in  the  heat  a-  it  becomes 
-lowed  through  lack  of  oxygen.  1  >o  yon  see  any  individual  cilia 
heating  which  arc  noi  attached  to  cells. J 

J^S.  Mount  a  piece  of  mantle  on  a  slide  between  two  noil  polar- 
i/able  electrodes  in  sea  water.  I 'lace  a  cover-glass  on  the  preparation 
and  study  with  the  high  power,  \\hat  i-  the  cited  ot  make  and 
break  of  the  gaKanic  current:  Make  and  break  single  induced  and 
interrupted  -In >ck-  ' 


239-  Paramecium  is  a  good  form  for  a  study  of  ciliary  action. 
Study  carefully  if  you  have  not  already  done  so.  Can  the  effective 
stroke  of  the  cilium  he  reversed? 

240.  l:ffcct  of  NH4OH.     Cvtolvsis — Place   Paraniecia  in  n/iooo 
XH4()H.      Xote   immediately   the  changes   undergone   hy   cilia   and 
vacuoles.     Dees  swelling  occur?     Note  especially  if  the  surface  of 
the  animal  is  lifted  oft'  while  the  cilia  still  remain  beating  on  the 
surface.     Describe  the  changes  during  cytolysis. 

y.       PROTOPLASMIC   ROTATION 

A  type  of  movement  closely  allied  to  amoeboid  movement  (see 
p.  39).  The  leaves  of  Vallisneria,  Chara,  Nitella,  Elodea,  and  the 
stamen  hairs  of  Tradescantia  are  well  adapted  for  study  of  proto- 
plasmic rotation.  Use  Elodea  leaves  in  the  following  experiments. 

241.  Study  under  the  high  power.      Note  time    for  a  complete 
revolution  and  the  direction  of  rotation  in  adjacent  cells.     Draw  a 
diagram  indicating  the  direction  by  arrows.     Try  warming  slightly 
the  slide.     Effect  on  rate? 

242.  Place  a  leaf  in  isotonic  sugar  solution.    Does  the  movement 
cease?    Cut  leaf  in  half  with  sharp  scissors  and  note  if  the  cells  near 
the  cut  edge  are  affected  in;  any  way.     Can  you  observe  any  proto- 
plasmic fragments  moving  in  the  fluid? 

243.  Place  a  leaf  in  isotonic  sugar  solution  and  add  a  few  crys- 
tals of  sugar.     What  is  the  effect  of  the  resulting  plasmolysis  on 
rotation?     Does  it  finally  cease? 

244.  a.     Place  leaves  in  water  one  sixth  saturated  with  chloro- 
form.    Effect  on  rotation?    Remove  to  pure  water  again.    Result? 

b.  Try  also  ether  water.  Note  that  practically  all  vital  processes 
are  slowed  or  abolished  by  these  anaesthetics  and  that  the  effect  is 
reversible. 

245.  Mount  a  leaf  on  a  slide  in  physiological  salt  solution.     Ef- 
fect?    Place  non-polarizable  electrodes  at  the  ends  of  the  leaf  and 
determine  any  effects  of  stimulating  by  galvanic  and  faradic  shocks. 
Result  ? 


I'AKT  IV 
PHYSIOLOGY    OF    NYrumox    (  Ixt  i.rmxv,    CIRC  n..vnox 

AM"    Kl'.Sl'IK. \TIOX  ) 

./.     METABOLISM 


A  verv  important  division  of  metabolism  (the  action  ot  cnxyino  i 
i>   already 
considered. 


ha-   already  been   >tudied.     Certain   general   features  remain  to  be 


I.     HOLOPHYTIC  METAB<  »LISM. 

_'4<>.  O.vytjcn  formation.  1'lace  in  a  tot-tube  with  clean  water 
a  healthy  branch  of  Elodea  (or  some  other  water  plant  that  has 
not  finely  divided  leaves).  Do  not  use  water  from  the  hydrant,  for 
this  contains  too  much  air;  use  if  possible  water  in  which  the  plants 
a i\-  found,  or  other  water  that  has  stood  for  a  time  in  tanks,  and 
be  careful  not  to  get  any  bubbles  of  air  in  the  tube.  Invert  the 
test-tube  in  a  vessel  of  the  same  water.  Place  in  a  bright  light,— 
where  the  direct  rays  of  the  sun  reach  it. 

At  the  same  time  prepare  another  experiment,  in  exactly  the  same 
way.  but  place  this  in  complete  darkness. 

Try  the  same  experiment,  but  using  water  that  has  been  boiled 
thoroughly  and  cooled  (|tiicklv  without  disturbance.  \\ash  the 
plant  in  this  water  before  placing  in  the  tube.  I  Mace  in  sunlight 
as  before.  Any  difference  from  above  result  :  Explain. 

Allow  the  three  experiments  to  stand  for  some  hours  or  for  a  day, 
if  Decenary.  In  which  one  are  bubbles  of  gas  produced?  If  in  all 
three,  which  -hows  the  larger  •|uaiitity?  Test  for  oxygen  by  the 
>park  test. 

j  |~.  I'liotosvnthcsis  in\  plants.  Kxamine  Spimgyra  lilameiit- 
that  have'  been  well  exposed  to  light.  Study  the  chlorophyll  band-. 
Sketch.  Then  run  under  the  cover-glass  of  a  second  preparation  a 
little  iodine  -ulutioii  and  examine.  Compare  carefully  with  the  un- 
treated preparation.  .Vote  the  distribution  ot  March.  Examine 
similarly  filament-  that  have  been  kept  in  dark  some  day-.  Xote  any 
diff. Ti-nrr  '  Kxplain. 

7- 


248.  Photosynthesis  in  leaves,  a.  Pin  two  flat  pieces  of  cork- 
together  over  a  portion  of  a  suitable  green  leaf  (to  exclude  light). 
Place  the  plant  in  a  bright  place  and  leave  two  days.  1).  Then  pick 
leaf;  dip  in  boiling  water  for  a  minute  or  so;  extract  chlorophyll 
with  95 f/f  alcohol  (some  time  will  be  necessary),  and  treat  with 
weak  iodine  solution.  Note  distribution  of  starch.  Kxplanation? 
c.  Perform  the  same  experiment  with  a  leaf  enclosed  in  a  bottle 
containing  strong  KOH  to  absorb  CCX.  The  petiole  is  passed  through 
a  slit  in  the  cork  and  the  whole  closed  airtight  with  vaseline.  Note 
difference  from  b?  Explanation?  d.  Note  distribution  of  starch 
in  a  variegated  leaf.  Place  the  leaf  (after  momentary  boiling)  in 
8oc/c  alcohol  to  extract  the  chlorophyll  and  treat  as  before.  Result? 
Conclusion  ? 

II.     HOLOZOIC  METABOLISM. 

The  following  study  of  certain)  of  the  processes  of  metabolism 
in  a  number  of  organisms  will  be  carried  on  partly  as  laboratory 
work,  partly  as  seminary  work.  The  processes  are  to  be  observed 
by  the  student  as  far  as  possible.  Where  this  is  not  possible,  descrip- 
tions of  them  are  to  be  read,  in  the  references  that  are  given.  The 
essential  point  is  to  have  after  study  a  clear  idea  how  the  process 
in  question  takes  place ;  be  ready  to  describe  and  explain  to  the 
instructor.  The  books  and  papers  referred  to  will  be  placed  on  the 
desk  in  the  laboratory,  and  are  to  be  referred  to  as  a  part  of  the 
regular  work. 

In  organisms  in  general  we  can  distinguish  a  number  of  factors 
and  processes  concerned  in  metabolism.  These  are  listed  in  the 
following,  together  with  suggestions  for  their  study  in  the  organisms 
examined. 

a.  The  taking  of   food.     Organs  or  processes  involved.     Make 
sketches  and  descriptions  if  possible. 

Most  organisms  have  either  some  process  of  bringing  food  to  the 
month,  or  of  going  to  the  food.  Determine  which  is  true  in  the 
given  case  (or  whether  both  or  neither  are  true),  and  describe. 

b.  The  digestion  of  food.     This  usually  takes  place  in  an  en- 
closed region,  the  alimentary  canal.     Make  a  diagram  of  this  when 
possible.     The  processes  involved  are  usually  the  subjection  of  the 
food  to  certain  chemicals.     This  is  usually  not  directly  observable, 
but  has  been  imitated  experimentally.     Such  experiments  we  have 
already  carried  on  in  our  study  of  enzyme  action.     Sometimes  the 
changes  in  the  food  can  be  traced ;  this  should  be  done  where  possible. 

73 


In   certain  case-   reference-   will   be  given.  t»  descriptions  of   the-e 
processes. 

c.  Tile  di-charge  nf  the  unused  parts  nf  the  fund   I  defaccati'  «n  i . 
<  >h-erve  and  de-cribe  it"  pn-<ible.      In  snme  cases  there  i-  a  definite 
opening  for  this  purpn.-e.  in  nther  eases  not. 

d.  Absorption,  assimilation  and  dissimilation.     These  are  u-ually 
m  »t  observable. 

e.  1  )i-trilmtinn  of   fn,  >d  \\-ithin  the  hndy.     There  is  usually  -nine 
method   .  f  carrying  the    fond   almut   within   the  Imdy  :   -nmetinic-  a 
definite  -et  nf  nrgaiis  fnr  this  purpose  (  circulatnry  sv-tem  of  higher 
animal-  i . 

f.  Respiration,  the  taking  nf  oxygen  and  giving  ;  ft"  of  O  >_..    The 
processes  involved  are  usually  movements  produced  in  the  -urnmnd- 
ing  atnn>s]ihere  or  water,  tn  hring  (  )._.aiid  carry  away  O  L.and  inter- 
nal current-  or  ninvement-   { -ame  as  mentiniied  in  last  paragraph). 
Study  both  carefully.     (  >fteii  special  organs  are  pre-ent. — respiratory 
nrgan-   (gill-,  lungs,  tracheae,  etc.).     Draw  and  ile-crihe. 

g.  Kxcretion, — the  discharge  nf   the   wa-te   products  of   dissimi- 
lation  (distinguish  clearly   from  defaecatinn  i.     Study  the  nrgan-  in- 
\nlved  and  how  they  act;  make  drawings.     Study  al-n  the  pmce--e- 
invnlved.     Xntice  that  often  currents  are  produced,   fnr  carrying  of! 
the  wa-te  materials  (as  in  f ). 

It  will  not  he  possible,  to  study  all  these  pn>ce--e-  in  each  organism 
we  take  up.  Some  are  lacking  in  various  organisms,  and  other-  arc 
unfavorable  for  study.  In  each  case  suggestions  will  he  made,  or 
<|Ue-tion>  a-kul,  indicating  the  points  to  he  studied. 

A.        I  '  \K  \M  El    i  I'M. 

Study  1'aramecium  tir-t,  a-  a  type  showing  how  nm-t  of  the-e 
prnce--es  are  carried  on  in  a  -imple  way. 

In  man\-  ca-es  in  the  -tndy  of  Paramecium,  the  animals  mav  he 
ninunted  to  advantage  in  gum  tragacanth,  which  makes  their  move- 
ment- -ln\\rr. 

247.  a.  The  tnkiiK/  of  fund.  I'.y  tin-  use  of  india  ink  ohserve 
hnw  Paramecium  hring-  fund  tn  it-  nioutli.  Sketch  a  ijuiet  indi- 
vidual. -ho\\  ing  the  current-,  in  relation  to  tlu-  nmuth  and  oral 
groove.  <  Jh-scrve  the  pa--age  of  the  ]»artick'-  intn  the  nmnth.  and 
the  formatinii  ()f  tlii'  f 1  \-aciinle-. 

h.  Distribution  <>f  i»,nl.  (  )h-erve  the  circulatinn  nf  the  fond 
vacuolc-.  Dd  the  vacuolc-  alone  mo\e.  or  does  tin-  internal  proto- 
|ila-m  mn\r  with  them?  I*'ollow  the  circulation  of  the  vacuolr-. 

7-1 


Make  a  drawing  of  Paramecinm,  showing  mouth,  oral  groove  and 
food  vacunles  and  indicate  the  path  of  circulation  of  the  food 
vacuoles  by  means  of  arrows.  (  Watch  carefully  for  defaeca- 
tion  ;  if  observed,  indicate  in  your  figure  where  it  occurs.  ) 

c.  Digestion.      Feed   the  animals  on  green  alga  cells.     (  )l>serve 
the  changes  in  color  in  the  food  vacuoles  after  they  have  beem  some 
time    in    the    body.      They    become    yellowish    in    place    of    bright 
green.     The  difference  in  color  among  the  different  food  vacuoK> 
is  commonly  easily  seen. 

Stain  some  of  the  living  Paramecia  with  neutral  red,  in  the  follow- 
ing way.  Make  a  i/iOOOf/f  solution  of  the  neutral  red.  To  a  small 
quantity  of  this  add  twice  this  quantity  of  culture  \vater  containing 
many  Paramecia,  and  allow  to  stand  for  ten  minutes  or  more.  The 
animals  will  now  be  found  to  be  partly  stained  red.  Since  this  sub- 
stance stains  only  structures  having  an  acid  reaction,  the  staining 
gives  an  opportunity  to  determine  the  nature  of  some  of  the  chemical 
processes  in  development.  It  will  be  found  that  some  of  the  food 
vacuoles  are  strongly  stained,  indicating  the  presence  in  them  cf  an 
acid.  Others  will  be  found  colorless,  while  others  are  of  a  palt- 
yellowish  tinge.  The  latter  are  those  in  which  digestion  is  about 
finished,  and  the  acid,  together  with  the  nutritious  parts  of  the  food, 
have  been  absorbed,  and  the  remaining  material  has  taken  an  alkaline 
reaction  (to  which  the  yellow  color  is  due).  Such  a  mass  is  usually 
found  in  the  posterior  part  of  the  body,  about  half  way  between 
the  mouth  and  the  posterior  end.  It  consists  of  waste  matter  ready 
for  discharge,  the  discharge  taking  place  at  about  this  point. 

Note  the  red  stained  zymogen  granules  about  the  vacuoles.  These 
are  thought  to  be  the  substances  giving  rise  to  the  ferments  of 
digestion. 

The  reaction  of  the  vacuoles  can  also  be  followed  by  feeding  on 
finely  divided  litmus  particles.  Try  this  experiment  as  a  check  on 
the  above. 

d.  Dcfaecation.     Observe  this  in  Paramecia  that  have  been  fed 
with  India  ink.    With  a  little  patience  in  watching  it  is  usually  easily 
seen  in  such  cases.     Represent  the  place  where  it  occurs  on  your 
figure. 

e.  Excretion  takes  place  by  means  of  the  two  contractile  vacu- 
oles.    Observe  the  method  by  which  these  are  filled  ;  draw  two  or 
three  stages  in  the  process,  showing  the  canals,  etc.     ( )bserve  the 
discharge  of  the  vacuoles  in  the  following  way.    Place  the  Paramecia 
in  a  thick  solution  of  India  ink,  so  that  they  appear  white  against 


a  tiark  background.  Withdraw  enough  <>f  the  water  with  filter  paper 
&O  that  the  cover  -hall  rest  upon  them  and  hold  them  still.  Kind  a 
-pecmien  with  contractile  vacuoles  near  one  edge  (not  above  or 
bel  w).  L'nder  these  circumstances  the  discharge  of  the  vacu»le 
content-  into  the  -nrnunxling  hlack  fluid  is  easily  seen.  Sketch. 
I-  the  discharged  fluid  carried  away  in  any  way.  or  does  it  remain 
again-t  the  surface  of  the  animal? 

f.  AV.v pirat  ii'ii.  '1'his  is  difficult  to  observe  or  demonstrate.  It 
ha-  been  done  in  the  following  way.  Make  a  weak,  slightly  reddish 
solution  of  rosolic  acid  (  ro-ol  ).  Place  a  dense  ma--  of  I'aramecia  in 
this,  on  a  slide,  with  a  supported  cover-glass.  <  )ften  the  animal- 
gather  in  dense  groups.  When  this  occurs,  enough  carbon  dioxide 
may  be  given  off  in  the  respiration  of  the  large  number  to  decolorize 
the  rosolic  acid,  \\hen  this  occurs,  if  the  preparation  is  placed 
on  a  white  background  the  region  where  the  I'aramecia  are  gathered 
appears  like  a  white  spot  in  the  red  preparation. 

Is  there  any  method  in  1'aramecium  of  continually  renewing 
the  water  that  is  in  contact  with  the  body,  so  as  to  have  at  all 
times  a  fresh  supply  of  oxygen? 

250.  Types  of  substances  digested.      Paramoecia  which   feed  on 
algae  or  bacteria  must  digest  proteins.     Determine  if  starch   is  di- 
gested, a-   f,  Hi  >ws  : 

Add  a  little  dilute  corn  starch  paste  to  -ome  of  the  organisms  in 
a  covered  watch-glass.  At  intervals  remove  a  few  Paramecia  to 
a  slide  and  add  iodine  solution.  Does  the  reaction  indicate  diijc-- 
tion  of  starch?  Keep  for  a  day  or  two  and  test  with  i  dine  again. 

The  dige-tion  of  fat  is  difficult  to  demonstrate  but  it  is  probable 
that  Paramecium  contains  lipolvtic  cn/.vme-. 

Pi.     A.Moi-.r.A. 

It  material  and  time  permit,  make  a  study  of  the  same  pn>ce--es 
in  Amoeba.  If  you  are  unable  to  see  the  facts  yourself,  read  the 
references  jjiven  below.  Komi  an  idea  of  the  way  each  of  the 
processes  mentioned  on  pp.  ~2  and  73  occur. 

251.  a.      The    (akin;/    of    finnl.      This    can    n-uallv    be    observed, 
with    -ome    patience,    in    a    culture    where    Amoebae   are    numerous. 
If  ob-crved,  describe  and  sketch.      If  you  do  not  -ee  it  yourself,  read 
the  accounts  given  in  the  following: 

(  arneiMe  Institution.  Publication  i<>,  Jenning-:  Contributions  to 
the  Studv  of  the  Behavior  of  the  Lower  <  )rganisms.  pp.  IIJ^-JDJ. 

l.ridy.  Kre-h  water  RhlZOpods  of  \orth  America,  pp.  S^-Xij  (in 
I  >inamoeba.  \\hicb  is  practical!)  an  Amoeba  I.  See  also  Plate  VI. 


1).  Digestion.  If  you  have  mu;iy  favorable  specimens,  stain 
with  neutral  red.  in  the  same  way  as  with  1'aramecia;  determine 
whether  an  acid  is  present,  and  if  it  disappears  before  the  waste 
matter  is  discharged. 

c.  Defaccation.      This    is   not    easily   observed.      !);>    y>u    know 
how  it  occurs? 

d.  E.vcretion.      Contractile   vacuole,   as   in    Paramecium.      With 
many   favorable  examples  the  external  discharge  may  be  observed 
by  the  use  of  India  ink,  as  in  Paramecium. 

e.  The   following  questions  you  may  not  be  able  to  answer  by 
direct  observation,  but  you   should  be  able  to  answer  them   from 
what  you  have  seen  or  read:     Is  there  any  special  method  of  dis- 
tributing the  food  within  the  body?     How  does  it  probably  occur? 
How   is  oxygen   probably   taken?     Carbon   dioxide  given   oft?     Is 
there  any  way  of  producing  a  current  of  water,  to  bring  food  and 
oxygen,  and  carry  away  waste  matter? 

C.  HYDRA  OR  A  SEA  ANEMONE. 

252.  a.  Study  and  describe  the  taking  cf  food,  by  feeding 
Hydra  a  small  animal  or  a  bit  of  meat.  Keep  the  specimen  in  a 
vessel  with  a  bit  of  plant,  to  furnish  oxygen,  and  determine  if 
you  can  about  the  length  of  time  taken  for  digestion. 

1).  Does  Hydra  exercise  any  choice  of  food  ?  Try  feeding  pieces 
of  plant,  or  bits  of  paper.  Are  they  taken  ? 

c.  After  a  Hydra  has  been  well  fed,  will  it  take  more  food,  or 
do  hungry  Hydras  act  differently  from  well  fed  ones  in  this  respect  ? 

d.  If  you  have  opportunity,  observe  the  egestion  of  the  waste 
matter.    How  does  it  take  place?    Make  a  diagram  of  the  alimentary 
canal  in  Hydra.     Is  there  any  way  of  distributing  the  food?     Ex- 
amine the  cavity  within  the  tentacles   with  high  power ;  .are  there 
any  internal  currents? 

e.  Are  there  any  external  currents,  for  bringing  food  and  oxygen, 
or  carrying  away  waste  matter? 

f.  How  do  respiration  and  excretion  probably  occur?    Are  there 
any  special  respiratory  or  excretory  organs?     Determine  whether 
oxygen  is  required,  by  leaving  Hydra  in  a  vessel  of  boiled  water 
and   keeping   it   tightly   closed.      Does   it   live   as    long  as   in    other 
water  ? 

D.  ANNELIDS. 

Study  the  processes  and  organs  of  metabolism,  so  far  as  it  is 
possible,  in  two  of  the  lower  annelids,  as  follows : 


_'5.v  .  Iciosonni.  a.  <  thservc  the  straight  alimentary  canal,  with 
an  opening  at  each  c-nd.  Distinguish  mouth,  -h<  >rt  muscular  pharynx, 
narrow  oesophagus,  wide  stomach,  and  narrow  intestine,  and  the 
ami-.  If  necessar)  stupefy  the  animal-  partly  hy  placing  them 
for  a  time  in  a  dish  containing  a  \\eak  solution  i  alxmt  '  j  saturated  i 
<>f  chloretone  i  u-e  a  weaker  solution  if  this  injures  them.  I  Xotice 
vlie  ciliary  movement  within  the  alimentary  canal;  in  what  parts  is 
it  found:  Are  there  any  peristaltic  movements?  Xotice  especially 
the  large  glandular  cells  which  make  up  the  walls  of  the  stomach. 
Are  there  any  separate  glands  connected  with  the  alimentary  canal? 

h.  Is  there  any  method  of  causing  an  external  current  in  the 
water  for  hringing  oxygen  and  carrying  away  carhon  dioxide?  Test 
with  India  ink.  Mow  are  the  currents  produced,  and  where  are  tluv 
f.  und  ?  Make  a  figure  of  the  animal,  showing  the  alimentary  canal, 
and  indicating  hy  arrows  the  external  currents. 

Are  there  any  indications  of  internal  currents.  i.e..  of  a  circula- 
ti  >ry  system  ? 

c.  In  Aelosoma  we  mid  definite  excretory  organs,  of  a  very  simple 
character.  Thev  c  n>ist  of  small  coiled  tuhes,  which  are  found  in 
pairs  at  intervals  attached  to  the  hody  wall  helow  the  alimentary 
canal.  Study  these  carefully.  Thev  can  hest  he  seen  when  the 
animal  is  placed  with  ventral  side  up;  and  in  a  region  where  the 
alimentary  canal  is  not  too  wide  (oesophagus  or  intestine). 
Xoiice  that  one  end  of  the  tuhe  is  open  and  hears  c'lia.  which  can 
he  seen  in  lively  movement;  this  end  is  called  the  funnel.  Are 
there  cilia  elsewhere  in  the  tube?  The  opposite  end  of  the  tuhe  i- 
attached  t  the  hody  wall,  and  opens  to  the  outside.  The  whole 
organ  is  a  nephridium.  Make  as  complete  a  drawing  of  the  nephri- 
dium.  on  a  large  scale,  as  you  can  and  indicate  the  ciliary  m  .vement. 
The  nephridium  takes  up  waste  siihstances  from  the  rluid  ot  the 
hody  cavitv.  through  its  walls.  These  are  washed  out  hy  the  ciliary 
.•'.ction,  a  certain  amount  of  fluid  which  aids  the  \\ashing  out  heing 
introduced  into  the  tuhe  through  the  opening  of  the  tunnel. 

_'5).  Dero.  a.  This  is  studied  particularly  for  its  yery  pecu- 
liar ri  spiratory  organs.  Xotice  however  the  simple  alimentary 
canal,  like  that  of  Aelos(,ina,  hut  perhaps  -till  simpler.  (  )li>erve 
also  that  there  i-  a  method  of  producing  internal  currents,  i.e..  a 

circulatory  system,  with  reddish  hi 1.      I  low  is  this  hlood  caused  to 

m<  >ve  ? 

h.  Arc-  there  any  external  current-:  ilVtcrmine  with  India 
ink.  )  (  (hservc  that  the-e  are  at  the  posterior  end  in  this  case 
(  compare  with  Aelosr  ,ma  i .  Studv  carelully  the  larijc  p"-terior 


extension.  Notice  the  linger-like  extensible  and  retractile  poim>, 
covered  with  a  sort  of  hood.  Ho\v  many  of  the  points  are  there? 
Are  they  ciliated?  Observe  if  possible  that  the  whole  structure  is 
Mipplied  with  much  blood,  which  circulates  through  it.  In  some 
species  the  points  are  much  longer,  extending  out  like  ringers. 
What  is  the  function  of  this  structure?  \Yliy  is  it  ciliated?  Why 
is  it  so  well  supplied  with  blood?  Draw  the  structure  carefully. 
c.  Xephridia  are  present  in  Dero,  but  are  not  favorable  for 
.^ttldy. 

255.  Study,  if  opportunity  is  given,  the  living  nephridium  of  the 
earthworm.     Observe  especially  the  ciliary  movements  in  the  funnel. 
Distinguish   funnel,  secretory  portion,  and   reservoir.     Observe  the 
ciliary  movement  within  the  secretory  portion.     Observe  also,  if  it 
is  possible,  the  blood  vessels  covering  the  nephridium.     Examine  the 
figures  in  Beddard's  Monograph  of  the  Oligochaeta   (or  copies  of 
these )  ;  note  in  these  the  blood  vessels. 

E.  CRUSTACEA. 

256.  Study  the  respiratory  and  circulatory  processes  in  Daphnia 
Where    does    respiration   occur?      How   is    the    respiratory   current 
produced?     (Use  India  ink  if  necessary.)     Observe  the  beating  of 
the  heart,  and  the  circulation  of  the  blood.     See  the  movements  of 
the  blood  corpuscles  in  the  head,  appendages,  etc.     By  figure,  dia- 
gram, or  description,   indicate   the   external    (respiratory),   and   in- 
ternal  (circulatory)   currents.     What  uses  do  these  currents  serve? 

Can   you   see   the   alimentary   canal   in    Daphnia?      If    so,    sketch 
or  describe. 

257.  Study,  if  opportunity  is  given,  the  respiratory  current,  by 
means  of   India  ink,  in  a  crayfish  or  crab.     Where  does  it  occur. 
and  how  is  it  produced?     Examine  in  a  preparation  the  structures 
producing  it.     Examine  also  the  gills.     Observe  that  they  are  simply 
expansions  of  the  body  wall,  that  have  taken  a  complicated  form. 
Compare  them  with  a  figure  on  the  chart,   showing  their   relation 
to  the  blood  vessels.    Draw  one  of  the  gills. 

2^8.     Examine,  and  describe,  if  there  is  opportunity,  the  respira- 
tory organs  and  respiratory  movements  in  Limulus. 

F.  IXSI-:CT. 

260.  Examine  and  draw  the  respiratory  organs  of  an  insect  larva. 
In  what  fundamental  way  do  they  differ  from  the  other  respiratory 
organs  we  have  studied  ? 

G.  FROG. 

261.  Examine  the  alimentary  canal  of  the  frog,  and  identify  the 

79 


different  part-*.  Measure  its  total  length,  and  compare  with  the 
length  i  it  the  hody.  Kxamine  the  alimentary  canal  of  a  tadpole. 
What  is  it-  length,  mea-nred  in  unit-  of  length  nf  the  hody  (with- 
out the  tail)?  How  docs  it  compare  in  relative  length  with  that 
of  the  fr  g?  \Vhy  the  difference  ? 

l-'.\amine  also  the  lungs  of  the  frog.  Inflate  them.  Note  their 
-ac-like  character,  and  the  internal  ridge-,  which  contain  hlood 
\  essels. 

li.     CIRCULATORY  SYSTEM 

I.     PHYSK  >L<  ><;V  (  >F  Till-;  1IKART   BEAT. 

jo_>.  The  fi'oi/'s  heart.  I'ith  a  frog,  heing  careful  to  av<  id 
loss  ->f  hlood.  Plug  the  hrain  with  cotton  if  any  occurs.  \<  >w 
c\po>e  the  heart  hy  cutting  through  the  hody  wall  to  one  side  of 
the  middle  line  i  to  avoid  abdominal  vein  >,  turn  the  pectoral  girdle 
to  one  side  and  remove  if  necessary.  Note  the  pericardium  sur- 
rounding the  heart.  Carefullv  cut  through  the  pericardium  expos- 
ing tlie  heart.  X<  >te  a  slender  hand  of  connective  tissue  running 
het ween  the  dorsal  surface  of  the  heart  and  the  pericardium — the 
fracnum.  Tie  a  thread  tightly  around  this;  then  divide  the  fraeiium 
d"i'sal  to  the  ligature.  C-e  the  thread  f  r  turning  the  heart  over 
or  otherwise  handling  it.  Study  carefully  the  anatomy  of  the 
organ;  draw  fn  m  dorsal  and  ventral  aspects.  Note  right  and  left 
auricle,  junction  of  three  venae  cavae  to  form  the  -inns  venn-u-. 
-ingle  truncu-  arterio^us,  hifurcation  of  the  latter  and  suhdivision  of 
each  hranch  into  three  arterial  trunk- — carotid,  aortic  and  pulmo- 
cutaneous.  He  sure  you  understand  the  anatomy  of  the  organ, 
nhserve  the  whitish  crescent  at  the  junction  of  -inn-  and  right 
auricle. 

a.  Study  carefully  the  -c<|Ucncc  of  the  heats  of  the  different 
chamhers.  Close  ohservation  is  needed  here.  Xote  any  change 
in  color,  si/.i-  and  form  of  the  ventricle  during  sv-tole  and  diastole 
respectively. 

h.  Count  the  numher  of  heats  per  minute.  \<>w  excise  the  heart, 
cutting  wide  of  the  -inu-  after  lifting  hy  the  ligature.  Place  the 
'  rgan  in  a  -mall  gla--  ve--cl  with  Ringer's  solution.  I  la-  e\ci-ion 
altered  the  rate  of  heat?  Kee|>  the  heart  for  use  in  experiment 
21  5  h. 

_''>}.  '/'//r  turtle's  heart.  l;.\pose  the  heart  of  a  turtle  without 
lo--  df  hlodd  and  familari/e  your-elf  \\ith  il-  -tructure  and  sei|iience 
of  heat  as  in  the  frog.  That  niav  he  done  as  follows:  Pith,  hv 


go 


making  a  transverse  slit  through  the  heavy  muscle^  on  the  hack 
of  the  neck.  Continue  the  slit  through  a  joint  between  the  verte- 
brae and  destroy  the  brain  with  a  wire.  I 'lug  the  cavity  with 
cotton.  Sever  the  union  between  plastron  and  carapace  at  the  sides 
with  bone  shears  and  cut  the  skin  and  muscles  as  near  the  plastron 
as  possible  so  as  to  remove  the  plastron  from  the  body.  ( )n  pulling 
the  forelimbs  straight  the  heart  will  be  seen  beating  and  by  a 
little  careful  dissection  can  be  freed  from  the  pericardium.  The 
latter  is  attached  to  the  tip  of  the  ventricle,  and  this  strand  should 
be  used  to  take  hold  of  in  handling  the  organ. 

264.  Conduction    of    impulse    in    the    heart.      In    the    frog    and 
turtle  the  impulse  originates  in  the  sinus  and  spreads  to  the  auricle 
and  ventricle;  in  the  mammal  the  impulse  starts  in  the  right  auricle 
near  the  venae  cavae  and   spreads   to  auricles  and  ventricles   and 
also  to  a  certain  distance  over  the  veins  opening  into  the  auricle. 
On    reaching    the    auricle-ventricular    junction    there    is    a    distinct 
pause  termed  the  auriculo-ventricular  interval ;  finally,  the  excita- 
tion reaches  the  ventricle,  and  the  contraction  wave  is  seen  to  tra- 
verse, the    ventricular    muscle.      The    auricuLo-veaitricular    interval 
may   be   lengthened  by  any   natural   or  artificial   hindrance   to   the 
excitation  wave. 

a.  Place  the  Gaskell  clamp  about  the  auriculo-ventricular  junc- 
tion.    Very  carefully  turn  the  screw  until  the  rubber  edge  makes 
a  gentle  pressure  on  the  cardiac  tissues  at  that  point.     With  care- 
ful work  a  degree  of  pressure  will  be  reached  that  diminishes  the 
conductivity  of  the  muscle  fibres  joining  the  auricle  and  ventricle 
so  far  as  to  allow  only  every  second  or  third  excitation  to  pass. 
The  auricle  will  beat  without  change  of  frequency,  but  the  ventricle 
will  beat  only  when  the  excitation  succeeds  in  passing  the  block. 

b.  Repeat  experiment  a,  but  place  the  screw  clamp  across  the 
middle  of  the  ventricle.     The  passage  of  the  excitation   from  one 
part  of  the  ventricle  to  another  will  be  delayed  or  interrupted  by 
the  lowering  of  the  conductivity  in  the  compressed  portion.     Many 
irregularities   in  the  frequency  and  force  of  the  heart  can  be  ex- 
plained by  variation  in  conductivity  of  its  several  parts. 

265.  Automaticity  of  different  chambers  of  the  heart,    a.     Care- 
ful  observation   is   required   to   detect   contractions   in   small   pieces 
of   the  heart.     Determine   the   rate   of   the   whole   heart.      Cut   off 
the  sinus  venosus.     Does  it  beat?     Rate?     Does  the  remainder  of 
the   heart   beat?     Rate?     Cut   the    sinus    into   small    pieces.      Rate 
of  each  piece?     It  is  best  to  tie  a  ligature   (of  Stannius)   between 

81 


sinus  and  auricle  while  the  heart  is  in  the  body  and  full  of 
-o  that  sinus  can  be  distinguished   fnnn  auricles  when  excised. 

Separate  (  I  )  the  two  auricles  fnnn  the  ventricle.  (  2)  the  auricles 
from  each  other,  (^)  the  tip  of  the  ventricle-  from  the  base,  and 
determine  in  each  ca-c  if  the  piece  of  heart  isolated  is  automatically 
rhythmic  and  its  rate.  In  which  region  is  the  beat  fastest?  Are 
the  now  automatically  beating  regions  independent  of  each  other  in 
rhythm. 

b.  Repeat  the  above  experiment  with  the  frog's  heart  Used  in 
experiment  262.  Does  the  conns  arteriosus  of  a  frog's  heart  beat 
when  i-olated? 

II.     IMIYSIOLOCY  (  )!•'  HEART  MUSCLE. 

jdo.  Graphic  record.  Use  the  frog's  heart  still  beating  in  the 
body.  I'ass  a  bent  pin.  to  which  has  been  fastened  a  fine  wire, 
through  the  tip  of  the  ventricle.  Fasten  the  wire  t«>  the  heart 
lever  by  wax,  and  adjust  the  lever  on  the  support  against  a  slow 
moving  drum.  Record  the  contractions. 

jfij.  Refractory  period  and  compensatory  pause.  Place  die 
signal  magnet  in  the  primary  circuit  of  the  inductorium  and  arrange 
the  latter  for  single  induced  shocks.  Attach  one  wire  from  the 
-ecoiidary  posts  of  the  inductorium  to  the  heart  lever  and  place 
another  about  the  auricles.  Record  the  normal  beat  of  the  heart 
on  a  slow  moving  drum,  and  stimulate  at  various  phases  of  the  beat 
with  make  or  break  shocks.  From  your  record  determine  at  what 
period  the  heart  is  non-irritable  (i.e.,  refractorv  toward  stimuli). 
.Vote  the  compensatory  pause.  At  what  pha-e  is  the  maximum 
extra-contraction  obtained?  Is  there  any  difference  in  the  latent 
I  eriod-  of  the  extra  contractions?  Trv  tetani/ing.  Result? 

jf'cS.  . ///  contractions  are  ina.viinal.  Inhibit  the  heart  by  a 
ligature  or  a  (  laskell  clamp  placed  at  the  auriculo-ventricular  junc- 
tion. Find  the  least  strength  of  stimulus  that  will  cause  the  ventricle 
to  contract.  Increase  the  -trength  of  the  -timnlu-,  but  do  not 
stimulate  oftencr  than  mice  in  ten  seconds  (to  avoid  the  stair-ca-c 
contractions  described  below).  Record  the  contractions.  1  )oes  the 
force  of  ventricular  contraction  remain  the  same,  notwithstanding 
the  increased  stimulus?  I  low  i-  this  expressed  in  words? 

Jin,.  Stair-case  effect  or  treppe.  Find  the  lea-t  stimuli!-  that 
will  cause  the  ventricle  t»  contract.  Repeat  tin-  minimal  Stimulus 
after  everv  relaxation,  recording  the  contractions  on  a  slow  moving 
drum.  I  low  dues  tin-  n  ult  agree  with  the  above  experiment? 

82 


270.  Inhibition    of   ventricle    by    constant    current.      X<>    record 
need  be  taken.      Place  an  indifferent  electrode  in  contact   with   the 
muscles  of  the  frog's  throat  or  other  indifferent  region  ;  the  other 
electrode  is  placed  in  contact  with  the  tip  of  the  ventricle  by  means 
of  a  thin  strip  of  cotton  soaked  in  7^   NaCl.    The  two  electrodes  are 
connected  through  a  key  and  pole-changer  with  the  battery.     With 
the  anode  in  contact  with  the  ventricle,   make  the  current.     Note 
the  change  in  appearance  of  the  ventricle  ;  explain.     Then  reverse 
the  current  and  break  just  before  the  beginning  of   systole.     The 
cathode  is  now  in  contact  with  the  ventricle.     Any  result  ?     At  what 
poles  does  the  inhibition  appear   (a)   at  make  and   <b)   at  break  of 
constant  current?     Compare  with  the  conditions  for  stimulation. 

271.  Stimulation    by   constant    current.      Bring   the   ventricle   to 
rest  by  a  ligature  at  the  auriculo-ventricular  junction.     Using  the 
same  arrangement  as  before,  determine  at  which  pole  stimulation 
appears  (a)  at  make,  and  (b)  at  break  of  constant  current. 

III.     INHIBITION  OF  HEART. 

272:  Rcfle.v  inhibition.  Etherize  a  frog  lightly  by  placing  under  a 
glass  jar  with  a  piece  of  ether  soaked  cotton.  Fasten  the  animal 
in  the  holder  ventral  surface  upward.  Apply  ether  at  intervals  if 
needed.  Expose  the  heart,  preventing  loss  of  blood.  Now  tap 
the  abdomen  rapidly  with  the  handle  of  a  scalpel,  noting  any  change 
in  the  number  of  beats  per  minute.  The  normal  rate  of  the  heart 
should  be  determined  before  tapping  begins.  Now  expose  the  intes- 
tine and  try  the  effect  of  direct  stimulation  of  the  intestine,  both 
mechanical  and  electrical.  What  is  the  effect  on  the  rate  of  heart 
beat?  If  the  above  method  of  reflex  inhibition  fails,  expose  the 
sciatic  nerve,  ligature,  and  stimulate  the  central  end.  Effect  on  the 
heart? 

273.  Situation    of   cardio-inhibitory    mechanism    in    central   ner- 
vous system.      (Cardio-inhibitory   center.)      Remove   carefully   the 
cerebrum  and  optic  lobes.     The  medulla  is  letft  intact.     Now  stimu- 
late the  intestine  as  before,  after  allowing  time  for  shock  to  pass 
off.    Result?    Stimulate  the  medulla  directly  by  platinum  electrodes. 
Effect  on  the  heart  beat?     Now   destroy   the   medulla   and   repeat 
reflex  stimulation.     Result? 

274.  Iiitra-cardiac    inhibitorv    mechanism.      Stimulate   the   heart 
directly   at   the   "white   crescent"   marking   sinn-auricular   junction. 
Result?     After  inhibition  note  carefully  the  manner  in   which   the 
beat  is  resumed. 

83 


_'75-  Inhibition  />v  direct  stimulation  of  'raijus.  Kxpose  the 
vagus  nerve  in  frog  as  follows:  1'a—  a  rather  wide  glass  tube  down 
the  oesophagus  i  to  put  the  tissues  on  the  stretch  ).  This  will  expose 
three  large  ner\  e-  at  the  side  of  the  neck;  these  are.  in  order  from 
above  down,  glosso-pharyngeal,  -I'lu/ns.  liv^oi/lossus.  Stimulate  the 
vagus  with  a  weak  tctani/.ing  current,  noting  the  effect  on  heart. 
If  no  ettect  is  shown  try  a  stronger  current,  <>r  try  tire  vagus  on  the 
oilier  sido  ot  the  body,  since  the  two  are  often  unequal  in  their 
action.  Xote  (  i  )  latent  ])eriod  of  inhibition.  (2)  duration  of  inhibi- 
tion. (  ^  i  manner  in  which  the  beats  are  resumed. 

Take  a  record  of  vagus  inhibition,  placing  a  signal  magnet  in  the 
circuit  to  record  the  moment  of  stimulation.  Then  connect  the  signal 
magnet  with  the  desk  binding  post  and  revolving  the  drum  at  the 
same  -peed  take  a  second's  time  curve. 

1 1  the  frog's  heart  is  weak  tise  a  turtle.  The  vagus  lies  in  the 
side  of  the  neck  and  may  be  exposed  bv  putting  the  tissues  mi  the 
stretch,  and  recognized  by  stimulation. 

To  make  sure  that  the  effect  is  not  reflex,  ligature  the  vagus  as 
near  the  central  end  as  possible,  cut  centrally  to  ligature,  lift  by 
ligature  and  stimulate.  Test  the  direct  irritability  of  the  heart  while 
in  a  state  of  inhibition.  Does  it  respond  readily? 

IV.     EFFECT  OF  VARIOUS  FACTORS  ON  Till-:  CHARAC- 
TER  <  )!•'  THE   RHYTHM. 

2~(i.  (ini[>luc  record  of  the  influence  of  temperature  on  the  rate 
of  heart  heat.  Kxpose  the  heart  of  a  frog.  Pass  a  small  hook  at- 
tached to  a  thread  through  the  tip  of  the  ventricle.  Then  excise  the 
whole  heart,  cutting  widely  around  it.  and  pin  the  tissues  surrounding 
its  base  to  a  small  cork  plate,  hasten  the  plate  to  a  glass  rod  by  in- 
serting the  latter  into  a  hole  cut  with  a  cork  borer.  The  heart,  thus 
attached  to  the  rod.  may  then  be  immersed  in  anv  desired  solution, 
and  its  action  recorded  by  the  thread,  which  is  tied  to  the  end  of  the 
short  arm  of  the  heart  lever. 

The  heart  is  immersed  in  Kingcr's  solution  contained  in  a  glass  or 
beaker  supported  by  a  block,  as  in  the  experiments  with  voluntary 
muscle.  Take'  tracings  of  the  beat  on  a  slow  drum  with  the  heart 
surrounded  bv  Kinger's  solution  at  the  temperatures  5  .  15  .  25  . 
Proceed  thus;  Have  ready  a  glass  filled  with  the  cold  Kinger,  e.g.. 
5°;  bring  the  heart  into  the  solution  in  the  Usual  manner  (  bv  re- 
moving the  block,  bringing  the  solution  up  from  below,  and  then 
replacing  block).  Let  the  heart  make  a  tracing  at  tin's  tempera- 

> 


ture  for  two  or  three  minutes;  mark  the  minute  intervals  on  the 
drum  immediately  helow  the  writing  point.  Then  replace  the  solu- 
tion hy  Ringer  at  a  temperature  of  10°  higher  (i.e.,  15 '  ),  and  let 
the  heart  record  the  heats  at  this  temperature,  marking  the  minutes 
as  before.  After  two  or  three  minutes  replace  this  second  solution 
by  a  third  10°  warmer  than  the  second  (i.e.,  25°  ),  and  take  a  simi- 
lar record  at  this  temperature.  Count  the  number  of  beats  per 
minute  at  each  temperature.  What  is  the  relative  increase  of  rate 
between  5°  and  15°?  Between  15°  and  25°?  What  is  the  average 
''temperature  coefficient  of  acceleration"  for  a  rise  of  10°  ?  How 
does  this  compare  with  the  temperature  coefficient  of  chemical 
reactions  ? 

2/7.  Actions  of  salt-solutions  on  the  heart-beat.  Take  records 
of  the  following:  Bring  the  heart,  arranged  as  before,  from  Ringer's 
solution  ( at  room  temperature )  into  pure  m/8  XaCl ;  after  a 
minute  change  this  solution  for  fresh  to  remove  all  traces  of  K 
and  Ca.  Note  any  changes  in  the  rate  and  character  of  the  beat 
in  this  solution.  After  some  minutes  change  the  m/8  NaCl  for  a 
mixture  of  100  vols.  m/8  NaCl  -(-  2  vols.  m/8  CaCL.  Is  there 
any  change  in  the  beat?  After  a  few  minutes  return  to  pure  m/8 
NaCl  and  note  the  effect.  Then  transfer  to  a  mixture  of  100  vols. 
m/8  NaCl  -(-  2  vols.  m/8  KC1.  Leave  in  this  solution  for  some  five 
minutes;  note  any  differences  from  pure  m/8  NaCl.  Finally  return 
the  heart  to  Ringer's  solution.  What  is  the  importance  of  Ca  and  K 
for  the  heart? 

278.  Action  of  C02,  acid,  alkali,  alcohol,  and  KCN  on  heart. 
Using  the  same  arrangement  as  before,  test  the  action  of  the  fol- 
lowing solutions  on  the  frog's  heart.  Take  records. 

a.  Ringer's  solution  saturated  with  carbon-dioxide.     The  solu- 
tion should  be  drawn  from  the  Sparklet  siphon  bottle  shortly  be- 
fore using. 

Try  also  half-saturated  and  third-saturated  solutions.  Return 
the  heart  to  Ringer's  solution  soon  after  definite  effects  have 
appeared. 

b.  Ringer's  solution  plus  11/400  HC1. 

c.  Ringer's  solution  plus  11/400  NaOH. 

d.  Ringer's  solution  plus  11/400  KCN. 

e.  Ringer's  solution  plus  4  vol.  7f  etnyl  alcohol. 

In  all  cases  determine  if  the  effects  produced  are  reversed  by 
return  to  normal  Ringer's  solution. 

Effects  of  some   alkaloids   on   the   heart.      Pith   a    frog  or   turtle 


without  lo-s  of  blood  and  expo-e  the  heart.  Determine  which  vagus 
contain-  the  inhibitory  liber-. 

_'7<).  .Iction  of  nicotine.  Apply  nicotine  solution  i  o._"  ,  i  to  the 
ventricle.  After  a  few  minute-,  stimulate  the  trunk  i  f  the  vagus 
nerve.  Xo  curve  need  be  written.  I-  the  heart  inhibited?  Xo\v 
lift  the  heart  with  a  i;las-  rod,  and  stimulate  the  intra-cardiac  inhi- 
bitor) nerve-,  i.e..  at  simi-auricular  junction  or  white  crescent. 
l\e-ult?  Nicotine  parali/.e-  -onie  inhibitory  mechanism  between 
the  vagus  and  the  intra-cardiac  inhibitory  nerves.  I  hit  it  is  known 
that  nicotine  docs  not  paralyze  nerve  trunks.  Hence  it  is  probable 
that  the  cardiac  inhibitory  liber-  in  the  vagus  do  not  pass  to  the 
cardiac  mu-cle  directly  but  end  in  o  ntact  with  nerve  cells  which 
take  up  the  impulses,  and  transmit  them  through  their  proces-e-  to 
the  muscular  libers  of  the  heart. 

jSi).  Atropine.  \\'itli  a  clean  pipette  apply  a  few  drops  of  a 
solution  of  atropine  (o.$f<  i  to  the  heart.  After  a  few  moments  lift 
the  ventricle  and  stimulate  the  crescent.  Is  the  heart  inhibited: 
Atropine  paralyses  the  intra-cardiac  inhibitory  nerves. 

281.  M nsairinc.  With  a  line  pipette  put  upon  the  ventricle  a 
tew  drops  of  salt  solution  containing  a  trace  of  muscarine.  Kffect  ? 

jSj.  Antagonistic  action  of  muscarine  and  atropine.  With  a 
fresh  pipette  apply  a  little  salt  solution  containing  atropine  (0.5',  ). 
Result? 

/'.     rRl-SSi'kl-    AND    VELOCITY    CONDITIONS   IX   Till-'. 
CIRCULATION. 

283.  Circulation  in  the  <\v/>  of  the  fi'oi/'s  foot.  Kthcri/e  lightlv 
a  trog  and  adju-t  on  the  t  rog  board  with  the  web  between  the  toes 
Stretched  over  the  hole  in  the  board.  Study  the  circulation  under 
the  microscope  and  draw.  (  >b-crvc  the  following  pi  tints: 

a.  Veins,  arteries,   capillaries      I  b>\\-  can  yon   distinguish   them? 

b.  \\hich    pulsate   and    in    which    i-    the    vclocitv     fa-test    and    in 
which  -1'  \\c-t  ?     Why  ? 

c.  <  >b-ervc   under  the  high   power  movements   of    individual    red 
and    white    corpn-clc-.      Are    tbe-e    found    in    particular    regions    of 
the  vessel?     Why? 

d.  Watch    for   a    white    corpuscle    passing   through    the    wall    of 
the  capillarv. 

_'K|.  I 'lace  a  tiny  drop  of  glacial  acetic  acid  (from  the  point 
of  a  pini  on  the  web.  Note  the  effect  of  the  irritant,  change-  in 
-i/c  of  \e--cl-.  collection  ,,f  leucocyte-,  etc.  l^o  not  //<'/  acid  on  the 
lens  of  the  inicroscope. 


285.  Artificial  circulation  scheme.     E.xamine  the  scheme.  follow- 
ing the  description  given  in  the  Harvard  Apparatus  Company  Cata- 
logue,  and  make  a  diagram  labelling  the  features  presented.      Fill 
with   distilled  water,  tipping  the  tube  so  as  to  allow  escape  of  air 
through   the  arterial   path,   and   attach   the   manometers   filled    with 
Hg.  connected  with  the  tubes  of  the  scheme  by  water.     Note  espe- 
cially the  following:    Effect  of  (  i  )  increasing  the  rate  of  heart  beat 
on    (a)    arterial  pressure,    (b)    venous  pressure,    (c)    character   of 
flow.      (2)    Pulse    in    the    aorta.       (3)    Action    of    the    mitral    and 
aortic  valves. 

286.  Graphic    record    of   blood   pressures.      Ulood    pressures   of 
living  animals  may  be  estimated  and  recorded  by  various  methods. 
The    principles    involved    can    be    determined    from    records    given 
by  the  artificial  circulation   scheme.     Place  a  writing  lever   in   the 
arterial  manometer  from  which  the  thistle  tube  has  been  removed, 
and    adjust    against    a    drum.       (See    demonstration.)       Take    a 
record   showing  the   effect  of    (  I  )    a   rapidly   beating  heart   on   the 
blood  pressure,  (a)  with  high  and  (b)  with  low  capillary  resistance; 
(2) -a   slow   beating  heart   with    (a)    high    and    (b)    low    capillary 
resistance.     A  base  line  should  be  drawn  showing  the  point  of  zero 
blood  pressure,  and  a  time  curve  (in  seconds)  taken  from  the  desk 
binding  posts. 

287.  Pulse  record.     Sf>h\gmograph  tambour.     Examine  the  in- 
strument, and  set  up  as  in  the  demonstration,  cementing  the  alu- 
minum angle  to  the  rubber  membrane  and  using  a  straw   for  the 
writing  lever.     Draw. 

Cover  a  small  thistle  tube  with  rubber  membrane  on  which  has 
been  cennented  a  bone  button.  Connect  the  tambour  by  rubber 
tubing  with  a  side  branch  and  clamp.  Place  the  thistle  tube  over 
the  "aorta"  of  the  circulation  scheme  and  take  a  record  of  its  pulse 
with  (  i  )  slow  heart  with  ( a  )  large  and  ( b )  small  capillary  resis- 
tance. (2)  Rapid  heart  with  (a)  small  and  (b)  large  capillary 
resistance.  A  time  curve  (seconds)  should  be  recorded  at  the  same 
time. 

288.  Human  pnlsc.     Place  the  thistle  tube  without  the  button 
over  the  carotid  artery  just  below  the  angle  of  the  jaw,  having  the 
side  branch  of  the  connecting  tube  open.     Adjust  against  a  slow 
moving    drum.      Now    close    the    side    and    record.      If    no    pulse 
shows,   adjust   the   thistle   tube   until   the   correct   spot    is   obtained. 
Compare  the  curve  with  that  obtained   from   the  artificial   circula- 
tion scheme. 


87 


C.    RESPIRATION 

I.  RESPIRATK  ».\   KV  LUNGS. 

289.  l\'cspinitiou  scheme.  Study  the  mechanics  <>f  mammalian 
ie-piratioii  in  the  artificial  respiration  scheme,  following  the  descrip- 
tion given  in  the  Harvard  Apparatus  Company  C'atalogue,  p.  74. 
The  manometer-  -hoiild  he  tilled  with  distilled  water  and  the  prc-- 
sure  condition  >hi  nld  he  such  that  the  lung  is  alwav-  -lightlv 
stretched  even  in  expiration.  This  i>  done  hy  closing  the  cavity  in 
the  ]>leural  tuhe  with  the  water  level  near  the  lung. 

Xote  especially  the   following: 

a.  I're-siire  relations  in  thorax  and  lung  cavities  during  inspira- 
ti  n  (lowering  water  level)  wtih  tracheal  tuhe  open.  Same  dur- 
ing expiration. 

1).  Same  as  ahove  hut  with  tracheal  tuhe  partially  closed.  l\ai-e 
and  lower  the  water  level  rapidly  and  note  the  effect  on  intrapul- 
monic  pressure.  C"lo>e  the  tracheal  tuhe  more  and  more  and  note  the 
effect^  on  intrapulmonic  pressure. 

c.  During    ins])iration    o]ieii    the    picural    tuhe.      Xote    the    effect 
on    intrathoracic    pressure.      This    is    what    happens    when    a    hullet 
enters  the  chest. 

d.  Coughing  or  sneezing  and   hiccough   can   he   imitated   in   the 
artificial  scheme.     1  )o  vou  -ec  how? 

II.  <>xii)ATI()X  [N  THE  TISSUES. 

-''/I.  (iascs  i/ircn  off  iii  respiration.  Fxpire  through  a  gla-- 
tuhe  and  I '.a  (  <  >H  ),  solution  in  a  bottle.  \\"hat  does  the  precipitate 
indicate;-  To  make  sure  that  the  O  )_,  is  actually  increased  in  expired 
;,ir  arrange  another  bottle  with  the  same  amount  of  Ba(OH), 
solution  and  draw  air  through  it  by  inspiration  the  same  number  of 
times  as  in  the  previous  experiment  on  expired  air.  Which  bottle 
contain-  the  m«>-t  precipitate? 

Breathe  against  a  cold  plane  pane  of  glass.  What  gas  is  indi- 
cated? Is  this  an  oxidation  product? 

291.  I ittlcpcinlcncc  of  C<).  production  mid  ()._.  consumption. 
"Intramolecular  respiration."  ("arefullv  remo\-e  the  seed-coats  from 

'•  pra-  that  have  been  snaked  in  water  over  night.  Fill  a  small  vial 
with  mercury ,  and  invert  in  a  small  vessel  containing  mercury,  taking 
care  to  admit  no  air.  Xow  place  the  peeled  peas  ,  me  b\  one  under 
the  rim  of  the  \  ial  s,,  that  they  tloat  to  the  top.  Let  stand  one  or 
tuo  day-,  oh^i-rve  the  production  nf  gas.  Test  hv  introducing 

88 


a  little  strong  KOH  with  a  bent  pipette.  Since  no  free  (  )..  wa^ 
present  in  the  vial  what  must  we  conclude  the  origin  of  the  C<  >.. 
to  have  been?  This  phenomenon  is  a  f/cncral  characteristic  of 
metabolism. 

292.  Review  Pasteur's  Yeast  Experiment   (p.  27)   which  shows 
very     clearly     the     difference     between     aerobic     and     anaerobic 
respiration. 

293.  Organisms   can  oxidize   substances   through   oxidizing   en- 
zymes.    Review  the  experiments  on  oxidation  under  the  head  of 
enzyme   action    (p.   23).     Organisms   have  also  a   strong  reducing 
power  as  indicated  in  the  following  experiment : 

Cut  thin  slices  of  the  tissues  (muscle,  kidney,  sex  organs,  brain 
or  spinal  cord,  etc. )  of  a  frog  and  stain  in  salt  solution  plus  methy- 
lene  blue.  Kill  a  thin  piece  of  muscle  tissue  in  hot  water  to  serve 
as  a  control  and  stain  in  the  same  way.  Place  all  on  slides  with 
salt  solution  under  cover-glasses.  Does  the  blue  color  disappear 
after  a  time,  thus  indicating  reduction?  Note  if  the  color  is  local- 
ized in  any  region.  Lift  the  cover-glass  and  determine  the  effect 
enzyme  action  (p.  23).  Organisms  have  also  a  strong  reducing 
power  ? 

294.  Simultaneous  reducing  and  o.vidiziny  actions.    Inoculate  the 
beef-broth-dextrose   culture-medium    with   a   collection   of   bacteria 
from  sewage  and  place  in   a   fermentation   tube.     Allow  to   stand 
a  day  or  two   and  note  the   collection   of   gas.     Gas   formation   is 
due  to  Bacillus  coli. 

Fill  the  tube  with  io(/c  KOH  solution,  excluding  air,  place  your 
ringer  over  the  end  and  invert.  Any  absorption?  Allow  the  gas 
to  collect  in  the  upper  part  of  the  tube  and  estimate  the  per  cent 
absorbed.  Again  till  the  tube  with  water  to  exclude  air.  Allow 
the  gas  to  collect  at  the  open  end  of  the  tube,  covered  by  your 
finger.  Remove  your  finger  and  quickly  apply  a  match.  What 
gases  are  indicated  by  the  tests? 

295.  Effects  of  lack  of  o.vvi/cn  on  Paramecium.  The  Engelmann 
gas  chamber  is  used  in  this  experiment.  The  Paramecia  are  ex- 
amined in  a  hanging  drop  of  culture  fluid  on  the  under  surface 
of  a  cover-glass  placed  over  the  aperture  of  the  gas  chamber. 
Oxygen  is  removed  by  passing  a  current  of  hydrogen  through 
the  chamber.  The  gas  is  generated  from  zinc  and  dilute  H,SO4, 
and  is  passed  through  two  wash  bottles,  one  containing  KMnOt  solu- 
tion, the  other  2Oc/f  NaOH,  and  then  through  the  chamber;  from  the 
exit  tube  of  the  chamber  a  rubber  tube  opens  under  the  surface 


89 


(if   water  to   serve  a>   indicator  of  the   rate  of  gas   flow.      All   junc- 
tions must  he  rendered  gas-tight.  Using  paraffin  where  neee--ary. 

Vote  the  effects  of  lack  of  oxygen  (  1  )  on  the  activity  of  the 
ulia  and  contractile  vaciiok--  ic«.ni]>are  at  interval  with  the  "con- 
trol", i.e..  Paraniecia  in  normal  culture  fluid;  (  _'  i  on  the  cou-i>- 
tcncy  of  the  protoplasm  ;  1^1  on  the  absorption  of  water  1>y  the 


I  >etennine  the  degree  of  reversibility  of  the  effects.  After  the 
movement-  have  alnio>t  ceased.  expos^  the  drop  to  air  by  removing 
the  >liding  t  ]>  from  the  ga>  chamber.  Are  the  movements  renewed  ? 
Try  the  same  experiment  with  I'aramecia  that  have  entirely  ceased 
movement. 

j<)i>.  l-'ficcts  nf  curium  tiio.viilc  on  I'araniccinni.  (  ienerate  the 
gas  with  marble  and  dilute  IK'l.  Pass  through  two  wash  bottles 
with  water,  and  through  the  chamber  as  before. 

Study  the  effects  of  (.  '(  )..  as  abnve.  -Vote  carefully  any  differences 
from  the  IL  experiment.  Determine  the  reversibility  of  the  effects 
as  before. 

297.  liffcct.f  «f  lack  of  o.vyt/cn  and  carl'on  dio.vidc  on  the 
ciliatcil  epithelium  of  the  oyster  /////.  Mount  jiortions  of  gill  fila- 
ments in  sL-a-water  in  the  Kngelmann  gas  chamber  as  above,  and 
study  the  effects  produced  by  a  stream  of  hydrogen  and  of  O  ), 
respectively.  .Vote  carefully  all  differences  between  the  effects  of 
these  two  gas(.-~.  (  "oinpare  with  the  conditions  in  1'aramecium.