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.