An introduction to general biology online

change is found to have taken place. The liquid, originally
clear, has become cloudj, and a drop of it examined microscop-
ically wall be found to be swarming with bacteria. A day or
two later, the cloudiness meanwhile increasing, the microscope
generally reveals not only swarms of bacteria, but also numerous
infusoria. At the same time the color of the liquid has deep-
ened, it begins to appear turbid, a scum forms on the surface,
and the odor of hay, which was present at the outset, is replacec*
by the less agreeable odors of putrefaction. The simple ex-
periment of bringing together hay and water has, in fact, set in
motion a complicated series of physical, chemical, and biological
phenomena.

The Composition of a Hay Infusion. A hay infusion consist?
of two principal constituents, hay and water. But neither i)\
these is chemically pure. Hay is only dried grass which for
weeks, and even months, was exposed in the field to wind and
dust. Covered with the latter — often the pulverized mud of
roads and roadside pools — hay is richly laden with dried bacteria
and other micro-organisms; while water, such as is ordinai-ily
drawn from a tap, frequently contains not only an abundance of
free oxygen and various salts in solution, but also numerous bac-
teria, infusoria, algae, diatoms, and other micro-orgamsnis iu
suspension. In the making of a hay-infusion, therefore, numer-
ous factors co-operate, and a series of complicated reactions

follow one another in rapid succession. At the start both

201



202 A HAT INFUSION.

liaj and water are in i state of comparative rest or equilib-
rium, but upon bringing them together action and reaction
begin. First, the dust on the hay is wetted and soaked,
and any micro-organisms in it or adliering to the hay are set free,
and float in the water ; next, the water finds its way into the
stems and leaves of the hay, causing them to swell and resume
their orio;inal form. At the same time various soluble constitu-
ents of the daad grass, such as salts, sugars, and some nitrog-
enous substances, diffuse outward into the water, while from
such cells as have been crushed or broken open during drying
or handling, solid proteid or starchy substances may pass out and
mingle with the water. These simple physical reactions obvi-
ously involve a disturbance of the chemical equilibrium of the
water. Originally able to support only a limited amount of life
(such as exists in drinking-waters), it is now a soil enriched
by what it has gained from the hay. The bacteria, extremely
sensitive to variations in their environment, and especially to
their food-supply, immediately proceed to multiply enormously,
so that a biological reaction follows closely on the heels of the
chemical change. But as a result of their metabolic activity the
bacteria set up extensive chemical changes, which in their turn
involve physical disturbances. For example, the dissolved oxy-
gen with which the liquid was saturated soon disappears, so that
more oxygen must, therefore, diffuse into the liquid from the
atmosphere. Carbonic acid is generated in excess, and some
may pass outwards to the air. Also, as a result of the vital
activity of the micro-organisms the temperature of the infusion
may rise a fraction of a degree above that of the surround-
ing atmosphere.

We are concerned, however, chiefly with the biological
results. In consequence of the exhaustion of the oxygen supply
in the lower parts of the liquid, many of the bacteria which
require abundant oxygen for their growth {aerobes) find
their way to the surface, where some pass into a kind of
resting stage {zoogloea) and form a scum or skin {mycoclerm) on
the surface of the liquid. Others, for which free oxygen is not
necessary or to which it is even prejudicial {anaerobes)^ live and
thrive in the deeper parts of the beaker. But, meantime, an-



PHYSIOLOGICAL CYCLE IN THE INFUSION. 203

other plienoinenon lias occnrred. The infusijria, originallv few
in number, iiiiding the conditions favorable, liave niuhi[)liLMl
enormously, and after a day or two may be seen dartini^ in jind
out among the bacteria, especially near the surface, and feeding
upon them. Among the infusoria, liowever, are some wliich
feed upon their fellows, so that we soon have the herbivorous
infusoria pursued by carnivorous forms, the whole scene illus-
trating in one Held of the microscope that struggle for existence
which is one of tlie fundamental facts of biology.

Obviously, this chain of life is no stronger than its weakest
part. The hay is the source of the food-su])ply for all these
forms, and this supply must eventually become exhausted.
"When this happens, the bacteria cease to multiply, the herbivo-
rous infusoria which depend upon them perish or pass into a rest-
ing stage, the carnivorous infusoria likewise starve, and all the
biological phenomena must either come to an end or change
their character.

Up to this point the action is purely destructive. But sooner
or later microscopic green plants may appear on the scene, —
Protococcits^ it may be, or its allies, — and a constructive action
begin, the waste products of the animals and of the bacteria be-
ing rebuilt by the green plants into complex organic matter. 1 )y
this time, also, the dissolved organic matter will have been
largely extracted from the liquid, the l)acteria for the most
part devoured by the infusoria, and the latter may more or less
completely have given way to larger forms — to rhizopods, roti-
fers, small worms, and the like. The putrefying infusion has
run its course, and the ordinary balance of nature has been
restored.

Thenceforw^ard an approximate equilibrium is maintained.
The green plants build complex organic matter and store uj)
the energy of light. The animals feed upon the j)lants, or
upon one another, break down the complex matter, and dissi-
pate energy. The ever-present bacteria break down all the
refuse, extract soluble organic matter from the water, decom-
pose the dead bodies of the animals or plants, and in the end,
it may be, themselves fall victims to devouring infusoria. The
physiological cycle is complete.



204 A HAT INFUSION.

A liaj infusion tlius affords in miniature a picture of the liv-
ing world. The green plants are constructive, and in the sun-
light build up matters rich in potential energy. These as foods
support colorless plants (such as bacteria) or animals. On these,
again, herbivorous and carnivorous animals feed ; and so, in the
world at large, as in the hay infusion, omnivorous as well as
carnivorous animals, in the long run, feed upon herbivorous
animals, and the latter upon plants — either colorless or green —
which thus stand as the bulwark between animals and starvation.



APPENDIX.



SUGGESTIONS FOE LABOEATORY STUDIES AND

DEMOXSTEATIONS.

The "Laboratory Directions in General Biology," published
and copyrighted by Prof. E. A. Andrews of Jolms Hopkins
University, will be found extremely useful and ijractlcal. Also
the following : Huxley and Martin's "Practical Biology " (Howes
and Scott), and the accompanying ' ' Atlas of Biology, • ' by Howes ;
Marshall and Hurst's "Practical Zoolofirv," Colton's "Practical
Zoology," Bumpus's "Invertebrate Zoology," Dodge's "Ele-
mentarv Practical Bioloo-v," Brooks's "Handbook of Inverte-
brate Zoology." According to our experience, the periods for
the course should be so arranged as to afford laboratory work
and recitations or quizzes in about the proportions of three to
two (for examj)le, three periods of laboratory work and demon-
stration to two of quiz), for a half-year.

Chapter I. (Introductory.)

It is convenient to give at the outset one or more practical

lessons on the microscope, affording the student an opportunity to

learn its different parts, use its adjustments, test the magnifying

power of the various combinations, etc. A good object for a

first examination is a human hair, which serves as a convenient

standard of size for comparison with other things. Other good

objects are starches, the scales from a buttertly's wing (sketch

under different powers), a drop of milk or blood, and ]i(nv(h^rod

carmine or gamboge rubbed up in water (to show the Brownian

movement). The student should compare the same object as

seen under the simple and the compound microscope (to show

205



206 APPENDIX.

reversal of the image in the latter), and should during the course
learn the nse of the camera kicida (Abbe's camera, of Zeiss, tlie
best). The stage-micrometer may also be examined at this time
or later, and the student taught to prepare a scale (see Andrews)
by drawing the lines, with camera, on a card under different
p>owers (A + 2, D + 2, D + 4, of Zeiss), and labelhng each
with the names of lenses and actual size of the spaces, as stated
on the micrometer.

Pencil- drawing should begin as soon as the first sj^ecimen is
in focus, and sketches should be made, from the very first exercise
onward, of everything really studied. It is absolutely indis-
pensable to heej) a laboratory note-hooh^ which ought at any time
to give tangible evidence that the laboratory study is bearing
fruit ; and in the very first laboratory exercise a beginning should
be made in this direction.

The preliminary microscopy of one or two laboratory peri-
ods, corresponding to the time spent in conferences U2)on the first
chapter of the text-book, leads naturally up to the easy micro-
scopical studies required in connection with the second chapter.

ChAPTEK II. (STilUCTURE OF LiVING ORGANISMS.)

The laboratory work may be made very brief and simple,
and the facts shown largely by illustration. Tlie principal
organs of a j)lant and of a five or dissected animal may be shown
and some of the more ob^dous tissues pointed out. A frog under
a bell-glass, and a flowering plant (geranium) in blossom, j)laced
side by side on the demonstration-table will serve to suggest
materials for the lists of organs and the comparisons called for.

The skin of a Calla leaf is easily stripjDed off and demon-
strated to the naked eye as one form of tissue. It may then be
cut up and distributed for microscopic study and for j^roof that
it is composed of cells. (During this process air is apt to replace
water lost by evaporation, and must be displaced by alcohol,
which in turn must be removed by w^ater.)

For a first microscopical examination of tissue there is no
better object than the leaf of a moss (a species having thin broad
leaves should be chosen) or a fern prothalHum. Otlier good
objects are thm sections of a potato-tuber from just helow the



LABORATORY STUDIES AND DEMONSTRATIONS. 207

surface (stained witli dilute iodine to sliow nnolei and ^itarcli-
grains), and frou-'s or newt's l>lood, mixed with nuniial salt scJu-
tion, and examined either fresh or slightly stained with dilute
iodine.

Thin sections of pith (elder, etc.), from which the air lias
been displaced by alcohol, give good pictures of tissue c<>jn]>used
of empty cells. Fresh or alcoholic nniscle from the frog's leg,
gently teased out, shows muscular tissue to be composed of elon-
gated cells (iihres). Finally, the student may prove that he
Inmself is composed of cells by gently scraping the inside of his
lip or cheek with a scalpel, mounting the scrapings on a slide,
and after adding a drop of Delalield's h^ematoxylin, covering,
and examining in the usual way.

To show the lifeless matter in living tissue it suffices to ex-
amine frog's blood or human blood; sections of potatoes, es-
pecially if lightly stained wdth iodine ; sections of geranium stems.
{Pelargonium)^ which usually show crystals in some of the more
peripheral cells ; cartilage, stained with iodine, in which the life-
less matrix remains uncolored ; or prepared sections of bone, in
which the spaces once filled by the living cells are now black and
opaque, being filled with dust in the grinding, or with air.

Chapter III. (Protoplasm and the Cell.)

Naked-eye Examination of Protoplasm. A drop of ]u'oto-
plasm is readily obtained from one of the long (internodal) cells
of Nitella^ after removing the superfluous water and snipping off
one end of the cell with scissors. The cell collapses and the
drop forms at the lower (cut) end. It may be transferred to a
(dry) slide and tested for its viscidity by touching it with a
needle. Microscopically it is instructive chiefly by its lack of
marked structure.

The Parts of the Cell. The structure of the cell is beauti-
fully shown in properly stained and mounted preparations of un-
fertilized star-fish or sea-urchin eggs, or of apical buds of j^iUUa.
If these are not availal)le potato-cells or cartilage cells do very
well; or sections of epithelium, glands, etc., may be shown.

The class may also mount and draw frog's or newt's blood-
cells, prepared and double-stained as follows. The blood is spread



208 APPENDIX.

out evenly on a slide and dried cautiously over a flame. Stain
witli hsematoxylin for three minutes ; wash thoroughly with water,
add strong aqueous solution of eosin, allow to stand one minute ;
wash this time very rapidly, remove the excess of water quicMy
with filter-paper pressed down over the whole slide ; dry rapidly,
and examine with low power. If successful mount in balsam ; if
the specimen is not pink enough add more eosin and wash still
more rapidly than before. In good specimens the cells keep
tlieir form perfectly, the cytoplasm is briglit pink, and the nucleo-
plasm is light purple.

Epidermis from young leaves of hot-house lilies ('' African "
lily, ' ' Chinese ' ' lily, and especially lily-of -the-valley) yields
cells showing finely the cell-wall, nucleus, and (in favorable
cases) cytoplasm. If stained with acetic acid and methyl-green
the nuclei are highly colored; with Delafield's hsematoxylin the
cytoplasm is more easily seen.

Cell-divisions or Cleavage are easily observed in segmenting
ova or in fresh specimens of Protococcus (Pleurococcus) de-
taclied from moistened pieces of bark which bear these algge.
(See p. 178).

Stages in the cleavage of the ovum may be seen in the seg-
menting eggs of fresh-water snails {Pliysa^ Planorhis) which
are easily procured at almost any tune by keeping the animals in
aquaria. The old egg-masses should be removed so as to ensure
the eggs being fresh. Or a supply of preserved segmenting eggs
(star-fish, sea-urchin) may be kept for demonstrating the early
stages.

Protoplasm in Motion. The best introduction to j)rotoplasm
in motion is afforded by a su23erficial examination of Amoeba
(for procuring A?nceha see above. Chapter XII). If A^inoeba is
not available young living tips of Nitella or Chara may be used.
Anacharis and Tradescantia are useful, and often very beautiful,
l)ut less easy to manage, as a rule. In mounting Nitella or
Chara care must be taken not to crush the cells, and as far as
possible pale fresh specimens rather than darker and older ones
should be chosen. If Anacharis is to be studied the youngest
leaves should be selected from the budding ends, and not, as is
sometimes recommended, leaves which are becoming yellow.
The movement in the cells of Anacharis leaves often besrins



LABORATORY STUDIES AND DEMONSTRATIONS. 209

only after tlie leaf lias been mounted for a lialf-liour or more ;
but when once established affords one of the most beautiful and
striking examples of protoplasmic motion. If Tra(.U Heard at is to
be used, care must be taken to have, if possi])le, tiowers just open
or opening. The morning is therefore preferable for work uu
this plant. High powers are necessary.

In all these forms the movements may often be stinnilated l)y
placing a lamp near the microscope or l)y cautiously warming
the slide over the lamp-chhnney. Ciliary action is easily shown
in bits of the gills taken from fresh clams, mussels, or oysters, or
in cells scraped from the inside of the frog^s oesophagus. A
striking demonstration is easily given by slitting open a frog's
(or turtle's) oesophagus lengthwise, pinning out flat, moistening
with normal salt solution, and placing tiny bits of moistened cork
on the surface. The progressive movement of the cork-bits is
then very obvious. Muscular contractility is easily shown l)y
removing the skin from a frog's leg, dissecting out the sciatic
nerve, cutting its upper end, and then stinmlating the lower end,
if possible, by contact with a pair of electrodes, otherwise by
pinching it with forceps. If the necessary apparatus is available
the regular muscle-nerve preparation may be shown (see Foster
and Langley's "Practical Physiology'').

Food-stuffs Contain Energy. This may be shown (in dem-
onstrations) by sprinking finely ])owdered and ihorou^jhl y
dried starch, sugar, or flour upon a fire, or upon a platinum dish
or piece of foil heated to redness over a small flame. Oils and
dried and powdered albumen (proteid) may be similarly made to
burn with almost explosive violence if apj^lied in a state of fine
division in presence of air.

The Chemical Basis, {ci) Proteids\ Coagulation'^ Blijor Jfor-
tis\ Rigor Caloris. White-of-egg may be shown (in demonstra-
tion) and made to coagulate in a test-tube hung down into a
beaker of water under which is put a flame. A therm(Mnetcr in
the test-tube may be read off from time to time as the ex])eri-
ment advances, until finally coagulation begins, when the temper-
ature is noted. The death-stiffening [rigor rnortin) comes on
very quickly in frogs killed with chloroform. Ileat-stilfening
{rigor caloris) is well shown by immersing one leg of a deca])i-
tated frog in a beaker of water at 40° C. The other leg re-



210 APPENDIX.

mains normal and affords a vahiable means of comparison. It
is not wortli wliile to make many chemical tests of proteids at
this point.

(b) Carholiydr cites. A useful demonstration may be made
of various starches, sugars, and glycogen. The iodine-test may
be applied if desired. If time allows, the microscopical appear-
ance of potato-starch, corn-starch, Bermuda arrowroot, etc.,
may be dwelt upon in the laboratory-work. Cellulose is well
shown in filter-paper or absorbent cotton.

(g) Fats. A demonstration of animal fats and vegetable oils
may be made if time allows. They may be examined microscop-
ically in a drop of milk, in an artificial emulsion made by shak-
ing u}) sweet oil in dilute white-of-egg, or in fresh fatty tissue
{from subcutaneous tissue of mouse, or fat-bodies of frog). It is
hardly worth while to examine these substances chemically, but
â– a few simple tests may be applied if desired.

Dialysis. A demonstration of dialysis is easily made by in-
verting a broken test-tube, tying the membrane over the flaring
€nd, filling the tube to a marked point with strong salt or glu-
•cose solution, and immersing it in a beaker of distilled water.
After an hour or so the fluid will be found to have risen in the
test-tube ao^ainst oTavitv.

Temperature and Protoplasm. The profound influence of
temperature on protoplasm is well shown by the frog's heart.
Decapitate a frog and destroy tlie spinal cord. Expose the
heart and count tlie beats at the room temperature. Then pour
upon the heart iced normal salt solution. Again count the beats.
Next pour upou it normal salt solution heated to 35° C. The
number of beats will follow the fall and rise of tempeiTvture.

Chapters IY to YIII. (The Earthworm.)

Large earthioorms must he used or satisfactory results can-
not be expected. Pains should therefore be taken to procure
the large L. terrestris {not the common Allolohojpliora mucosa)^
which is readily recognizable by the flattened posterior end.
This species is not everywhere common ; hence a supply should
be procured and kept in a cool place in barrels half full of earth,
on the surface of which is placed a quantity of moss. They will



LABORATORY STUDIES AND DEMONSTRATIONS. 211

thus live for inoiitlis. Z. Urrestris mav be «jl)t;iinc»l in irreat
numbers between April and November, l)y .searoliiiii:; for tlieni
at niirht with a lantern in localities where imnierous castinors
show them to abound (a rather heavy but rich s(jil will be found
most productive). They will then be f(jund extended fr(^in their
burrows, lying on the surface of the ground, and may be seized
with the fingers. Considerable dexterity is needed, and it is
necessary to tread very softly or the worms take alarm and in-
stantly withdraw into their burrows.

For dissection fresh specimens are far preferable for most
purposes, though jyroperly preserved ones answer the purpose.
Fresh specimens should be nearly killed by being })laced for a
short time (about five minutes) in TO^ alcohol, and then stretched
out to their utmost extent in 50^ alcohol in a dissecting-pan,
the two ends being fastened by j^ins. They should then be at
once cut open along the middle dorsal line with scissors, the
flaps pinned out, and the dissection continued under the 50^
alcohol. (They must be comjpletely covered with the liquid.)
By this method the mimitest details of structure may l)e ob-
served, and many of the dissections should be done under a
watchmaker's lens.

For preservation (every detail of which should be attended
to) a number of living worms are placed in a broad vessel filled
to a depth of about an inch with water. A little alcohol is then
cautiously dropped on the surface of the water at intervals until
the worms are stupefied and become perfectly motionless and re-
laxed (this may require an hour or two). They are then trans-
ferred to a laro-e shallow vessel containinoj iust enouf^h 50^
alcohol to cover them, and are carefully straightened out and
arranged side by side. After an hour the weak alcohol is re-
placed by stronger (70^), which should be changed once or twice
at intervals of a few hours; they are finally placed in 9(>^
alcohol, wdiich should be liberally used. The trouble demanded
by this method will be fully repaid by the results. The W(^run:
should be quite straight, fully extended, and plump, and they
may be used either for dissection or for microsco2)ic study.

For the purposes of section-cutting worms should be carefully
washed and placed in a moist vessel containing plenty of wet
filter-paper torn into shreds. The worms will devour the paper,



212 APPENDIX.

which should be changed several times, until the paper is voided
perfectly clean. The worms are then preserved in the ordinary
way, and when properly hardened are cut into short pieces,
stained with borax-carmine, imbedded in paraffin, and cut into
sections with the microtome.

The living worms should first be observed — their shaj)e,
movements, behavior to stimuli, pulsation of the dorsal vessel
(time the pulse and vary the rate by temperature changes).
"Well-preserved specimens should then be carefully studied for
the external characters (draw through tlie fingers to feel the setse).
(Sketch.) Observe openings. The nephridial openings cannot be
seen, but if preserved worms be soaked some hours in water and
the cuticle peeled off tliey may be clearly seen in this. A
general dissection of a fresh specimen should now be made,
and the positions of the larger organs studied. (Make partial
sketch, to be filled out afterwards, as in Fig. 24.) The alimentary
canal and circulatory organs should now be carefully studied.
Even the smallest of the blood-vessels may easily be worked out
under the lens by using fresh specimens (killed in 70^ alcohol
and afterwards dissected under water) and carefully turning aside
the alimentary canal.

The alimentary canal should afterwards be cut through be-
hind the gizzard and gradually dissected away in front, exposing
the nerve-cord and the reproductive organs (wash away dirt with
a pipette). ISo great difficulty should be found in making out any
of the parts, excepting the testes. These are difficult to find in
mature worms, but may be found with ease in those which have
no median seminal vesicles (usually the case with specimens hav-
ing no clitellum).

The contents of the seminal receptacles and vesicles from a
fresh worm should be examined with the microscope. Kemove
an ovary (with forceps and small curved scissors), mount in water,
and study. (Stained in alum-carmine and mounted in balsam
the ovary is a beautiful object.) The student should also re-
move a fresh nephridial funnel and jDart of a nephridium, and
study with the microscope. (This may have to be shown by the
demonstrator, but should never be omitted, as the ciliary action
is one of the most striking things to see.) A careful dissection
of the anterior part of the nervous system should also be made.