An introduction to general biology online

LABORATORY STUDIES AND DEMONSTRATIONS. 213

If time presses, tlie detailed study of iiiicroscopieal sections
may be omitted, but a series of prepared sections shcnild be kept
on liand and a demonstration given.

The embryological development is too difficult to study, but
very instructive demonstrations may be given l>y those who have
had some experience. In the neighborhood of Philadelphia egg-
capsules may be found in great numbers in old manure-heajis,
in May and June. One end of the capsule should be slicc(| olf
with a very sharp scalpel and the contents drawn out, under
water, with a large-mouthed pipette. The mass may then be
mounted in water under a supported cover-glass and studied
with the microscope. The embryos may be preser\ed in
Perenyi's fluid, and either studied whole in the preserving fluid
or hardened in alcohol and cut into series of sections.

Chapters IX to XI. (The Common Brake.)

Except when the ground is frozen Pteris may be dug up and
brouii:ht into the laboratorv in a fresh state. Fronds mav be
cut and dried in midsummer and considerably freshened (by a
moment's immersion in warm water) when needed to be used (in
the oj^ening exercise) to illustrate the aerial portion of the plant.
Rhizomes may be obtained at convenience and kept in weak
alcohol (50^).

The Morphology of the Body. To illustrate this, one whole
and entire plant should, if possible, be at hand for examination.
The aerial and the underground portions may then be sketched
in their normal relations. Branches, roots, and old leaf-.<talks
should be pointed out, identified, and sketched.

The Anatomy of the Rhizome should first be made out \\\\\\
the naked eye. The lateral ridges will be detected by the class,
which should be asked to draw the cross-section as seen M'ith
the naked eye. For this preliminary work each student should
have a piece of rhizome two or three inches in length. (Care
should afterwards be taken that the drawing has been correctly
placed dorsoventrally.) A rough dissection with jack-knife or
large scalpel may next follow, with inferences as to the characters
of the several tissues found (as fibrous, pidpy, woody, etc.).

The Microscojy'iG Anatomy of the Bh home is interesting, and,



214 APPENDIX.

for the most part, easy, but demands mucli time. If time al-
lows, cross-sections of roots may be made and mounted in balsam.
They are readily cut in pith. Sections of the rhizome may be
made freehand with a razor or, better, with a microtome : but
the old stems are exceedingly hard and liable to injure the
knives.

The Frond or Leaf may be obtained in fruit in July and
August and preserved in alcohol. From it sections of leaflets
may easily be got by imbedding in pith. Epidermis is obtained
with some difticulty (by beginners) after scraping. Fresh fern-
leaves from hothouses answer the purpose as well, are easier to
get, and more attractive. Really good sections of fern-leaves are
not easy for beginners to make. They should be kept on hand.

Sporangia may be obtained in abundance from alcoholic
specimens of Pteris^ or upon hothouse ferns, even in midwinter.
Some of the many species of Pteris found in hot-houses answer
every purpose. The thin edge of a scalpel slipped under tlie.un-
ri^^e indusium removes the latter, and generally also long ranks of
sporangia in all stages of development. In some sporangia spores
may be found. Sporangia and spores are always readily got,
but care must be taken to select fruit-dots which are not too old
or too young.

Sprouting the Spores. To obtain good specimens of s]3rout-
ing spores and i^rothallm free yro?n dirt^ we can recommend the
following procedure : Fill several small flower -pots, which have
been thoroughly cleaned inside and out, with clean fine sand.
Sterilize the whole by baking in an oven or a hot-air sterilizer.
Set the pots into large (porcelain) dishes capable of holding water,
and keep the bottom of these dishes covered to the de]3th of one
inch with water; cover the pots completely ^\itli bell-glasses.
After twenty-four hours, or after the sand and the pots have be-
come thoroughly wet, inside and outside.^ dust thickly the sand
and the outsides of the pots T\dth spores (obtained from fern-
houses by shaking fertile fronds over white paper). Care must
be taken to get spores^ and not merely empty sporangia. After a
Aveek or longer (sometimes several weeks) a bit of the surface-
layer of sand is removed to a drop of water on a slide and exam-
ined for sprouting spores. These will often be found in various
stages of development. After a month or two prothallia will ap-



LABORATORY STUDIES AND DEMONSTRATIONS. 2] 5

pear on the outside of the pots; and as tliese are clean, tlicy may
be removed and examined (bottom side upwards) free of all
dirt.

Failing these, prothallia may almost always be found in fern-
houses on the tops or sides of the pots, and especially on the
moist earth under the benches. Care should be taken not to
confound prothallia with the lighter green and relatively coarse
liverwort {Lunularicb) often found in hotliouses.

The Sexual Organs "of Prothallia. AVith good clean speci-
mens these are easily found with a rather low power. Iliglier
powers are needed to make out details. If the archegonia and
and antheridia are young they are green ; if old, brown. On
young prothallia antheridia only are often found, and on very
old ones archegonia only.

Fertilization. This is not easy to observe, but the attempt
may be made by examining successively a number of very fresh
and vigorous prothallia in different stages. They must be
mounted carefully (not flooded with water), and spermatozoids
are generally more easily found swimming about after the speci-
men has been mounted a little while.

Embryology. Except in its general features, this is too dif-
ficult for the beginner. He may, however, obsei've the later
stages by studying old prothallia with the young fern just ap-
pearing, and young ferns with the old prothallia still adherent.

Chlorophyll and Starch. Vigorous prothallia afl:"ord excellent
examples of cells bearing chlorophyll-bodies in which starch is
easily detected. Some of the marginal cells should be examined
with the highest power, attention being given to the chloro-
phyll-bodies and their arrangement. In favorable cases one may
observe the opaque rod-like or oval grains inside the latter,
and prove by reagents that they are starch grains.

The student should also examine, at this point, tlie large
chromatophores of Nitella^ which may be obtained ]>y pressing
out a drop of the contents from an internodal cell, adding dilute
iodine solution, and examining with a high })ower. In favor-
able cases as many as a dozen starch grains, stained blue, may bo
found inside a single elliptical chlorophyll-body.



216 APPENDIX.

Chapter XII. (Amceba.)

Amoeba is one of the most capricious of animals, appearing
and disappearing with inexpHcable suddenness, and as a rule it
cannot be found at the time when needed , unless special prepara-
tions hav^e been made in advance. It is never safe to trust to
chance for a supply of material. It is equally unsafe to trust to
the methods usually prescribed. Amoebae may, however, often
be procured in abundance and with tolerable certainty as follows :
A month or six weeks beforehand collect considerable quantities
of water-plants (especially Nitella or Chara) from various pools
or slow ditches, with an abundance of sediment from the bottom.
It is important to select clear, quiet pools containing an abun-
dance of organic matter (such as desmids, diatoms, etc., in the
sediment) — not temporary rain-pools or such as are choked with
inorganic mud (dirt washed in by rain). The material thus pro-
cured should be distributed in numerous (10 to 20) open shallow
dishes (earthenware milk-pans) and allowed to stand about the
laboratory in various places — some exposed to the sun, others in
the shade. The contents of many, perhaps all, of the vessels
will undergo putrefactive changes and swarm with life — first with
bacteria, later with infusoria — and will then gradually become
clear again as in a hay-infusion. The sediment should now be
examined at intervals, and Amoebce are almost certain to appear,
sooner or later, in one or more of the vessels. Usually the small
A. radiosa appears first, but these should only be used if it is
found impossible to procured. Proteus., which is far larger, clearer,
and more interesting. Experience will show that particular
pools always yield a crop of Amcehm^ while others do not.
When once a productive source is found all trouble is ended.

If possible a sediment should be selected that swarms with
AmoebcB. It is very discouraging for students to pass most of
their time looking for the animals instead of at them. Large
cover-glasses should be used, and the material taken with a
pipette from the very surface of the sediment (not from its
deeper layers). When first mounted the animals are usually con-
tracted, and only become fully extended after a time. Outline
sketches should be made at stated intervals, the structure of the
protoplasm carefully studied, the pulse of the contractile



LABORATORY STUDIES AND DEMONSTRATIONS. 217

vacuole timed (vary by vanning temperature), and the effect of
tapping the cover-glass noted. It is practically useless to look
for iission, for encysted forms, or for the external opening of the
contractile vacuole; and the ingulhng of food or passing out
of waste matters is rarely seen. The formation of pseiid(»i)odia
should he carefully studied. After exanunhig the living aTiimals
they should be killed and stained with dilute iodine.

Arcella is almost always, and Diffliujia sometimes, found
Avith Arnceha. These forms may be examined for comparison.

It is desirable also to compare white l)lood-corpuscles, which
may be obtained either by pricking the finger or, better, from a
frog or newt. A drop of blood, received upon a slightly warmed
slide, should be covered and sealed with oil aroimd the edtre of
the cover-glass. The white corpuscles are at first rounded, l)ut
soon begin to show change of form. (Xo contractile vacuole, no
differentiation into ectoplasm and entoplasm, often no nucleus
visible.)

Chapter XII. (Infusoria.)

Parammcia are almost certain to ap2:>ear in the earlier stages
of the ATYioeha cultures, and in similar decomposing licpiids or
infusions, and to ensure having them a large number of vessels
and jars containing an excess of vegetable matter should be i)re-
23ared a month or more beforehand. Their successful study is
very easy if they are procured in "cery large miirLbers (the water
should be milky with them), otherwise it is practically im]")ossible.
Three slides of them should be pre2)ared and set aside for a short
time (under cover, preferably, in a moist chamber) to allow the
animals to become quiet. One slide should contain simply a
drop of the infusorial water ; a second the same, with the addi-
tion of a little powdered carmine; to the third add a (h*oj) or two
of an aqueous solution of chloral hydrate (made by droi)i)ing a
crvstal or two into a watch-o-lass of water). The tirst slide
should be studied iirst; and it will usually be fiamd that after a
time the animals crowd about the edges of the cover, often lying
nearly or quite still. If this is not the case, the sj)ecimens j)ara-
lyzed by chloral may be studied. The carmine sjiecimens will
show beautiful food-vacuoles tilled witli carmine; and bv careful
study the formation of the vacuoles may be observed.



218 APPENDIX.

The general strncture sliould be carefully studied, the con-
tractile vacuoles particularly examined (they are seen best in dying
specimens or in those paralyzed by chloral), and dividing or con-
jugating individuals looked for (they are often abimdant). The
only really difficult point is the nucleus, which cannot be well
seen in the living animal. It may be clearly seen by mounting
a drop, to which a little dihite iodine or 2^ acetic acid has been
added. The former shows the cilia well, the latter the tricho-
cysts. Osmic acid and corrosive sublimate also give good preser-
vation. The internal changes during fission and conjugation
must be studied in prepared specimens mounted in balsam. Such
preparations are often of great beauty and interest.

Vorticella must be sought for on duck-weed or other plants,
or on floating sticks, and the like. Zodthamnion^ Carchesium,
etc. , are liable to appear at any time in the aquaria. All these
forms are easily studied. Conjugation is very rarely seen, but
fission and motile forms are common. The macronucleus is
especially well shown in dead or dying specimens.



Chapter XI Y. (Pkotococcus.)

Protococcus {PleurocoGGus) is found in abundance on the
northerly side of old trees in many parts of the United States.
In case it cannot be obtained in any region it may be procured,
during 1895 and 1896, from Prof. Sedgwick, Institute of
Technology, Boston, Mass. , by mail. The laboratory- work with
it is too easy to require comment. See, however, Arthur,
Barnes & Coulter's "Plant Dissection" (Henry Holt & Co.,
ISTew York).

Chapter XY. (Yeast.)

Bakers', brewers', compressed, and dried yeast may be had
in the markets. Brewers' yeast is to be preferred, as com-
pressed yeast-cakes contain starch, bacteria, and other extraneous
matters. All of the kinds may be cultivated to good advantage
in wort (to be obtained at breweries) or in Pasteur's fluids. (See
Huxley and Martin, chapter on Yeast.) Wild yeasts may be



r



LABORATORY STUDIES AND DEMONSTRATIONS. 219

found by examining sweet cider niicroscoi)ically. For the fol-
lowing methods of demonstrating nuclei in yeast and ohtannn*
ascospores we are indebted to Mr. 8. C. Keith, .Ii-.

To Demonstrate Nuclei in Yeast. Any <;«)«jd activelv-irrowint''
yeast will answer, but a large (brewers') yeast is preferaljlc. .Mix
a little of the yeast with an equal amount of tap-water in a test-
tube and shake thoroughly. Add an equal volnme of llermanirs
fluid and shake again. As soon as the yeast has settled j)our off
the supernatant liquid and wash the yeast by decantation. Trans-
fer some of the cells to a slide, flx by drying, stain by Ih'ick'ii-
hain's iron-hsematoxylin method (see Centralhlatt fi'ir Barirr't-
ologie^ xiv. (1893), pp. 358-300), wash, dehydrate with alcuhul,
follow with cedar-oil, and mount in balsam. In successful speci-
mens the effect is very satisfactory. (See Fig. 96.)

A Simpler Method. To demonstrate nuclei in yeast more
quickly and very easily the following method may be used : Pxjil
(in a test-tube) for a moment an infusion of vey^y vigorous yeast
in water, place a drop of the boiled infusion on a slide, add a
drop of 'y<?/'y dilute "Dahlia" solution, cover, and after one or
two minutes examine with a high power. The nuclei in most of
the cells will be easily discoverable.

To Obtain Ascospores in Yeast. It has been usually recom-
mended to employ for this purpose blocks of plaster-of-Paris.
"We have found the following method more trustworthy :

The yeast to be used should be the " top" yeast used in ale-
breweries. It should also be actively growing and fresh. If
fresh yeast cannot be obtained, some may be revived by culti\a-
tion for 2-1: hours at 25° C. in wort, and a little of the thick sedi-
mentary portion may then be placed in a very thin layer on dry
filter-paper which has previously been sterilized by baking. The
filter-paper is then j^laced on a layer of cotton about \ incli in
thickness lying on a plate or saucer, the cotton having })revit»usly
been thoroughly wetted with cold sterilized tap-watiM-, The
whole is covered by a bell-glass and set in a ratiier warm place
(25° C). In the course of two or three days spores will be found
in many of the cells. The lower the tenq)erature the longer ia
the time required for spore formation. If "bottom" yeast is
used instead of "top" yeast a nnich longer time is required, and
the results are far more uncertain.



220 APPENDIX.



Chapter XYI. (Bacteria.)

For the study of Bacteria it is very desirable to have a largo
species, and for this purpose there is none better than Bacillus
megaterium, which may be obtained from almost any bacteriologi-
cal laboratory and grown in the bouillon used by bacteriologists.
During 1895 and 1896 it may be obtained from Boston (see
above). Tliis form is very large, and produces spores readily.
(See De Bary, " Lectures on Bacteria;" Sternberg, ''Bacteriol-
ogy;" Abbott, "Principles of Bacteriology; " etc.) The pro-
longed study of bacteria is not suited to beginners. Vinegar
bacteria may be seen in the mother- of -vinegar by pressing a bit
of it out under a cover slip and examining with a high power.
The jelly of mother-of- vinegar is a good example of zooglma.
The white scum which apj^ears on aquaria and infusions is of
the same general character (zooglma').



Chapter XYII. (A Hay Infusion.)

To make a successful hay infusion care should be taken to
nse water containing numerous and various organisms, and tliere-
fore distilled v^rater, spring-waters, and well-waters, are in general
to be avoided. Tap- water should also be avoided if it is derived
from springs or wells. The l)est water for the purpose is that
drawn from ponds, rivers, lakes, or other surface sources.
Clean ditch or pool water is excellent. The choice of hay is less
important, but it is well to avoid old hay and hay that is very
woody. The infusion should be warmed, but not lieated or
boiled. It may be kept in a beaker in diffuse daylight, e.g., in
a north window, the beaker being loosely covered.

mSTKU]\IENTS AND UTENSILS.*

The student should liave access to the following articles :
A compound microscope with tvro eyepieces and low and
high power objectives (i.e., about 1 in. and \ in., or objectives

* Most of the apparatus and reagents here mentioned may be obtained from
any first-class dealer in physical and microscopical apparatus, e.g., from The



LABORATORY STUDIES AND DEMONSTRATIONS. 221

A and D of Zeiss, or J and ^ incli of Bauscli and Lonib ; still
higher powers are desirable).

A simple dissecting microscope; a desirable f(jrm is an ordi-
nary watchmaker's lens provided with a support. An (>r(lin;iry
pocket-lens; glass slides (3 X lin.), cover-glasses, watch-crystals,
small gummed labels, needles with adjustable handles, camel' s-
hair brushes, blotting and lilter paper, a good razor, pipettes
(medicine-droppers), glass rods and tubes, glass or pon-elain
dishes for staining, etc., a set of small dissecting instruments
(small scalpel, forceps, and straight-pointed scissors), a section-
lifter, pieces of pith for section-cutting, thread, a shallcnv tin pan
lined with wax, long insect pins for pinning out dissected s])eci-
mens, drawing materials, and a note-book for sketches and other
records.

Each table should be furnished with a set of small reafjent-
bottles, a Bunsen burner, wash-bottle, test-tubes, beakers, and a
bell-glass for protection from dust. Thermometers, a balance,
microtome, drying oven, and a paraffin water-bath should also be
accessible.

REAGENTS AND TECHNICAL METHODS.*

Alcohol. — Since biological laboratories belonging to incorpo-
rated institutions obtain alcohol duty free, it should be UheniUy
supplied and freely used. Alcohol of 100°, i.e., "absolute"
alcohol, may be purchased in 1-pound bottles. "Squibb's"
absolute alcohol may be obtained of any druggist, f but ordinary
alcohol of 90-95^ answers nearly every purpose. ''Cologne
spirits," i.e., alcohol of about 94^, may be obtained from the
distillers at 60c., or thereabouts, per gallon. It may then be

Bausch & Loinb Optical Co., Rochester, N. Y.; the Fnmkliu Educational
Co., Hamilton Place, Boston; or Queen & Co., Chestnut Street, Phihulelphia.
Chemical and other apparatus may be obtained from Eimer & Amend, 205-211
Third Avenue, N. Y.

* Every laboratory should be supplied with some of the standard books upon
this subject, e.g., Strasburger's Botanisclie Practicinn, Jena; Whitniun'a
Methods of Research in Microscopical Anatomy and Embryology, Boston: Lee,
The Microtomist's Vade Mecum, last edition; Zimmerman's Botanical Micro-
technique (Humphrey), Holt, N. Y.

f See also Whitman, 1. c, p. 14.



222 APPENDIX.

diluted to 80^, 70^, 50^, etc., as needed. For tins purpose an
alcoliolinieter is very convenient.

Acetic Acid. — One or two parts glacial acetic acid to 100 parts
water.

Acetic Acid and Methyl-green. — This is valuable for staining
nuclei in vegetal tissues. Dissolve metlijl-green in one or two
per cent acetic acid until a rich, deep color is obtained.

Borax-carmine. — Add to a 4^ aqueous solution of borax 2-3^
carmine, and heat until the carmine dissolves. Add an equal
volume of 70^ alcohol, and filter after 24 hours. After staining
(6-12 hours, or more for large objects, a few minutes for sec-
tions) place the object in acidulated alcohol (100 c.c. 35^ alcohol,
3-4 droj)s hydrochloi'ic acid) and leave until the color turns from
dull to bright red (10-30 m.). Afterwards remove to 70^
alcohol.

Canada Balsam, Mounting in. — This invaluable substance may
be obtained in the crude condition, dried by prolonged heating,
and then dissolved in chloroform, benzole, or turpentine, for
use. The benzole solution is perhaps the best, and may be ob-
tained from most of the dealers. The principles of mounting in
balsam are veiy simple. It does not mix with water or alcohol,
but mixes freely with clove-oil, chloroform, benzole, etc. Ob-
jects are therefore generally treated, first with very strong alco-
hol, 95-100^, in order to remove the water ; then with clove-oil,
chloroform, or turpentine to remove the alcohol, and afterwards
mounted in a drop of balsam. This should usually be placed on
the cover-glass, which is thereujDon inverted over the object.
The balsam gradually sets and the preparations are permanently
preserved.

Carmine. — Carmine may be obtained as a powder, which
when rubbed up thoroughly with water in a mortar passes into a
state of very fine subdivision. . This proj)erty makes it available
for experiments with cilia, etc.

It is more often used in solution, as a staining agent. (See
Borax- carmine.)

Cellulose- test. — Saturate the object in iodine solution, wash in
water, and j^lace it in strong sulphuric acid j)repared by carefully
pouring 2 volumes of the concentrated acid into 1 volume of
water.



LABORATORY STUDIES AND DEMONSTRATIONS. 22^

Collodion and Clove-oil. — Used for fixiiiij: sections to the slide
in order to prevent the displacement of deUcate or isolated part&
in balsam-monnting. Mix one part of ether-coll()di<»n and tliree
parts of oil of cloves. In niountiiii^, varnish a sHde with the
mixture by means of a camel' s-hair brush, lay on tlie sections,
and place the slide for a few minutes on the water-hath (i.e.,
until tlie clove-oil evaporates). Transfer the slide trj a wide-
mouthed bottle of turpentine (to dissolve tlie ])arathn), remove it
and drain off the turpentine, place a drop of Canada balsam on
the middle of a cover-glass, and invert it over the object.

Dahlia. — Dissolve in water.

Eosin. — Dissolve in water until a bright-red solution is ob-
tained. It should be diluted when used.

Glycerine, dilute. — Two parts glycerine, one part distilled
water.

Haematoxylin (Delafield's). — Add 4 c.c. of saturated alcoholie
solution of hgematoxylin to 150 c.c. of strong aqueous solution of
anmionia-alum ; let the mixture stand a week or more in the
light, filter, and add 25 c.c. of glycerine and 25 c.c. of methyl
alcohol. The fluid improves greatly after standing some weeks
or months.

Haematoxylin (Kleinenberg^s). — To a saturated solution of cal-
cium chloride in 70^ alcohol add an excess of pure alum ; filter
after 24 hours and add 8 volumes of 70^ alcohol, filtering again
if necessarv. Add a saturated alcoholic solution of lueniatoxylin
until the liquid becomes purple- blue. The longer the liquid
stands before using, the better. It should be diluted for use
with the alum-calcium-chloride solution in 70,^^ alcohol.

Hermann's Fluid. — See Lee's Vade Mecum.

Iodine Solution. — Dissolve jiotassium iodide in a small quantity
of water, add metallic iodine until the mixture assumes a dark-
brown color, and then dilute to a dark-sherry color. The solu-
tion should be kept from the light.

Magenta (Aniline Red). — Dissolve in water.

Methyl Green. — Used in aqueous or alcoholic solution or

with acetic acid.