Charles Wesley Hargitt.

Outlines of general biology ; an introductory laboratory manual online

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if the position of the heart is determined the carapace and
heart may be pierced with the cannula of the syringe.

For preservation cut away a small piece of the carapace
on the dorsal side to permit the entrance of the preservative.
Alcohol is a better preservative than formalin since the acid
in the latter dissolves out some of the mineral matter of the
shell, and leaves an undesirable scum on the liquid and
about the animals.

Clam. The fresh water clam is much easier and better
for study than the salt water forms. Use the larger forms.
If the valves are forced open and a wedge of wood, or a
pebble, is placed between to prevent closing, the animals
may be thrown directly into formalin for preservation. They
may be kept alive for some time in a cool place with running
water.



152 GENERAL BIOLOGY

To harden for making macroscopic sections across the
body., .wedge -the valves open, place in .1 per cent chromic
acid for a day or so. Wash in water and preserve in alcohol.
When ready, for sectioning remove the valves carefully,
place the body of the clam on a piece of cork or wax and
with a razor or brain knife make sections from | to \ inch
thick.

Snail. If the edible snail (French or Roman snail) is used
it must be purchased from dealers.

Frog. Any of the common frogs will do, though they
should be as large as possible. For the study of living
specimens in the winter, material must be obtained from
dealers unless it has been collected earlier and kept alive
in a tank. . If the animals are placed in a tank and kept in
a cool place in running water, or the water frequently changed,
and there is a platform or float on which they may rest, it
is possible to keep them alive all winter. During cold
weather food is not needed. To preserve, anesthetize with
ether or chloroform and place in 5 per cent formalin. Al-
cohol is not so good as a preservative for these forms.

For demonstration of circulation in the web, narcotize
the frog with chloretone, place on a plate of glass and cover
body with damp cloth. Hold the toes spread apart with
bent pins held in place against the glass with masses of
putty or wax.

To inject the bloodvessels for the study of the arterial
system proceed as follows: Anesthetize the frogs with
chloroform or ether (the latter leaves them in somewhat
better condition), remove a piece of the pectoral girdle from
over the heart, and remove the pericardium. With the
scissors cut a slit in the ventricle, insert the cannula through
the ventricle into the truncus arteriosus, and with a steady,
gentle pressure fill the arteries with the injection mass



APPENDIX 153

(formula for injection mass on page 159). No ligature will
be needed since the valves of the truncus prevent the back-
ward flow of the mass. During the injection of the arteries
the blood has been forced into the veins, and because of
this it is possible to follow the veins fairly well without in-
jecting them. After the injection is complete, the blood
and excess injection mass should be w r ashed off, and the
frogs preserved in formalin.

For the study of the histology of the frog examine fresh
tissues in normal salt solution. For sections of the spinal
cord fix in 10 per cent formalin, embed in paraffin and make
rather thick sections. Borax carmine or hematoxylin are
good stains to show the general form and something of the
cells. For a sharp distinction of the white and gray sub-
stances Weigert's hematoxylin method for medullated
nerves gives beautiful results. (See directions in books on
technic. The mordant of copper acetate may be used after
the sections are on the slide.) For sections of the stomach
cut the organ into two parts, rinse in salt solution and fix
tw T enty-four hours in Zenker's fluid. Hematoxylin and
Congo-red are good stains to differentiate the tissues.

In the spring get spawn from ponds and place small masses
in jars for the study of the development. Water plants in
the jars will help to aerate the water. If the water at any
time appears turbid, change. The tadpoles may be kept
for some time after hatching if the aquaria are kept supplied
with algae or other water plants.

Preparation and Mounting of Slides.

To get the best results in mounting small objects or
sections, the animal or tissue must be properly prepared.
It must be killed quickly so that the constituents of the



154 GENERAL BIOLOGY

cells are fixed in a normal condition. Usually some harden-
ing of the tissue takes place during the fixation, but if not
the alcohol used for preservation will also accomplish this
purpose.

Killing and fixing reagents are numerous; some are good
for a special purpose only and others have a wider use.
Those mentioned among the reagents are good, easily used,
and satisfactory for any of the work demanded in a begin-
ning course.

After fixation the tissue must be washed to remove the
excess of the reagent, and then preserved in 80 per cent
alcohol until wanted for use. Since this procedure has a
bleaching effect on the tissues, dyes are used to stain the
object to make it more easily seen. If sections are desired
it is necessary to embed the tissue or organ in paraffin or
other medium to support the softer mass while the sections
are being made. Directions for this must be had from books
on histological technic.

For examination of living tissues normal salt solution is
the medium used; for fresh water animals, if alive, water
is used; preserved animals are examined in the solution
in which they are preserved. Glycerin is used if it is desired
to render the objects somewhat transparent. They may
be placed in the glycerin from water, alcohol, or formalin
directly.

If a permanent mount is desired the object is usually
first stained, then dehydrated by placing for about an hour
in alcohol of various grades (35 per cent, 50 per cent, 70
per cent, 80 per cent, 95 per cent). If absolute alcohol is
at hand they should be placed in this for an hour to com-
plete the dehydration. To remove the alcohol some fluid,
usually an oil, is used which will mix with the alcohol and
also with the balsam which is used to complete the mounting.



APPENDIX 155

Cedar-wood oil, clove oil, creosote, xylol and benzol are some
of the common fluids used for this purpose. Not only do
they remove the alcohol but they also render the object
transparent and are therefore called clearing agents. Cedar-
wood oil and xylol are the most commonly used of the oils
mentioned. When the object has become transparent it
is placed on a slide, the excess of oil allowed to run off, or
removed with filter paper, then a drop of Canada balsam
placed on the object and this is covered with a thin cover
glass which is allowed to gently settle on the object. When
the balsam has hardened the object is permanently fixed.
If -absolute alcohol is not at hand for the final dehydration,
place the object from 95 per cent alcohol directly into a
mixture of one-third beechwood creosote and two-thirds
xylol, or into the same proportions of xylol and melted car-
bolic acid crystals. Cedar oil and oil of cloves will also clear
directly from 95 per cent alcohol. After the object has
been cleared proceed as directed in the previous paragraph.
Before a slide is laid away it should be labeled as to the
animal, or part, or tissue; how fixed; how stained; date
and name of person making the preparation.

Reagents.

Acetic Acid. A solution of 0.2 per cent to 1 per cent
applied to fresh tissues will render the nuclei visible. %

Alcohol. This is the most used reagent in the laboratory
and should be kept on hand in abundance, made up in
various strengths. Alcohol is rather expensive if bought in
small amounts from a druggist or ordinary dealer, but it
can be purchased, for scientific purposes, with the revenue
tax remitted. The collector of internal revenue of the
district, or the Federal Treasury Department can give



156 GENERAL BIOLOGY

directions for its purchase. Denatured alcohol may be
used and is cheaper, and about as satisfactory, as the regular
alcohol. Grain or ethyl, and not wood or methyl alcohol,
should be used ; the latter is poisonous.

Commercial alcohol is ordinarily about 95 per cent in
strength. To make various grades from the commercial
product, fill a graduate with 95 per cent to the mark which
indicates the desired strength, and then add water up to
95 cc (e. g., to make 70 per cent from 95 per cent take 70
cc of alcohol and add water up to 95 cc) . It is well to have
alcohol of the following grades prepared: 35 per cent, 50
per cent, 70 per cent, 80' per cent, 95 per cent. These will
be used in the preparation of slides. For preservation 70
per cent or 80 per cent is used.

Acid Alcohol. Of 70 per cent alcohol take 99 cc and of
concentrated hydrochloric acid Ice. Used chiefly for
destaining tissues.

Anilin Dyes. For staining, these may be dissolved in
water, or in alcohol of any grade. They, are most com-
monly used in aqueous solutions. It is better to stain for
some time with a weak solution, than for a brief period
with a strong mixture. Some of the stains will be removed
by alcohol, and experience, or reference to some book on
technic, must show the best methods of use. The com-
mon dyes used for the staining of the cytoplasm are: eosin,
congo-red, acid fuchsin, light green, orange G.

Benedict's Solution. Used in the same way as Fehling's
solution for demonstrating the presence of grape sugar.
This solution is said not to deteriorate on long standing.

Sodium citrate 173 g.

Sodium carbonate 100 g.

Dissolve in 600 cc water, using heat. Filter and make up
to 850 cc with water. Dissolve 17.3 g. of copper sulphate



APPENDIX 157

in 100 cc water and make up to 150 cc with water. Add
the cupric sulphate solution to the carbonate-citrate solution
slowly, stirring. The mixed solution is ready for use.

Borax Carmine. Dissolve 4 g. borax in 100 cc water. Add
1 g. carmine and dissolve it with heat. Cool and add to
the solution 100 cc of 70 per cent alcohol. Filter after
twenty-four hours. A good stain for large objects and for
tissues in bulk. Use the stain twenty-four hours, and
differentiate with acid alcohol.

Bouin's Fluid.

Saturated aqueous picric acid 75 cc

Commercial formalin 25 "

Glacial acetic acid . 5 "

It is best to add the acetic acid just before using. Kill
the tissue four to twenty-four hours, wash in 50 per cent
alcohol, preserve in 80 per cent alcohol. This is one of the
best of the killing fluids for general use.

Chloretone. Make a. saturated solution in water. Used
for narcotizing animals. In some cases it may be sprayed
on top of the water which contains the animals. For nar-
cotizing frogs for demonstrating the circulation of the blood
in the web the following method has proved satisfactory:
With a pipette inject about 2 cc into the stomach through
the esophagus. If after twenty minutes the animal is not
quiet repeat the dose. The animal will remain quiet for
hours, but will be recovered within twenty-four hours.

Chloriodide of Zinc.

Chloride of zinc 30 . g.

Potassium iodide 5 . g.

Iodine 0.89g.

Dissolve the above ingredients in about 15 to 20 cc distilled
water. The solution does not keep long, and should be
kept in the dark. Used as a test for cellulose.



158 GENERAL BIOLOGY

Fehling's Solution.

A. Copper sulphate 34.65 g. dissolved in ... 500 cc of water

B. Sodium or potassium hydroxide .... 125 g.
Sodio-potassium tartrate (Rochelle salts) . 173 g.

Dissolve in 500 cc of water.

Keep solutions separate until ready to use. Just before
using mix equal parts of A and B. This solution is used to
test for the presence of grape sugar.

Formalin. Commercial formalin is a solution of formalde-
hyde gas in water (40 per cent). To use, this is considered
as absolute and a 5 or 10 per cent solution made (e. g., to
make 5 per cent formalin mix 5 cc of commercial formalin
and 95 cc of water). For practically all organisms, plant
and animal, used in the laboratory or in the museum 5 per-
cent formalin is an efficient preservative and for most things
as satisfactory as alcohol. If the tissues are very watery
the solution should be changed after twenty-four hours.

Glycerin. Use pure or diluted with equal parts of water
or alcohol. Useful as a clearing agent and as a temporary
mounting medium.

Hematoxylin (Delafield's). Hematoxylin crystals 1 g. di,s-
solved in 10 cc of strong alcohol. Add this slowly to 100 cc
of saturated aqueous solution of ammonium alum, stirring.
Expose this solution to the air for several weeks to ripen.
Filter and add 25 cc of glycerin and 25 cc of methyl
alcohol.

Hematoxylin is one of the most satisfactory stains for
tissues; it is a nuclear stain. To use, dilute the stain and
let it act until the tissue is dark and overstained. Destain
with acid alcohol or with very dilute aqueous hydrochloric
acid (1 per cent of acid or less, in water). The slides hold-
ing the sections must now be washed for at least five minutes
in running water, to remove the acid and restore the blue



APPENDIX 159

color. Now counterstain the sections, if desired, dehy-
drate, clear and mount.

Injection Mass. The following is one of the best masses
for injecting bloodvessels.

Dry corn starch 1 Ib.

Chloral hydrate (2 per cent) 600 cc

Alcohol (95 per cent) 150 "

Color 150 "

The chloral hydrate and alcohol of the above may be re-
placed by 750 cc of 5 per cent formalin; the resulting mass
appears to act as well as the one above. To make the
color take about 50 g. of dry insoluble color, such as chrome
yellow or vermilion, grind in a mortar with 50 cc of glycerin
and 50 cc of alcohol (95 per cent). Mix the color and the
liquid and slowly stir in the starch to make a homogeneous
mass. If the mass appears to be getting too thick use less
starch, if too thin add more starch. Before using the mass
should be strained through two thicknesses of cheesecloth,
to remove all particles which might clog the cannula or
artery. The injection mass does not spoil upon standing,
but must be well stirred before using.

Iodine Solution. Dissolve potassium iodide in distilled
water to saturation, then saturate this solution with metallic
iodine. To use dilute with several volumes of water. May
be used as a stain for protoplasm, but chiefly used for starch
test.

Lime (or Baryta) Water. Shake up a little quick lime
(or barium oxide for baryta water) in water and allow the
mixture to settle. Decant or filter the clear liquid. In the
presence of carbon dioxide this clear liquid will become
milky, or will show a white precipitate.

Lyons Blue. This is one of the anilin dyes. An alcoholic
solution should be made. This is used as a contrast stain
with borax carmine.



160 GENERAL BIOLOGY

Methyl Green. A strong aqueous solution (1 per .cent)
with 1 per cent of acetic acid. An excellent stain for fresh
tissues. Stains the nucleus only, stains rapidly and does
not overstain.

Methylene Blue. Pure methylene blue added to water to
make a light blue tint will stain parts of the living animals
contained in the water. Several hours are usually neces-
sary. Does not affect the animals, but the stain should
be pure for this purpose.

Normal Salt Solution. Dissolve 7 g. of common salt in
1000 cc of water. This solution is used as a medium for
the examination of fresh tissues. The concentration ap-
proaches that of the lymph, and tissues and cells in it are
not distorted as they would be in water.

Pasteur's Solution.



Ammonium tartrate .
Potassium phosphate
Calcium phosphate .
Magnesium sulphate
Cane sugar
Water to make



50 g.

10 g.

lg.

1 g.

750 g.

5000 cc



This solution is used as a culture medium for yeast, moulds
and bacteria. The Pasteur's solution without sugar is the
same wdth the sugar omitted.

Worcester's Fluid. This fluid is a saturated solution of
corrosive sublimate (mercuric chloride) in 10 per cent
formalin. For small animals and small pieces of tissue kill
for fifteen minutes to an hour. Wash in water, preserve in
formalin or alcohol. Especially good for killing Paramecium
and Protozoa. If used hot Protozoa and hydra are killed
instantly.

Zenker's Fluid.



Corrosive sublimate (mercuric chloride) . , 5 g

Potassium bichromate ... 2 o'e

Water .'.".' l<X/cc

Just before using add glacial acetic acid ... 5 cc



APPENDIX 161

Fix for six to twenty-four hours, wash in running water for
twelve to twenty-four hours. Preserve in alcohol 80 per-
cent.

This is one of the standard killing mixtures, and for general
histological use it is excellent. It would be well to treat the
fixed tissue in alcohol with iodine for twenty-four hours to
remove all excess of the mercury salt. After this the iodine
must be thoroughly removed from the tissue by several
changes of alcohol.

Tests for Organic Substances.

Grape Sugar or Glucose. Into a test tube place the sub-
stance to be tested, add Benedict's or Fehling's solution and
boil. The appearance of a yellowish or red color indicates
the presence of the grape sugar.

Starch. If a substance containing starch is acted upon
by an iodine solution a blue color will result. The reaction
takes place more quickly if the substance has been boiled
and the starch grains swelled.

Cellulose. This substance is common in plant tissues, in
the fibers and cell walls; cotton is almost pure cellulose.
The section, or material, to be tested for cellulose is treated
with a drop or two of chloriodide of zinc. Cellulose is
colored violet, lignified membranes a yellowish brown,
membranes containing cutin or cork from yellow to yellow-
ish-brown.

Fat. Fats and oils are stained black by osmic acid. A
1 per cent solution acting on a thin piece of tissue will show
the reaction within a few minutes. Sudan III in aqueous
solution will stain fat or oil a yellowish color, and Scharlach
R will give a red color. These stains will also act upon fat
preserved in formalin. Ether or benzene may be mixed
11



162 GENERAL BIOLOGY

with the substance to be tested and allowed to remain for
a few minutes, then filtered and the filtrate evaporated in
a draft. The oil, if present, will be left behind in the dish.
Protein. Place the substance to be tested in a test tube,
add a few drops of strong nitric acid and boil. Cool and
carefully add a few drops of ammonia. A yellow color
appearing when the nitric acid is boiled and becoming a
deep orange on the addition of the ammonia is an indication
of the presence of the protein. This is one of the best and
most easily made tests for protein.



GLOSSARY



abdomen. In vertebrates that part of the trunk below or
behind the thorax; in invertebrates the posterior region
of the body.

aboral. Opposite the oral or mouth region.

achromatin. The material in the nucleus not colored by
certain dyes.

adaptation. The condition of a portion of the body such
that it is adjusted or fitted to its functions.

adductor. A muscle which draws some part toward the
middle line of the body.

alga. Simple plants, one or many celled; without definite
leaves, stem or roots.

alternation of generations. The alternation of sexual and
asexual forms in the life history of a plant or animal.

alveolar. Resembling little cells or sacs, a common appear-
ance of protoplasm.

amitosis. Simple or direct division of the nucleus, a mere
pinching into two parts. Opposed to mitosis or indirect
division.

analogous. Similarity of function.

anaphase. A stage in indirect division in which the chromo-
somes are pulled apart into two groups.

anastomosis. The interjoining or fusing of nerves or blood-
vessels.

animalcule. A microscopic animal.



164 GENERAL BIOLOGY

anterior. At or toward the front or head end.

anus. The posterior opening of the digestive tube.

aorta. In invertebrates the chief artery of the body; in

vertebrates the large artery supplying the main organs

of the body,
aortic arch. The arch or loop of the main arteries as they

leave the heart,
apopyle. In the sponge the opening of a radial canal into

the central cavity.

appendage. A subordinate part of an organ or body; es-
pecially an external organ or limb.

artery. A bloodvessel carrying blood away from the heart,
asexual. Non-sexual; reproduction by means other than

sexual, as by budding or fission,
aster. Star. The star-like structure found at the end of

the spindle during mitotic division of the nucleus,
asymmetry. Lack of symmetry, especially absence of bilateral

symmetry.

B

bacillus. A little staff. A rod-shaped bacterium.

barb. A hook or point extending backward, which pre-
vents the pulling out of the object.

bilateral symmetry. Symmetrical with respect to the right
and left sides.

biramous. Having two branches, as a typical crustacean
appendage.

bivalve. Shell composed of two lateral valves or pieces -as
the shell of the clam.

blastopore. The pore opening into the primitive gut of. a
developing animal.

blastostyle. That portion of the fleshy axis which pro-
duces medusae in certain hydroids.



GLOSSARY 165

blastula. A hollow sphere of cells produced by the develop-
ment of the egg.

body cavity. Coelom; the cavity between the digestive
tube and the body wall.

brachial. Pertaining to the arms.

branchial. Pertaining to the gills.

buccal groove. In infusoria the groove leading to the mouth.



caecum. A tube or sac open at one end only; usually applied
to appendages of the digestive tract.

calcareous. Composed of lime or calcium carbonate.

calciferous. Esophageal glands in the earthworm contain-
ing calcium carbonate.

capsule. A little case. In hydroids the cavity of a sting-
ing cell which contains the poison.

carapace. A shell or shield covering the head and thorax
of Crustacea.

cell. One of the structural units of a living body; a mass
of protoplasm containing a nucleus.

centrosome. The central body or region of an aster in
mitosis.

cephalothorax. The region of the body composed of the
fused head and thorax.

cerebral. Pertaining to the cerebrum or brain; in inverte-
brates cerebral ganglia or brain.

chela. The pincer-like claw of Crustacea and other arthro-
pods.

cbitin. The horny material forming the covering of insects
and certain other animals.

chlorogog. Gland cells surrounding the stomach-intestine
of the earthworm.



166 GENERAL BIOLOGY

chlorophyll. The green coloring matter of plants.

chloroplast. A chlorophyll body or granule.

chromatin. The ingredients of the nucleus which stain
deeply with certain dyes.

chromosome. A distinct body formed from the chromatin,
and present only at the time of nuclear division.

cilia. Minute, vibratory, protoplasmic processes on a cell.

circum-esophageal. About the esophagus. Refers to the
nerve connectives joining the ventral cord and cerebral
ganglia of invertebrates.

cleavage. Splitting or division of the egg cell at the be-
ginning of development.

clitellum. A thickened, glandular region of the earthworm
which secretes the egg case.

cloaca. A chamber into which empty the intestine, kidneys,
and reproductive organs.

coccus. A spherical bacterium.

ccelom. The body cavity; the space between digestive
tube and body wall.

coenosarc. The fleshy stalk or stem of hydroids, uniting the
various polyps.

colony. A group of animals living or growing together, the
various individuals being connected.

commissure. A band of nerves connecting ganglia in in-
vertebrates; tracts of nerve fibers within the central
nervous system of vertebrates.

conjugation. A temporary or permanent fusion of cells in
reproduction.

contractile vacuole. A pulsating vacuole in protozoa hav-
ing an excretory function.

corpuscle. A cell of an animal body floating in a fluid or
separated by an intercellular matrix.



GLOSSARY 167

crop. The pouch-like enlargement of the digestive tube
used to store food.

cuticle. The outer skin. In protozoa the cell wall.

cytology. The science of cell structure and function.

cytoplasm. The protoplasm of the cell proper, as con-
trasted with the nucleoplasm, or protoplasm of the
nucleus.

D

desmid. ) _

Y Minute unicellular algse.

digit. A terminal division of an appendage of vertebrates,

finger or toe.
distal. Away from the point of attachment, opposed to

proximal,
dorsal. The back or upper surface.

E

ectoderm. The outermost layer of cells.

ectoplasm. The outer, denser protoplasm in protozoa.

embryo. The young of an animal before its parts are fully
developed, and before the commencement of inde-
pendent existence.

embryology. The science of the development of animals
and plants.


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Online LibraryCharles Wesley HargittOutlines of general biology ; an introductory laboratory manual → online text (page 9 of 10)