QH 317. G 74 ed.3 LABORATORY DIRECTIONS IN GENERAL BIOLOGY BY EDWIN GRANT CONKLIN PROFESSOR OF BIOLOGY PRINCETON UNIVERSITY nj Oi I! £ si o m Third Edition CD m o • LABORATORY DIRECTIONS IN GENERAL BIOLOGY BY EDWIN GRANT CONKLIN PROFESSOR OF BIOLOGY PRINCETON UNIVERSITY Third Edition » INTRODUCTION v^ „ lt. A * * v^ THE BIOLOGICAL SCIENCES Read: Calkins, Biology, pp. 1-5; or Sedgwick and Wilson; General Biology, pp. 6-8; or Shull, Animal Biology, pp. 1-6; or Woodruff, Foundations of Biology, pp. 1-5. GENERAL BIOLOGY is the science which deals with the most general and fundamental characteristics of living things, whether plants or animals. The study of plants alone is known as Botany, of animals alone as Zoology. A thorough study of any plant or animal includes a knowledge of its (i) Physiology, which deals with its physics and chemistry, its functions and activities; (2) Morphology, which deals with its structure, whether gross (Anatomy), microscopic (Histology), or developmental (Em- bryology) and also with its classification (Taxonomy) ; (3) Ecol- ogy, which treats of its relations to its environment ; (4) Biogeny, which deals with the origin of the individual plant or animal (Ontogeny, Heredity, Development, Genetics, etc.) or with the origin of races, species and larger groups of individuals (Phy- logeny, Evolution). In studying any plant or animal it is desirable that it should be considered from all of these aspects, but some organisms are better suited than others for the study of one or another of these subjects. Accordingly this course is divided unequally into three parts, the first of which deals chiefly with the Physiology and Morphology of the organisms studied, the second with their Ecology and the third with Biogeny. [3] GENERAL BIOLOGY THE AIMS AND METHODS OF LABORA- TORY WORK IN BIOLOGY The purpose of all laboratory work is to study nature at first hand ; but in order to save time it is necesary to utilize knowledge slowly acquired by many generations of previous students. There- fore we do not follow Agassiz's motto: "Study Nature, not Books/' but, rather, "Study Nature and Books." The educational value of laboratory work lies chiefly in the cultivation of accuracy and independence both of observation and of judgment, and in the deeper and more lasting impression which is made of that which we have actually seen and handled. Each student is ex- pected to make for himself the observations and experiments hereafter indicated. The assigned readings which are given at the beginning of each topic should be carefully read before coming to the laboratory. In the laboratory these Directions must be stud- ied and followed. Only in this way can a great waste of time, effort and material be prevented. A record of every observation or experiment must be entered in the prescribed note book, under numbers corresponding to those in these Directions. This record should consist of drawings and descriptive notes, and every page should bear the name of its author and date. The record for each topic must be inspected and passed by an instructor before any new topic may be under- taken. To each student in the laboratory is assigned a locker containing a microscope, reagents, glassware, etc., for the safe keeping of which he is held responsible. The microscope is the most complex and delicate instrument in this outfit, and work with it should be preceded by a study of the following description of its parts and directions as to its use. I. THE MICROSCOPE. Read: "How to Use and Care for the Microscope." Spencer Lens Co. ; or "Use and Care of the Microscope," Bausch and Lomb Optical Co. A. DESCRIPTION. The body or tube bears the lenses and is supported upon a stand which also carries a mirror to cast light [4] GENERAL BIOLOGY upon the object examined through a hole in the flat stage upon which the object is placed. This hole can be made of various di- ameters by means of the iris diaphragm. A lens for concentrating light, and known as a condenser, is placed between the mirror and the stage. The lens at the upper end of the tube is the ocular or eye-piece ; there are two oculars, of different magnifying power. The combination of lenses at the lower end of the tube is the objective; in this microscope there are two objectives of different magnifying power, one marked 3, the other 6; the former (low power objective) is in focus, i. e., gives a clear image of the ob- ject examined when its lower end is about */$ in. above the object, the latter (high power objective) is in focus when it is about ^ in. above the object. The objectives are carried upon a nose-piece, by revolving which either one of them may be brought to lie at the lower end of the tube. The tube is really double, one tube being telescoped within another. By holding the body firmly in the left hand and taking hold of the projecting brass ring just below the eye-piece with the right, the inner tube may be drawn out some distance, thus lengthening the body and increasing the magnifica- tion. The length of the tube without lenses and nose-piece is 150 mm. and it can be drawn out to 195 mm. ; with the nose-piece the tube is 10 mm. longer. A table of magnifications of the different lenses with a given tube length is found on the inside of each mic- roscope case. B. USE. i. Reflect light, from white clouds if possible, upon the object. Where all the light is needed, use concave mirror; where light is intense and a low magnification is required, use plane mirror. 2. Use smaller diaphragms with higher powers. 3. To focus lenses upon an object, use the coarse, then the fine adjustment; the former movement is by means of a rack and pinion. The rack is the toothed plate along the back of the tube, the pinion is a smal cog wheel which fits into the rack and is turned by the two milled wheels on each side of the tube. The fine adjust- ment is by means of a milled screw head back of the pinion. If the fine adjustment screw is turned in the direction in which a clock's hands move the tube is lowered, turned in the reverse direc- tion it is raised. In using high power turn the tube down nearly to the object, and then, while looking through the microscope, bring the object into focus by slowly turning the tube upward. GENERAL BIOLOGY Never focus down upon the object, since by this method there is danger of crushing the lens into the object. Keep one hand on the fine adjustment when looking at an object and vary the focus con- stantly to bring all the fine details of structure into view. 4. Do not use higher power objective without cover glass over object examined. 5. Always use the lower power before the higher one ; and always use the lowest possible power sufficient for distinct vision. 6. Do not touch lenses with fingers. If the field is blurred or the object dim either the cover glass or the lenses are at fault. If the cover glass is dirty remove it and clean it; if the fault is in the eye-piece the particles of dirt revolve when the eye-piece is rotated. If the field is still dim the objective is dirty and must be removed and cleaned. In cleaning the lenses never use anything but clean tissue paper supplied for that purpose. If necessary, the lenses may be moistened by breathing upon them; if this is not sufficient, consult the instructor. 7. Keep both eyes open, using either the right or the left. The strain of microscopic work on the eyes is usually due to the fatigue of constantly closing one eye. If you cannot see the object with both eyes open use an eye-shade provided for that purpose. 8. Never leave the laboratory without first placing the micro- scope in its case and locking it and all your apparatus in your lo-cker. II. PREPARING OBJECTS. The preparation of objects for examination under the micro- scope is termed mounting. Objects are usually mounted on pieces of glass 3x1 in., known as slides. Observe the following direc- tions : 1. If the object to be studied is a mass of cells, separate it into very small pieces by means of teasing needles; if it is a fluid use only a very small drop. If too much fluid has been used it will run out from under the cover glass and the excess must then be soaked up with filter paper. Temporary preparations are usually mounted in water, permanent ones in balsam. 2. The lenses of the microscope, the upper side of the cover glass and the lower surface of the slide must be perfectly clean and dry. 3. Having placed the object in a small drop of mounting fluid [6] GENERAL BIOLOGY take a cover glass in your left hand, rest one edge of the cover on the slide near the drop, and support the opposite edge on a teasing needle ; lower the cover glass gradually over the drop, being careful to inclose no air bubbles. Do not press upon the cover glass. 4. Before putting a permanent preparation away label it care- fully with the name of the object and the method of preparation. 5. Never use reagents haphazard, but only when you have a definite purpose in view. Reagents are used for fixing, hardening, preserving, staining, dehydrating, clearing, embedding and mount- ing. Firing is the process of killing and hardening the living thing so that it preserves as nearly as possible its natural form. Staining is the dyeing of the object so that some parts are more deeply colored than others. Dehydrating is the process of removing the water from the object, usually by alcohol. Clearing usually consists in substituting some oil for the alcohol which is in the object. Embedding is the process of permeating and surounding the object with some substance such as paraffin, preparatory to cutting sections of it. 6. Miscroscopical slides which have been prepared in this way are valuable, sometimes very valuable, and when such preparations are given out for use they must be handled carefully. Do not crush the cover glass or slide by focusing down on it. Do not pick up your microscope with a slide on the stage, as it is very likely to fall on the floor and be broken. Do not leave slides on the table when you have finished with them but return them to the desk or to the box from which you received them. A charge will be made for every slide that is broken. III. NOTES AND DRAWINGS. i. Drawings should be made of every object studied; this is necessary not only as a record of what has been seen, but also as an aid to accurate observation. Make your drawings a record of what you actually see and if you cannot see what the directions call for consult an instructor. Do not attempt to make drawings without the object before you and do not make rough sketches and then finish them from memory. In general make outline draw- ings without shading. Where certain structures occur in large num- bers it is sufficient to represent them in only a part of the drawing. Label all important Structures by means of reference lines and marginal words. Use hard pencils (4!!), with very sharp points, [7] GENERAL BIOLOGY and make the drawings large enough so that all details can be represented without confusion. 2. To draw to scale : — Place paper at base of microscope and endeavor to trace outlines as seen with left eye while seeing point of pencil at same time with right eye. The pencil point must appear to coincide with the part of the object being drawn. Do not move the eyes. IV. EXAMINATION OF COMMON OBJECTS. 1. Mount a few fibres of wool, cotton, linen and silk in water on different slides, cover with cover glasses and examine first un- der a low power, then under a high one. How do the fibres differ ? Sketch and label one of each under the lower power and then under the high power of the microscope, drawing to scale. 2. Examine a drop of emulsion (oil suspended in water) and notice peculiar effects of refraction when lenses are focused upon different portions of a drop. 3. Examine bubbles of air in water. These may be obtained by running water under a cover glass supported at one side by a bit of paper and then tapping on the cover glass with a needle. What differences can you see between these and oil drops ? [8] .-:; • PART I GENERAL PHYSIOLOGY AND MORPHOLOGY A. CHEMICAL AND PHYSICAL CHARAC- TERISTICS OF LIVING THINGS Read: Sherman, Chemistry of Food and Nutrition, pp. 1-102; or Nutrition, pp. 1-102; or Mathews, Physiological Chemistry, pp. 1-187; or Hawk, Physiological Chemistry, pp. 1-147. The bodies of all living things are composed of about 15 chem- ical elements and a great number of chemical compounds : 97 per cent of the human body consists of carbon, hydrogen, oxygen and nitrogen, and 3 percent of n other elements. Three- fourths of all the hydrogen and nine-tenths of all the oxygen are combined to form water. In addition to water and mineral salts living things contain carbon compounds, or "organic compounds." Compounds of carbon, hydrogen and oxygen form Carbohydrates and Fats; compounds of carbon, hydrogen, oxygen and nitrogen form Pro- teins. I. CARBOHYDRATES (Starches, Sugars, etc.). Carbohydrates of physiological importance are : — Monosaccharids (C6 H12 O6) — dextrose, levulose, glucose. Disaccharids (C12 H22 O^) — cane sugar, malt sugar, milk sugar. Polysaccharids (C6 H10 O5)n — starch, dextrin, glycogen, cellu- lose. I. MONOSACCHARIDS (glucose, fructose, etc.). Cannot be split into simpler sugars. a. Dextrose. Take a one per cent solution and test solution as follows: Boil 5cc. of Benedict's1 or Fehling's2 solutions in a test tube. Result ? [91 GENERAL BIOLOGY Then add 8 drops of dextrose solution and boil again. Result? This is the "dextrose test." Copper sulphate (Cu So4 in an alkaline solution is "reduced" to yellow cuprous hydroxide (Cu OH) or to red cuprous oxide (Cu2O) when boiled with a reducing sugar. Test your urine for sugar in this way. 2. DISACCHARIDS (Sucrose, maltose, lactose) can be split into monosaccharids by hydrolysis. a. CANE SUGAR (Sucrose). Take a few drops of i% solu- tion and test with Benedict's or Fehling's Fluid. Is it a reducing sugar ? b. HYDROLYSIS OF CANE SUGAR. Boil some of the i % solution with a few drops of hydrochloric acid. Cool, neutralize, and apply Benedict's test. Is a reducing sugar present? 3. POLYSACCHARIDS. Native starch, a. Mount a scraping of potato in water and examine under microscope. Study and draw structure of starch grains. Run a drop of iodine solution under cover. What happens? b. Grind a little commercial starch in a mortar and shake with cold water. Filter and test nitrate with iodine. Explain result. c. Test solubility in boiling water. Note character of resulting solution. Dilute and add a drop or two of iodine solution. What results, and why? d. Cellulose (plant cell-walls). Cotton fiber is almost pure cellulose. Note insolubility in water and alcohol. Is it insoluble in acids ? Alkalies ? Does it react with iodine ? Treat with 40 per cent sulphuric acid and then add iodine. What results? Treat with Schultze's chlor-zinc-iodide.3 Explain results. This is known as the "cellulose test." 1 Benedict Fluid: Copper sulphate, 17.3 grams; Sodium or potassium citrate, 173.0 grams; Sodium carbonate, 200 grams; Distilled water to make 1000 cc. 2 Fehling's Fluid: — (i) Copper sulphate, 34.65 grams; Distilled water to make 500 cc. ; (2) Potassium hydroxide, 125.0 grams ; Rochelle salt, I73-O grams; Distilled water to make 500 cc. (3) Mix equal parts of (i) and (2) when needed for use. 3 Schultze's Chlor-zinc-iodide is made as follows: — (i) Dissolve no grams of zinc in 300 cc. hydrochloric acid and evaporate to 150 cc. ; (2) Dissolve 12 grams of potassium iodide in as little water as possible and add 0.15 grams iodine; (3) Mix (i) and (2), and filter if necessary. [10] GENERAL BIOLOGY 4. FERMENT ACTION: ENZYMES. a. Salivary diastase (ptyalin). Collect a few cc. of saliva in a test tube ; dilute with about five volumes of water and filter into two test tubes ; boil one of the tubes and leave the other unboiled. Add to each tube an equal volume of dilute starch paste and place tubes in incubator warmed to 40 C. for fifteen minutes. Then divide the substance in each tube into two portions and test one of each of these for starch (iodine) and the other for sugar (Benedict's). What effect does ptyalin have on starch? What effect does boiling have on ptyalin ? II. LIPINS (Oils, Fats, Yolk. etc.). 1. OILS AND FATS, (i) Note physical properties, differences in melting point, etc., of three fats — olive oil, butter, and tallow. Test solubilities of these fats in water, alcohol, chloroform, ether. (2) Shake a few drops of olive oil with water in a test tube. What happens ? Set tube aside for a few minutes. What happens ? Shake up a few drops of the oil with one percent sodium carbonate in- stead of water, examine with microscope, and note difference in results. (This is an emulsion.) (3) Rub a thin film of butter on a slide, and put on a drop of the dye known as Sudan III. Ob- serve under microscope what occurs. Stain thin sections of Castor bean and of Lima bean with Sudan III. Is oil present in both? 2. CHEMICAL TESTS FOR FATS. The reaction with ether and with fat stains are two well-known tests for fats. III. PROTEINS (Albumins, Peptones, Albuminoids, etc.). Use white of egg as type of protein. 1. Carefully pour white of egg into a dish. This is approxi- mately a 12 per cent solution of a protein (albumin) in water. Notice its consistency. Test its reaction with litmus paper; is it acid, alkaline, or neutral? A 10 per cent solution of this white of egg has been made by shaking it up with 9 times its volume of distilled water and filtering. 2. COAGULATION, (i) Coagulation by heat. Have a water bath with water at the boiling temperature. Put some of the un- diluted albumin in a test tube and place in the water bath. Does it coagulate? Try a little of the 10 per cent solution in the same way. Does it coagulate? What is the effect of dilution on coagula- GENERAL BIOLOGY tion by heat? (2) Coagulation by chemicals. To 5 cc. of the 10 per cent albumin add a few drops of 3 per cent copper sulphate. Try also strong nitric acid and sulphuric acid, allowing a drop or two to run down the side of the test tube. Try also 95 per cent alcohol. (3) Test urine for albumin as follows: a. Boil 10 cc. of urine in a test tube ; if turbidity appears add a drop or two of strong acetic acid; if turbidity disappears it was due to phosphates, if not to albumin, b. Put 2 or 3 cc. of strong nitric acid in a test tube, then add urine, allowing it to run gently down the'iside of the slanted test tube ; if albumin is present a ring of coagulum will form between the acid and the urine. 3. CHEMICAL TESTS FOR PROTEINS: Xantkoproteic Reaction. Dilute some of the 10 per cent albumin till it is about 2 per cent; place a small quantity in a test tube. Add a few drops of nitric acid. What occurs? Boil. What occurs as to color and other changes? Cool the solution and add ammonia until the acid is neutralized. Note color produced. (This is the essential feature of this reaction.) Try in the same way a weak solution of gelatin (albuminoid) ; does it coagulate? Does it give the xanthoproteic color ? 4. ACTION OF ENYZMES ON PROTEINS. Thin pieces of boiled white of egg were placed in artificial gastric juice, made by add- ing pepsin to a 0.2 per cent solution of hydrochloric acid and allowed to stand 24 hrs. Some of the digested albumin was placed in one dialyzer and some fresh, undiluted albumin in another. Test the water below each membrane for albumin. IV. ENZYMES (Organic Ferments). 1. Chemical substances probably allied to proteins though they have never been completely isolated. They are formed by animal or plant protoplasm and act as catalyzers in many chemical re- actions within living things. 2. They are classified, according to what they do, as : Amylo-lytic or Starch Splitting (Diastase, Ptyalian, etc.). Lipo-lytic or Fat Splitting (Lipase, Steapsin, etc.). Proteo-lytic or Protein Splitting (Pepsin, Tripsin, etc.). Sugar Splitting (Maltase, Invertase, Lactase). Alcohol forming (Zymase). Coagulating (Thrombin, Rennin). Oxidizing (Oxidase, etc.) [12] GENERAL BIOLOGY 3. You have already observed the action of ptyalin on starch, and of pepsin on albumen. Write the chemical formula for the former of these reactions. V. HORMONES (Chemical Messengers). Chemical substances, possibly enzymes, usually formed by duct- less glands and poured into the blood. They stimulate or inhibit many vital processes (Thyroidin, Adrenin, Secretin, etc.) VI. VITAMINS. Accessory food substances of unknown chemical nature, pro- duced by animals and plants. May act as enzymes or hormones. Essential to life, but minute quantities sufficient. (" Water-soluble B," "Fat-soluble A," etc.). VII. PROTOPLASM. (Living substance, material basis of life). Composed of all the preceding classes of substances together with water and various salts. Protoplasm is not a single chemical compound but is an organized mixture of many compounds, espe- cially proteins. There are innumerable kinds of protoplasm. B. MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF LIVING THINGS Protoplasm exists only in the form of cells, which are in- dividual masses of protoplasm, each containing a denser body, the nucleus. I. CELL STRUCTURES. Read: Calkins, Biology, pp. 6-29; or Sedgwick and Wilson, General Biology, pp. 20-40 ; or Parker, Elementary Biology, pp. 56-79; or Shull, Animal Biology, pp. 70-83; or Woodruff, Foundations of Biology, pp. 6-29. A. PLANT CELLS. i. Carefully tear in two a leaf and from the torn edge pick off a small piece of the transparent covering (epidermis). Mount in water and examine under the microscope. Draw about 10 ad- jacent epidermal cells, making each cell about half an inch in diameter. [13] GENERAL BIOLOGY 2. In prepared sections through the root-tip of an onion ob- serve the shape and size of the cells. Draw about 10 adjacent cells. Having found one or more complete cells with round nucleus ob- serve and make a drawing about 2 inches in diameter of a single cell showing the following parts: i. Cell Membrane. 2. Nucleus. 3. Cytoplasm (protoplasm surrounding nucleus). In the nucleus observe: 4. Nuclear Membrane. 5. Nucleolus. 6. Chromatin (stained granular part of nucleus). 7. Achromatin (unstained part of nu- cleus). B. ANIMAL CELLS. 1. With the handle of a scalpel gently scrape the inside of your lip or cheek and mount the scrapings in a drop of water. Draw several cells. Run a drop of aceto-carmine under the cover glass by placing the drop on one side of the cover and applying a bit of filter paper to the other side. The nucleus stains deeply owing to the fact that it contains chromatin. Draw one cell show- ing nucleus, cytoplasm, and cell-membrane; making the drawing about 2 inches in diameter. 2. In similar manner observe the cells and cell structures in prepared slides of the skin of a frog. 2. Mount a drop of frog's blood and examine under the low power and then under the high. Draw several of the red, also of the white, corpuscles. II. CELL FUNCTIONS. Each cell performs all the fundamental functions of life — it nourishes and reproduces itself, is contractile and sensitive, — though some cells are devoted more exclusively to one of these functions than to the others (Specialization). In this place we study only the functions of reproduction and movement. i. CELL REPRODUCTION. All cells reproduce by division; the nucleus first divides, in one of two ways, after which the cell body constricts into two. Nuclear division occurs by the indirect pro- cess (Mitosis) or by the direct process (Amitosis). (i). Indirect Nuclear Division (Mitosis). In prepared slides of the growing root- tip of the onion observe nuclei in the following stages of division : (a) Early Phophase, in which the nucleolus has disappeared and the chromatin granules have united to form threads; (b) Late GENERAL BIOLOGY Phophase, showing disappearance of nuclear membrane and formation of chromosomes (chromatic rods) from the threads; (c) Metaphase or Equatorial Plate, showing the chromosomes in the equator of the cell, each dividing by a longitudinal split; (d) Early Anaphase, showing the daughter chromosomes separating toward the poles of the cells ; (e) Late Anaphase, showing union of daughter chromosomes to form daughter nuclei; (f) Teleo- phase, in which the cell-body divides and the nuclei ; return to the "resting condition." Draw a cell in each of these stages of division. (2). Direct Nuclear Division (Amitosis). In prepared slides of the follicile cells surrounding the egg of the cricket observe various stages in the direct division of the nucleus. Draw cells in which (a) the nucleolus is dividing, but the nucleus is still spherical, (b) the nucleus is dumb-bell shaped, (c) the nucleus is divided into two. 2. PROTOPLASMIC MOVEMENT. With a pair of fine forceps pull off some of the hairs which grow on the stamens of the flower of the spiderwort (Tradescantia) and mount them in water under a cover glass. Observe : ( i ) The hair is made up of a succession of cells, with corrugated walls. (2) Just within the cell wall is the granular protoplasm, strands of which may be seen moving or circulating. (3) Within this protoplasm is a clear spherical or ovoid body, the nucleus. (4) Most of the center of the cell is occu- pied by a purple, homogeneous fluid, the cell sap. If the flowers of Tradescantia are not available use one of the leaflets of the water weed Elodea canadensis. Observe the green bodies (chloroplasts) within the cells. Do they circulate? Make a drawing showing these structures, and indicate by ar- rows the direction of the protoplasmic movement. C. CLASSIFICATION OF PLANTS AND ANIMALS Read: Calkins, Biology, pp. 162-166; or Parker, Elementary Biology, pp. 137-147; or Parker and Parker, Practical Zoology, pp. 215-228; or Shull, Animal Biology, pp. 260-274 ; or Woodruff, Foundations of Biology, pp. 348-351. All living things are classified as plants or animals depending upon certain peculiarities of structure and function. In general [15] GENERAL BIOLOGY plants have rigid cell walls of cellulose (C12 H20 O10), which is lacking in animals ; the food of plants consists of relatively simple chemical compounds, whereas that of animals is much more com- plex; also plants are usually less active than animals. The minor subdivisions of both animal and plant kingdoms are very numerous and are known as Orders, Families, Genera, Species and Varieties. The scientific name of any animal or plant consists merely of the name of the genus and species to which it belongs. This method of naming animals and plants is due to Linnaeus (1707-1778) and is known as binomial nomenclature. Inspect the specimens in the Museum, Herbarium and Vivarium, and become familiar with as many as possible of the subdivisions and classes named above. Enter in your laboratory notes in the following manner the scientific name (copied from the labels of specimens on exhibition) of some one member of each phylum and class of the animal kingdom, so far as represented in the ex- hibits : PHYLUM CLASS GENUS SPECIES Cnidaria Hydrozoa Hydra fusca The animals and plants which will be studied in this course are common forms which are found in the vicinity of the laboratory. The particular forms to be studied are chosen because they illus- trate especially well certain general principles or characteristics. Locate in the tables of classification the position and relation to other animals or plants of each organism studied in the labo- ratory, museum or field. D. METAPHYTA Metaphyta are many-celled plants, with more or less differentia- tion of the cells and tissues for particular functions. The lower Metaphyta belong to the Cryptogamia or flowerless plants, the higher ones to the Phanerogamia or flowering plants. Owing to limitations of time, it is not possible in this course to study more than one representative of the Metaphyta, and for this study one of the higher flowering plants is chosen, viz., the common bean. PHASEOLUS VULGARIS, String Bean (Subkingdom Phanerogamia, Division Spermatophyta, Sub- division Angiospermae, Class Dicotyledonae.) [16] GENERAL BIOLOGY Read: Bigelow, Applied Biology, pp. 66-121; or Coulter, Barnes and Cowles, Text Book of Botany, Vol. i, pp. 295-484; or Curtis, Nature and Development of Plants, pp. 1-129; or Duggar, Plant Physiology, pp. ; or Ganong, Text Book of Botany, pp. 1-178; or Physiology of Plants, pp. or Huxley and Martin, Practical Biology, pp. 460-481 ; or MacDougall, Plant Physiology, or Vines, Text Book of Botany, pp. 666-783, or Woodruff, Foundations of Biology, pp. 61-114. A. MORPHOLOGY. I. SEED. In beans which have been soaked for 24 hours in water ob- serve : — 1. Shape, Size, Color. 2. The Seed Coat, a tough outer membrane. 3. The Hilum, or scar, where it was attached to the parent plant. 4. Dry the surface of the seed and squeeze it gently; water will exude from a small hole near the hilum, the Micropyle. Draw a bean to show all of these features, in profile and also in face view. II. EMBRYO. Remove the seed coat and observe the Embryo, which fills the whole space within the seed coat ; note the following parts of the Embroyo : — a. Two Cotyledons or seed leaves, which constitute most of the bean seed ; they are attached to one another by their bases. Mount in water scrapings from one of the cotyledons and examine under microscope; also test scrapings with iodine and others with Benedict's Solution. What is the chief constituent of a Coty- ledon ? Separate the two Cotyledons and observe : — b. The Hypocotyl (stem and root) on the margin of the Cotyledons with its apex toward the micropyle. c. The Plummule (bud) between the Cotyledons, composed of two small primary leaves and a minute bud between them. Draw an embryo to show the inner face of a cotyledon with hypocotyl and plumule attached. [17] , en en cn cn CC /-v en /— s ^ ^ bfl £ £ £ £ •4-1 •*-< £ 2 £ 3 _, ^ ?% C Oi ' /—^ rt ^ IH ^O 1* 1* IH O 0 0 • ~- bfl OJ j- £ 0 £ ^> o GO GO >-^..»H r^ ^CU^? C c rt.S i - o U /— V s-^-*-* (Planari ; Flukes) peworms cn 'o cn 0 X! O 'O IH 5^2 ° S c GO ! > "C? rj .2 0 h_) <>-'^s— 'O . — ^ Kfi v^-'>>-'^ rt IH ^> "^ GO O CTj CJ ^ **^ cm cu C rt rt •§•§& T3 "*-* "S c $5 oj C 0 o 2 o 03 O O rt C3 j— » • ^-« O S'r; OH N /•! hj ^ *3 rt ^— ^t zz o "& N CU cn O P ^^S vi 0 w! o «« nl c3 2 jy x £ I'll cu rt «^j -e£? |- CJ in "cu J5 ^2 ""£ oj a rt cu cu UffiQ KGO< ^^ "J » U HHU J3 CU U O M M' CO T^- ~ (N co M oi to • • • 1-4 oi rt S *ri P rt cu •a cj rt cu K^ C ^ cu u ffi 0} IH 2 rt J3 O cu rt *3 "c PQ -t-> ~ £ cu £ % rt cu O cj P GO s * ^ O <• r^H ^ /-~s /""S x"-\ ^~^ ,•— s c4 ^Q ~ ^O cn "^-^ "*— ' s-x v»^ v-x PHYLUM (=TYPE}_ I. PROTOZOA Entire body consists of a sin- gle cell which may be inde- pendent or joined with others to form a colony II. SPONGIARIA (Sponges) Fixed forms, chiefly marine.Gas- trula attaches by oral pole. Colonies formed by budding. III. CNIDARIA Attached polyps, or free medu- sae with Stinging Cells, Gas- trula attached by apical pole. Colonies formed by budding. IV. CTENOPHORA. Jelly spheres, entirely marine ; 8 meriodional rows of swim- ming plates. V. PLATYHELMINTHES, Worms flattened dorso-ven- trally. Oral pole of gastrula •a« •^ C * ** s*o c rt O< as 3^- be VI. ROTIFERA. Microscopic animals with head (trochal disk) trunk and tail (foot). VII. NEMATHELMINTHES Thread Worms. Mostly par- asitic, with unsegmented bodies covered by dense cuti- cle. VIII. CHAETOGNATHA. Small marine Arrow Worms, body divided into head, trunk and tail; setae on sides of mouth. IX. ANNELIDA. Ringed worms with segmented bod- ies, each somite inclosing » jf A "^ T3 C cu _ j^ M 3 cn "H 5 ^ ^2 "S O ^ -M ql O -C ^ O C ^j rt o o ^ 0 ^ - C ^Jll o y 2 £ "§^-^13 >4 -^--> ^i i i * * cn cn x^ -^j ^ jj^ • ^^ ^^3 ^- CU ~v rt cu *^ ^C ' "* *"* 3&s" 3 £ e s=s •4-t y£ ill S ."73 ^ ^^2 *J 4-1 O i 3 *^ — T cu O ^"^ cn cn O cu I- «^ -M CU H .2 ? tt * ^ rt 5 S ^ c3 ""O O G S 2 rt *3 £"i.cj| "•3 " *>J ^ ^aj ^s >» ^ ^ 'c^*^ W^S rt r^ "cu /— \ II J> bo rt L cu ^Hu *5 P5 £ ^SvLL ^Q II >*, ^ •gS| I en i > >- C oJ c/i ctf co ^ °^ •- > ^"S crj ^° \i bo ojp* r^ O cj *"' cn *" S _c ">'O o o - •*-• C . J-i CU ^ »— x PH ^_» CT$ ^J* '"^ *ij ^> fj H <-] bo os GO • j> °— - • >"* rt -*^ •*-• cn bo PQ> ~ Cf> C . Crustacea (Crayfish) . Onychophora (Peripatus) . Hyriapoda (Thousand Leg . Insecta (Insects) Arachnida ( Spiders ) . Phororiida . Brachipoda , Polyzoa (Colonial forms) . Pelecypoda (Oyster, Clam^ . Amphineura (Chiton) , Gasteropoda (Snail) , Scaphopoda , Cephalopoda (Squid) Holothuroidea (Cucumber Echionoidea (Sea Urchin) Asteroidea (Starfish) Ophiuroidea (Brittle Star Crinoidea (Stone Lilies. Usually attached) nteropneusta (Balanoglossu; 'unicata (Ascidian, Sea Squi .crania (Amphioxus) Cyclostomata (Lamprey) Pisces a. Elasmobranchii (Sharks a. Ganoidei (Armored fist c. Teleostei (Bony fish) d. Dipnoi (Lung fish) Amphibia (Frogs, Toads) Reptilia (Reptiles) Aves (Birds) Mammalia (Mammals) — >-» 04 ro Tt w W c*3 hH OJ CO T}- IO i-i oJ w, -4-> -4-> « IH O 05 -a U OJ ^5 ? ^ 01 oJ •*-• co 3 ftJ3 u 0 ,2 U 0^ ,G*5 CJ * ^"* O O ,"tl ^0 jz To o> •d o 0. IH IH H~ < 0^ 5 U > rt 2" 3 e OS UD o S en • -• < C/5 tojQ o t^) «_• /*""^\ ^2 t^ ^ i §^ *3 c 01 << Crt -\ •« 5^ D o- •4-1 01 O OJ gj • — co rG G ^ T-l -M ill 3 a -> -it! ^*7; O t/3 H -> a; T P 3 C ' S « H C 3 ^*£ 3 *co p! X IH OJ 3J= O. 1^-° D "^ y< «J , M •*-• TH G H.'S,' %-& ~ -4-" T . t— t . ^ covered by < chitin ; withou I. MOLLUSC nsegmented a: attached by of tentacles around mouth. II. MOLLUS nsegmented an j.. . i i g i'>^_; o 5? " ^ 5J p, j^ r*~t ^ III. ECHINO arine animals apparently rad cal, but really ambulacral sy; vesels and tub IV. CHORDA 'gmented anim ^U«1^, / TVT _ StN-ClCLUIi ^INOl tubular nervo fmrcal cirli^ a * § 0 ; > 3 5 C W > ciS ) a buc ( o CO "rt "ol full of yeast which is actively giving off bubbles of gas. Insert a cork in this second tube and connect the two by a bent glass tube, one end of which passes through the cork into the air space above the yeast, the other end of which dips below the surface of the baryta water. What changes take place in the baryta water? This is a test for carbon dioxide, (d) Chemical reaction of fluid yeast. Determine by the use of litmus paper whether fluid yeast is acid or alkaline in its nature. What do you suppose the cause of this to be? Prepare a written statement giving as far as possible an explana- tion of all the facts you have observed in the experiments in this section. BACTERIA. (Division-Thallophyta, Subdivision Fungi, Class Schizomycetes.) Read: Calkins, Biology, pp. 34-43 or Parker, Elementary Biology, pp. 82-94; or Sedgwick and Wilson, General Biology pp. 192-204; or Woodruff, Foundations of Biology, pp. 44-53. [33] GENERAL BIOLOGY A. PREPARATION OF CULTURES FOR FUTURE STUDY 1. BACTERIA OF HAY INFUSION. Fill three test-tubes l/2 full of a fresh infusion of hay: (i) care- fully close one tube with cotton wool and boil a few minutes; (2) do the same with a second tube and then boil it again after 24 hours and repeat the boiling for several days if convenient; (3) leave a third tube open and do not boil it; set all three in a warm place where they can be observed from time to time. 2. BACTERIA OF THE AIR. Clean a smooth potato with a stiff brush and water, removing with a knife all injured portions as well as the buds ("eyes"). Ster- ilize the potato in boiling water for thirty minutes, then cut in slices by means of a knife sterilized in a Bunsen flame. Place the slices of the potato on a sterilized glass plate and leave the clean cut surface exposed to the air in the room for one hour. Cover with a sterilized bell jar, under which some distilled water is placed to maintain a moist atmosphere, and set aside for several days. If any organisms develop on the potato they must have come from the air of the room. 3. BACTERIA OF HYDRANT WATER. Take a sterilized gelatin culture plate in a Petrie dish, open the dish and quickly allow a few drops of hydrant water to run across the gelatin. Close the dish at once and set it aside for several days. If bacteria were present in the water one or more colonies of them will be found along the path of the drop. 4. BACTERIA OF MILK. Dilute i cc. of milk with 100 cc. of sterilized water. Add i cc. of this dilution to a Petrie dish of "litmus agar," cover and place in the incubator for 36 hours. [34] GENERAL BIOLOGY B. STUDY OF DIFFERENT STAGES, TYPES AND CULTURES OF BACTERIA I. ACTIVE STAGES. Study bacteria from various media, viz. : Pasteur's Solution, Beef Tea, Infusions of Hay and Peas, Potato and Gelatin Cultures, Sewage, etc., and observe and draw the following forms: 1. Cocci; rounded forms occurring singly or in bead-like rows ; without flagella. 2. Bacilli; rod-like or thread-like forms. 3. Spirilla; spiral forms which may consist of many turns (Spirillum Spirochaeta) or of only a fraction of one turn (Vibrio "Comma bacillus"). In these various forms observe the following points : First ; size, measure. Second ; structure. Can you notice any change of shape in an in- dividual? Any difference between the external and internal por- tions? Any peculiarity of the ends in the longer forms? Third; movements. Some vital, others purely physical (Brown- ian movements). The former progressive, the latter vibratory and irregular. Study the Brownian movements in particles of Chinese ink in water. Put a few drops of fluid containing bacteria on a slide, hold the slide over a Bunsen flame and kill the bacteria by boiling, cover and examine with high power. Can you notice any movement of the dead bacteria? Compare with movements of living ones. II. RESTING STAGES. Examine the scum ("Zooghca") from the surface of various liquids, especially the hay infusion ; it consists of myriads of bac- teria in a resting condition imbedded in a gelatinous substance, the "bacterial jelly." III. STAINING AND MOUNTING. Spread a small drop of liquid containing active bacteria on a clean cover glass and let it dry slowly ; then pass the glass through a Bunsen flame two or three times to coagulate and fix the bacteria upon the glass. Put a drop of Methylen Blue or Gentian Violet upon the glass. After five minutes rinse with distilled water and mount in a drop of water upon the slide. If a permanent mount is [35] GENERAL BIOLOGY desired thoroughly dry the glass after rinsing and mount4n Canada Balsam. Treat some of the Zoogloea in the same way and observe that the bacteria stain more deeply than the substance in which they are imbedded. IV. BACTERIA FROM THE MOUTH. Take some scrapings from the teeth, dilute with water, mount and study the various forms under a high power. How many kinds of bacteria can you find? V. STUDY OF CULTURES IN HAY INFUSION. Study the tubes of hay infusion which were prepared and set aside on a previous day (see (A. i). Note the changes which take place in each of the tubes. Does the infusion in any of the tubes become turbid and in which one is this most marked? Determine by microscopic examination what the cause of the turbidity is. How do you account for the differences between the tubes ? Where did these organisms come from and how did they get into the tubes? Keep the tubes under examination for several days or weeks and observe in what tubes a scum forms on the top of the liquid. Does the formation of this scum have any influence on the turbidity of the fluid? Study the scum under the microscope and determine what it consists of and whether it differs in the different tubes. What ultimately becomes of the scum? What changes, if any, are there in the odor of the fluid during the period of ob- servation, and how do you account for them? Are different kinds of bacteria found in the tubes? If so, make sketches to show them. Do the bacteria in the same tube differ in form fronvday to day? If so, sketch them in the order in which they appear. After one week what is the condition of the fluid and the bacteria found in each of the tubes ? Write up an account of the phenomena you have observed and give your explanation of them. VI. STUDY OF CULTURES ON POTATO. (See A. 2.) Observe on the potato variously colored spots or "colonies." Are all of these colonies bacteria? Are all the organisms in a colony alike? What is the significance of this fact? What kinds of organisms are most abundant in the air of the room? Can they undergo drying without being killed? Can they grow and multi- ply without food and moisture? [36] GENERAL BIOLOGY VII. GELATINE PLATE CULTURE FROM HYDRANT WATER. Are colonies present on the plate? If so, how many? Are they all alike in shape and color? What kinds of organisms are found in the different colonies? Are they more or less numerous than in air or milk? VIII. AGAR PLATE CULTURE FROM MILK. w*~- By means of a "counting plate" determine the approximate number of colonies present in the agar, and calculate the number of bacteria present in I cc. of undiluted milk (the I cc. of milk used in the culture was diluted 100 to i). Colonies which have the form of minute footballs belong to the group of "colon bacilli" and come from the intestinal tract of some mammal, in this case probably from a cow. If colon bacilli are numerous it indicates that the milk has not been taken under sanitary conditions. What does the changed color of the litmus agar indicate ? C. DEMONSTRATIONS. I. Living Bacteria Seen With Dark-Field Illumination., II. Prepared Slides of Pathogenic Bacteria. F. -PROTOZOA One-celled animals in which the entire body consists of a single cell, which may be independent or may be joined with others to form a colony. i. PARAMECIUM CAUDATUM, Slipper Animalcule (Phylum Protozoa, Class Infusoria, Order Ciliata.) Read : Calkins, Biology, pp. 60-75 '•> or Parker, Elementary Biology, pp. 106-120; or Parker and Parker, Practical Zoology, pp. 261-286; or Sedgwick and Wilson, General Biology, pp. 168-172; or Woodruff, Foundations of Biology, pp. 39-43, 244-248, 340-342. A. MORPHOLOGY. Put a small drop of water containing Paramecia on a slide; sur- round it with cotton wool to limit the movement of the animals and cover with a glass ; examine with the low power of the micro- scope and then with the high power. Note: [37] GENERAL BIOLOGY 1. SIZE: measure. 2. SHAPE: fusiform, rounded at the anterior end, bluntly pointed at the posterior end. 3. LOCOMOTION : due to cilia uniformly distributed over the whole surface. Note also movements of flexion (bending). 4. STRUCTURE. The two protoplasmic layers: (ectoplasm and endoplasm). a. Ectoplasm (Cortex) : the firm elastic outer layer; its deeper part marked by oblique myophan striations. ( i ) . The cuticle, a delicate superficial layer differentiated from the underlying protoplasm. (2). Cilia, delicate vibratile filaments arising from the ecto- plasm and protuding through openings in the cuticle. These open- ings can be seen on a specimen from which the water is allowed to evaporate. (3). Trichocysts : minute oval sacs in deeper part of the ecto- plasm arranged perpendicular to the surface; when the animal is irritated, e. g. by iodine, a stiff thread can be shot out and pro- jected beyond the cilia. They are probably defensive organs. (4). Two contractile vacuoles in the ectoplasm of the dorsal side about l/3 of the animal's length from each end. While dilating they are nearly spherical, but at the moment of contraction sep- arate canals can be seen radiating from them. (5). The oral groove begins at the anterior end of the left side and runs back to the mouth near the middle of the ventral side. The cilia of the groove drive food particles into the mouth. Run some Chinese ink in water under the cover glass and note that some of it is carried into the groove. (6). The mouth is an aperture in the ectoplasm at the posterior end of the groove through which food passes into a narrow tube, the gullet. Watch the ink collect at the inner end of the gullet into a ball which is suddenly passed into the endoplasm. Watch the course of the food ball within the endoplasm until it is finally ejected through the anus. (7). The anus is a temporary aperture between the mouth and the hinder end of the body — visible only at the moment of ejection of fecal matter. b. The Endoplasm (Medulla) is the more fluid protoplasm filling the central portion of the body. In it observe : — [38] GENERAL BIOLOGY (i). The food vacuoles, which are spherical spaces in the endoplasm filled with water containing food particles. (2). The circulation of the endoplasm is rendered obvious by the food vacuoles and the granules, which are carried round in a definite direction. (3) The nucleus is an elongated oval body near the center of the body of the animal. It is best seen after the death of the animal, or in stained specimens. (4). The micronucleus is a much smaller body applied to one side of the nucleus and resembling it in staining reactions. Make a full-page drawing of an animal to show the above- mentioned structures. B. PHYSIOLOGY. I. METABOLISM. 1. Ingestion of food. Place some Paramecia on a slide with powdered Chinese ink and watch the formation of food balls in the gullet and their ingestion. Study the formation of a food vacuole. 2. Circulation of Endoplasm. Observe and sketch the changes of position of the food vacuoles in the body and show by arrows the course of circulation. Time the circulation by noting the time at which the ink is added and that at which the first ink ball com- pletes the circuit. 3. Egestion. Observe, if possible, the egestion of ink from the body. Show by a drawing where and how this takes place. 4. Digestion. Observe and draw the changes in color, etc., of food as the vacuoles circulate through the body, also the changes in the size of vacuoles and their fluid contents. What do these changes indicate? 5. Nature of Food. Study the nature of the contents of the food vacuoles of normal Paramecia and find, if possible, what they feed upon. Is it animal or vegetable matter? Does Paramecium choose its food ? Stain with iodine and see if any starch is used as food. 6. Excretion. The contractile vacuoles are excretory organs for getting rid of water and nitrogenous waste (urea). Study and sketch a vacuole in various stages of contraction and expansion. Time the contractions and expansions and record the results in your notes. Place Paramecia in a thick solution of Chinese ink [39] GENERAL BIOLOGY and observe the extrusion of a clear drop of fluid at the moment of contraction of the contractile vacuole. 7. Respiration. Place a number of the animals in a drop of phenolpthalein (which loses its rose-color in the presence of carbon dioxide) and note the result; also note the manner in which the animals collect at the surface of a dish in which the water is very foul. What do these observations teach? Are there any organs of respiration? II. REPRODUCTION. 1. Fission. Observe a Paramecium in the process of division. In a stained preparation note what changes take place in the nuc- leus and micronucleus during this process. Can you detect any difference between the two daughter individuals? How do the contractile vacuoles, the buccal grooves and the gullets arise in the two ? Draw three stages in fission to show all of these points. 2. Conjugation. Study and draw living individuals in the act of conjugation. What portions of the body are in contact? Is there any distinction of size or sex in the two individuals? In stained preparations study the nuclear changes which takes place during conjugation. Draw three specimens to show different stages in the nuclear changes. III. IRRITABILITY. 1. Automaticity. Does the animal appear to act of its own accord or only through the influence of external stimuli? 2. Movement. How many kinds of movement does the animal exhibit? What are the organs of locomotion? With a dissecting lens observe the movements of an animal in a drop of water. Does it move in straight lines? Does it keep one side uppermost? How does it alter its course? Can it move backwards? By means of lines and arrows plot the movements of an animal during one minute. By means of powdered ink observe the direction of cur- rents over the body. What is the direction of the currents in the buccal groove? 3. Sensitivity. Is the animal sensitive to touch or pressure? How does it behave when in contact with a solid body? Place a small drop of salt solution colored with Chinese ink on a slide and note whether the animals are sensitive to this substance. In similar manner test them with 1/50 per cent and i/io per cent acetic [40] GENERAL BIOLOGY acid, and also with a bubble of carbon dioxide. Place animals in a tube heated at one end and cooled at the other. What results ? In a similar manner test them with the electric current. Also test their sensitivity to light and gravity. Record all of your results. IV. AMOEBA PROTEUS. (Phylum Protozoa, Class Sarcodina, Order Rhizopoda.) Read: Calkins, Biology, pp. 44-59; or Parker, Elementary Biology, pp. 1-22; or Parker and Parker, Practical Zoology, pp. 229-238 ; or Sedgwick and Wilson, General Biology, pp. 158-167. Place a small drop of sediment from a vessel containing Amoeba on a slide with a drop of water ; cover with cover glass and search for Amoeba with low power. If not easily found, prepare several such slides and examine them after they have been standing for some minutes, so that the Amoebae may crawl out of the sediment. When an Amoeba is found examine with a high power and note : A. MORPHOLOGY. 1. SIZE; is it visible to the naked eye? 2. SHAPE; is it regular? Constant? Are the pseudopodia of the same size and shape? Do they ever branch? How many do you find? Sketch at intervals of one 'minute for five minutes. 3. STRUCTURE: An outer clear layer, the ectosarc (ectoplasm), and an inner granular more opaque substance, the endosarc (endo- plasm). Is the boundary between the two layers a sharp one? Where is the ectosarc thinnest ? Is there a membrane outside this layer? Are all the granules of the endosarc of the same size? Which layer is the more fluid ? In the ectosarc a clear vesicle may be found which appears and disappears; this is the contractile vacuole. How long does it take to contract, how long to expand? Are there any visible contents ? Is there more than one contractile vacuole ? In the endosarc a round, clear body may be found, which does not change shape; this is the nucleus. Is it more solid than the surrounding protoplasm? What is its shape? Size? Is it always in the same place? There are often also in the endosarc various foreign bodies which serve as food, such as diatoms, desmids, green cells, etc. Draw to show structure. 4. Stained Specimens : In a prepared specimen, stained and mounted, observe nucleus, ectosarc, indosarc, etc. Draw. [41] GENERAL BIOLOGY B. PHYSIOLOGY. 1. Movements: Is Motion continuous? Regular? How is it produced ? Watch process of formation of a pseudopodium. What part does the ectosarc play in the process? The endosarc? Watch an active Amoeba and trace on paper its path of motion. Are there permanent anterior and posterior ends? Does there seem to be any difference in surface tension between the anterior and posterior ends? Are the currents in the endosarc constant? Indicate in a drawing the course of the currents by arrows. Where are the currents swiftest? Where slowest? Are cilia present on any por- tion of the body? 2. Nutrition: If possible watch the process of taking in food and of its egestion. What does the animal eat? How and where does it take in food ? Are food vacuoles formed ? Is there a definite course of circulation of food within the body? Where is the food digested? How distributed? How are gaseous, liquid and nitrogeneous waste substances expelled from the body? 3. Reproduction is difficult to observe and may be omitted from notes. 4. Irritability : Are there any indications that Amoeba is sensi- tive to stimuli ? Does Amoeba show any reflex movement? Is its behavior more or less varied than that of Parmecium? Enter answers to all these questions in your notes or drawings. G. METAZOA Metazoa are many-celled animals in which there is differentia- tion into at least two body layers, the Ectoderm and the Endo- derm; the former is the organ of relation, the latter the organ of nutrition ; in addition all have ova and spermatozoa. In all meta- zoa the fertilized ovum undergoes repeated divisions (Cleavage) which lead up to the formation of a hollow sphere of cells (Blastula) and from the latter arises a two-layered condition (Gastrula), the outer layer being the Ectoderm, the inner the Endoderm; between these two a third layer, the Mesoderm, is usually formed. Ectoderm and Endoderm consist of cells closely packed to- gether into a layer, such a grouping of cells being called Epithe- lium. Mesoderm, at its first appearance, usually consists of scat- tered cells with large spaces between them, such loosely connected [42] GENERAL BIOLOGY cells being called Mesenchyme; later closely packed layers of mesoderm cells may be formed that are known as Mesothelium. The cells of the different layers of the gastrula differ from one another, and in the course of further development differentiations appear among the cells of the same layer. In this way Tissues, i. e., differentiated groups of like cells and their products. From the two primitive tissues, epithelium and mesenchyme present in the blastula and gastrula, all other tissues are derived, as shown here- with. i. EPITHELIUM gives rise to a. Epithelial tissue b. Muscular tissue c. Nervous tissue d. Germinal tissue 2. MESENCHYME gives rise to : a. Connective tissue b. Skeletal tissue c. Vascular tissue d. Storage (reserve) tissue These different tissues will be studied in the laboratory in con- nection with each animal considered. The various functions of animal life, which in the Protozoa are all performed by a single cell, are performed in the Metazoa not only by many cells and tissues but also by groups of different tissues united to form Organs, each with a specific function, and by groups of organs united to form Systems, each having some one general function, as shown in the following table : FUNCTIONS I. METABOLISM II. REPRODUCTION III. IRRITABILITY Ingestion Digestion Egestion Respiration •{ Excretion Circulation Assimilation Growth Dissimilation f Asexual ( Sexual f Reception of Stimuli | Transmission of Stimuli Coordination Movement ORGANS Mouth, Teeth Stomach, Intestine Anus Trachea, Lungs, etc. Kidneys, Bladder, etc. Heart, Arteries, Veins, etc. Have no special organs or sys- tems No special organs or systems Ovaries, Testes, Uterus Sense Organs Nerves Ganglia, Brain Muscles SYSTEMS Alimentary Respiratory Excretory Circulatory Genital Nervous Muscular [43] GENERAL BIOLOGY These various organs and systems will be considered in detail in connection with each of the animals studied. DEVELOPMENT OF OVUM In prepared slides of Echinoderm eggs observe the following stages: — i. Cleavage; i-cell, 2-cells, 4-cells, 8-cells, i6-cells, 32- or 64-cells. Observe the appearance of a cleavage cavity after the 8-cell stage. 2. Blastula: — Observe the hollow sphere composed of a single layer of cells (Epithelium). Are there any indications that scat- tered cells (Mesenchyme) migrate into the cavity of the blastula (Blastocoel) ? 3. Gastrula: — Note the flattening and ultimate infolding of the blastula at one pole. Do the cells at this pole differ in appearance from the others? The infolded cells constitute the endoderm, the outer layer the ectoderm. The infolded cavity is the Gastrocoel, or digestive cavity; the opening to the exterior is the Blastopore. Draw and label the stages and structures named above. HYDRA VIRIDIS OR FUSCA, Freshwater Hydra. (Phylum Cnidaria, Class Hydrozoa.) Read: Calkins, Biology, pp. 76-102; or Parker, Elementary Biology, pp. 221-236; or Parker and Parker, Practical Zoology, pp. 2&g-3i4', or Woodruff, Foundations of Biology pp. 118-121. A Metazoan which throughout life remains in a two layered con- dition, like a gastrula. Observe with naked eye, or with pocket lens, the hydras in a jar of water where they have been undisturbed for some time. Notice the general habitus of body, method of obtaining food, etc. Transfer a hydra to a slide with plenty of water, and observe with the dissecting microscope ; afterward cover, supporting the cover glass so as not to crush the animal, and examine wtih the low power of the compound microscope. I. FORM. The body: What is the general shape? Do its length and breadth vary? It is usually attached at one end, the foot, by a kind of sucking disk and terminates at the other in a conical pro- jection, the hypostome, with the mouth at its summit. The mouth is a small aperture, but it can be greatly dilated to take in food. It opens into a central digestive cavity, the enteron. The tentacles [44] GENERAL BIOLOGY are hollow processes of the body wall. How many are there? Com- pare the number of tentacles in brown and in green hydras. Is there more than one circle of tentacles? Observe the knob-like swellings on the tentacles. Measure the length of the tentacles when expanded ; when fully contracted. For what purpose are the tentacles used? Buds: — Young hydras of various sizes and stages of development may be attached to the sides of the parent. Are colonies formed by budding? Why? Draw an entire animal, with all the parts named above. II. STRUCTURE. 1. The body wall of the animal is composed of two layers of cells, one within the other, (a) The Ectoderm is the outer layer. What is its color? How much of the thickness of the body wall does this layer form? (b) The Endoderm is the inner lining of the body cavity (digestive cavity). In the green species (Hydra viridis) it contains chlorophyll bodies; in the brown species, H. fusca, it contains "sooty corpuscles." Which layer is the thicker? (c) The supporting layer or Mesoglea is a thin gelatinous layer between the ectoderm and the endoderm. 2. The tentacles. Examine a tentacle with the high power. Of how many layers is it composed? Focus up and down so as to obtain views (optical sections) at various levels. Is the tentacle hollow or solid? The elements of the two layers can be most easily seen in the tentacles. Observe the following: (a) The ectoderm cells are large and conical with their apices directed inward. The boundaries of the outer ends form a mosaic, their inner ends rest directly on the supporting lamella. Do these cells vary in shape when the tentacle is extended or contracted? (b) The interstitial cells are small rounded cells placed be- tween the inner ends of the large ectodermal cells. (c) The cnidoblasts or "thread cells" are modified interstitial cells prolonged at the outer end into a cnidocil or "trigger" and containing an oval, highly refractive capsule, the nematocyst. The capsule is filled with fluid and contains a spirally wound filament formed by the doubling in of the wall of the capsule at one pole. The nematocysts form knob-like swellings on the tentacles. They are of two kinds : ( I ) smaller, more numerous ones situated at the bases of the longer cnidocils and containing short stout threads; (2) larger ones lying near the middle of each knob-like [45] GENERAL BIOLOGY swelling, globular in shape when seen from the face, flask-shaped when seen from the side; they contain long, slender filaments armed with barbs at the basal end. Run in a little iodine and ob- serve the ejection of the threads of the nematocysts. Note that the threads are turned inside out in the process of discharge, the basal portion being discharged first. What is the use of the barbs? The hollow thread ? The fluid in the cysts ? Do nematocysts occur anywhere else than on the tentacles ? (d) The endoderm cells line the cavity of the tentacles. They are large and some of them bear flagella by which currents are caused. Focus on the middle of the thickness of a tentacle and observe the flagella on the endoderm cells and the nutrient particles streaming up and down the cavity of the tentacle. What difference can you detect in the relative numbers of these elements (cells) in the various parts of the body? Make drawings of a tentacle to show the characteristic layers, and cellular elements. III. REPRODUCTION. 1. Asexual reproduction occurs by the formation of hollow out-growths from the sides of the body wall. Each of these acquires a mouth and tentacles at the distal end of its body and finally, constricting at the base, separates from the parent animal. Look for such buds in various stages of development. 2. Sexual reproduction: hydra is monoecious (hermaphro- ditic), the same animal producing eggs and spermatozoa. (a) The spermaries are swellings of the body wall produced by the local multiplication of interstitial cells, and covered on the outside by a cap formed of large ectodermal cells. The spermaries are situated just below the tentacles. How many do you find? Is the number constant? Find a ripe spermary and observe the movement of the spermatozoa within the capsule. By gentle pressure upon the cover glass break open the capsule and observe the swimming of the spermatozoa and their size and shape. (b) The ovaries usually develop later than the spermaries and are formed near to the base of the animal. How many do you find? Is the number the same in the brown and green species? Single cells of each ovary enlarge to form the ovum, while the other cells nourish it and form a capsule about it. Make drawings of buds and of the sexual or gams. [46] GENERAL BIOLOGY IV. STUDY OF PREPARED SECTIONS. Examine series of transverse and longitudinal sections of hydra prepared by the paraffin method, and note the large central enteron surrounded by a body wall of two layers of cells. 1. The ectoderm. Is it of uniform thickness? In it observe: (a) Large squarish or conical cells. Do they contain nuclei and vacuoles ? Their basal ends are continued into muscle fibres (Klein- enberg's Fibres) which are mainly longitudinal in direction, and in cross-section appear as a row of refractive dots on the sur- face of the supporting lamella. Over the outer surface of these cells is a thin cuticle. At the foot the ectoderm cells are more columnar and contain granules, (b) Interstitial cells are present over the body and tentacles but absent in the foot; they stain deeply, (c) Nematocysts, abundant in the tentacles, less numerous on the body and absent on the foot. Are they found in the endo- derm? 2. The supporting lamella. A thin, deeply staining layer be- tween the ectoderm and the endoderm. Is it composed of cells? 3. The endoderm cells; variable in shape and size. They are of two kinds: (a) Larger cells, irregular in shape and size, con- taining vacuoles, and with the nucleus flattened and near the basal end. In H. viridis the basal part of each cell contains rounded bodies, chloroplastids, coated with chlorophyll. In H. fusca similar bodies are present, "sooty corpuscles," devoid of chlorophyll. The basal ends of these cells are often prolonged into muscular pro- cesses like those of the ectoderm cells, but transverse in direction. (b) The smaller secretory cells, pear-shaped and lying between the bases of the larger ones. These last mentioned cells are num- erous in the walls of the hypostome but fewer elsewhere. Their protoplasm is granular and they stain deeper than the larger cells. Make a drawing of each section. V. STUDY OF ISOLATED CELLS. Place living hydra on a slide, draw off the water and cover for a few minutes with a drop of Haller's Fluid. Cover and tap gently upon the cover glass to separate the cells. Select and draw good examples of the varieties of cells men- tioned. . v « * * .".• ^ r , ML is [47] GENERAL BIOLOGY LUMBRICUS TERRESTRIS, The Earthworm (Phylum Annelida, Class Chaetopoda, Order Oligochaeta. ) Read : Darwin, The Formation of Vegetable Mould through the Action of Worms ; also Sedgwick and Wilson, General Biology, pp. 41-104, or Calkins, Biology pp. 131-161 or Parker and Parker, Practical Zoology, pp. 318-341 ; or Woodruff, Foundations of Biology, pp. 121-129. A. EXTERNAL CHARACTERS. Place a preserved worm in a dissecting dish, cover with water, and observe : — 1. General form, color, iridescence. 2. Anterior and posterior ends? How do they differ? Dorsal and ventral sides; how distinguished? Right and left sides; are they symmetrical? 3. Body divided into metameres, or somites by grooves around it. Count the somites. 4. Between the 2Qth and 35th somites, a swollen light-colored region, the clitelluni. How many somites does it cover? 5. The setae, stiff light-colored spines projecting from the sur- face of each somite, and easily felt with the fingers. How many are there on each somite, and how are they arranged ? Do they all point in the same direction ? Remove a seta, mount it in a drop of water, and examine it under the compound microscope. What is its general shape? Do its ends differ? 6. The cuticle. Soak an alcoholic specimen in water for a few minutes, and then strip off some of the cuticle. What is its color? Texture? Examine some from the ventral surface and note the cuticular sacs in which the setae are imbedded. What is their shape? Arrangement? Examine the cuticle under the high power and observe the striae crossing one another at right angles. At some of the intersections are pores to allow the escape of secre- tions of the epidermis. 7. Apertures, (a) The mouth, in front of the first somite, and below a protuberant lobe, the prostomium, which runs across the first somite on its dorsal surface, (b) The anus, a vertical slit at the end of the last somite. The following apertures are not easily seen, and must be looked for with a hand-lens, or a dissecting microscope. They can often be seen by drying the surface of the [48] GENERAL BIOLOGY worm, and then gently squeezing it, when a small drop will come out of the openings, (c) Sexual apertures, (i) Openings of spermaducts, or vasa def erentia ; two openings surrounded by swollen areas on the ventral surface of the i5th somite. From these openings, grooves are often found passing back to the clitel- lum. (2) Openings of oviducts ; two small pores on ventral surface of the I4th somite. (3) Openings of the seminal receptacles or spermatheca, two openings on each side between the 9th and loth, and loth and nth somites, in line with the outer row of setae and posterior to them, (d) Nephropores ; openings of the segmental organs or nephridia ; two openings in each somite, one on each side, just dorsal to the ventral pair of setae. Draw the anterior and posterior portions of the body to illus- trate all that you hcwe observed. B. INTERNAL ANATOMY. Extend the worm, ventral side down, in a dissecting pan, and fasten firmly by a pin at each end (the anterior one through the prostomium only) ; cover with water, and cut open carefully from behind forward with fine scissors making the incision along the dorsal side a little to one side of the dorsal median line. Do not cut deep, but merely through the body wall. Carefully cut through the partitions or septa along each side, stretch out the body wall to right and left, and fasten with pins. Observe the following structures, dissecting as little as possible to make them out : I. GENERAL FEATURES. 1. Body wall, thick and firm and composed of three layers: (a) A thin cuticle on the outside; (b) a more or less colored layer, the epidermis ; (c) a light-colored, and much thicker layer internal to the epidermis, the muscular layer. 2. Body cavity or coelum, with the digestive tract passing through it from mouth to anus, and septa or transverse partitions dividing it into as many chambers as there are somites. Each sep- tum passes from the digestive tract to the body wall. What is the relation of the septa to the external grooves? 3. Seminal vesicles, large lobed bodies between the io!h and 1 5th somites, partly covering the digestive tract. 4. Dorsal or supra-intestinal blood vessel, generally full of [49] GENERAL BIOLOGY blood, and seen on top of the digestive tract, along the dorsal median line. In the 7th to nth somites it gives off laterally 5 large pulsatile vessels, or "hearts," which pass around to the ventral side of the digestive tract. 5. Nephridia; or segmental organs, light-colored fluffy bodies attached to the posterior side of the septum, right and left, in each somite. Make a sketch to show the above organs in place. II. DIGESTIVE TRACT. Make out the following parts in the order named. 1. Pharynx, thick and muscular, extending back into the 6th somite and attached to the body wall by many radiating muscles. 2. Oesophagus, the narrow portion from the 6th to the I4th somites. On its sides in the nth and I2th somites are 3 pairs of light colored swellings, the calciferous glands. Place one of the glands in a watch glass of dilute hydrochloric acid ; explain results. 3. Crop. A large thin walled expansion in the I5th and i6th somites. 4. Gizzard, immediately posterior to the crop in the I7th and i8th somites, and with thick muscular walls. 5. Stomach-intestine, extending from the gizzard to the anus. It expands in each somite, and is contracted by each septum. Along its dorsal surface is a dark colored body, the liver, or pancreas. Cut open the intestine along one side and note the large ridge on its dorsal internal surface, the typhlosole. Cut open the gizzard and the crop, and note the lining of these structures and the character of the food contained. Make a sketch of the digestive tract, showing the above men- tioned structures. III. VASCULAR SYSTEM. The dorsal blood vessel and the "hearts" have been mentioned. To observe the other principal vessels, remove the crop, gizzard, and oesophagus. Cut the oesophagus away from the pharynx, pull it gently back while cutting the septa which hold it in position, and leaving all the other organs in place. This will lay bare the white nerve cord on the median ventral line of the body cavity. Upon it the supra-neural blood vessel may be seen. In removing these parts of the digestive tract, the sub-intestinal vessel may be seen on its ventral side. [50] GENERAL BIOLOGY IV. REPRODUCTIVE SYSTEM. 1. Seminal vesicles (for storage of own sperm) ; composed of 3 pairs of white sacs arising from a median portion below the oesophagus. This median portion is subdivided into an anterior and a posterior part. 2. Seminal receptacles (for receiving sperm from another worm) ; 2 light colored sacs on the ventral surface of the body- wall, on each side of the median line and attached to septa be- tween the 9th and loth and nth somites. 3. Ovaries ; very small light colored bodies with pointed tips and rounded bases on the anterior wall of the I3th somite, not very far from the middle of the ventral surface, one on each side, right and left. 4. Oviducts ; these are also not easily seen, but form what ap- pear as thickenings of the wall between I3th and I4th somites. 5. Cut off the lateral lobe of a seminal vesicle, cut open its median part and carefully wash out its soft contents to show the following structures ; great care in dissection and observation is necessary, (a) Vasa Efferentia; large folded or convoluted masses which form the funnel-like openings, one on each side of the median line in the loth and nth somites. From these, delicate thread-like ducts pass back on each side to unite in somite 12 to form the Vas Deferens, which passes along the body wall one on each side of the median line, as far back as somite 15 where it opens to the exterior, (b) Testes; four small white bodies, a pair in each somite, inside the seminal vesicles in part concealed by the funnels of the vasa efferentia, and attached to the posterior surfaces of the septa between somites 9 and 10, and 10 and n, two on the right and two on the left of the median line. Make a sketch of the reproductive system and explain the func- tion of each part. V. NERVOUS SYSTEM. 1. The nerve cord; extending the whole length of body on the median ventral line, lying in the body cavity but near the body wall. In each somite it expands to form a ganglion and gives off three pairs of nerves, (a) Two large pairs arise from the ganglion, (b) One smaller pair arises from the slender part of the cord (connective) near the anterior end of the somite. 2. Circum-oesophageal nerve ring. Raise the oesophagus and [51] GENERAL BIOLOGY trace the nerve cord anteriorly to its division into right and left halves which pass around the digestive tract to form a ring which unites with the brain on the dorsal side of the pharynx. 3. Brain ; connected as above shown with the ventral nerve cord, but lying dorsal to the pharynx. Note the nerves given off from the brain and also from the connectives on the side of the pharynx. Make a drawing of the nervous system. VI. BODY WALL. Pin out part of the body wall quite flat and note that the mus- cular layer of the wall is interrupted along four longitudinal lines in which are the setae in sacs or setigerous glands ; four of these occur in each somite. Between somites 12 and 13 some of these glands are conspicuously large; tease out one and note under the microscope the color, shape and hardness of the setae. C. ANATOMY AND PHYSIOLOGY OF LIVING SPECI- MENS. I. MOVEMENTS. 1. Place a worm upon moist filter paper and observe the direc- tion and method of movements. 2. In small light-colored worms note the contraction of the dorsal blood vessel and the movements of the blood toward the anterior. 3. Gently touch different parts of the body and note which are the most sensitive. 4. Place the worm under a glass vessel with some cotton satur- ated with chloroform, the vapor of which will render the animal insensible ; when motion has ceased remove the worm ana cut it open as in the specimen previously dissected, but only in the an- terior region and a little to one side of the median line. Keep the specimen wet with physiological salt solution. II. VASCULAR SYSTEM. If the specimen is not quite dead observe : a. The contraction of the hearts, dorsal vessel, and sub-neural vessel ; in the latter the wave of contraction passes backward. b. Small blood vessels passing from the dorsal vessel to the digestive tract, and from the ventral vessel to the body wall and to the septa. [52] GENERAL BIOLOGY c. Fine vessels seen upon the septa, body walls and the nephridia. III. COELOMIC FLUID. Puncture the body where not yet opened and take out in a fine pipette some of the fluid of the body cavity, examine under a high power and note : — 1. White amoeboid corpuscles. 2. Yellow granules, from the chlorogogue cells (See D II. i). 3. Bacteria or other foreign bodies, especially Gregarina, which are often present. D. HISTOLOGY. I. BODY WALL. Examine prepared transverse sections of the body ; observe body wall, now seen to be made up of five layers : 1. Cuticle, a thin non-cellular layer (membrane) often torn off. 2. Deric epithelium or epidermis, a single layer of cells many of which are swollen (gland .cells). 3. A thin outer layer of circular muscle fibres with blood ves- sels and connective tissue nuclei among them. 4. A thick layer of longitudinal muscle fibres or plates, ar- ranged in elongated groups of elliptical form. 5. Peritoneum or coelomic epithelium, a thin layer of granular protoplasm containing nuclei, lining the body cavity. II. DIGESTIVE TRACT. In its wall four layers are to be seen. 1. Chlorogogue cells, large and more or less elongated and irregular. 2. An outer layer of longitudinal muscle fibres cut across. 3. A layer of circular muscle fibres and of blood vessels (not easily made out). 4. Enteric epithelium; a single layer of elongated cells with stained nuclei and a thin cuticle over their central ends through which fine cilia project into the lumen of the gut. III. NERVOUS SYSTEM. In a transverse section of the ventral cord, note : 1. An outer muscular sheath or coat. 2. Large ganglion cells in groups or clusters. [ 53 1 GENERAL BIOLOGY 3. A mesh work of fine fibres. 4. Very large clear "giant fibres" each in a definite sheath. 5. In some of the sections the nerves are to be seen as they pass from ganglion cells to the body wall. IV. BODY CAVITY. Some of the sections will show the following structures : 1. The corpuscles of the coelomic fluid. 2. Blood vessels cut across and filled with coagulated blood. 2. Mesenteries or dorsal and ventral membranes connecting the digestive tract with the body wall on the median line. 4. Septa and nephridia cut at various angles. Make a full-page outline of a cross section to show all of these organs and in the ventral sector fill in histological details of (a) Body Wall, (b) Body Cavity and Nervous System, (c) Digestive Tract. CAMBARUS, The Crayfish. (Phylum Arthropoda, Class Crustacea.) Read: Huxley, The Crayfish, pp. 1-226; also Parker and Parker, Practical Zoology, pp. 346-37-; or Calkins Biology, pp. 166-186. A. GENERAL CHARACTERS. I. BODY. Note that the animal has a body proper and a series of paired appendages. The body is bilaterally symmetrical and divided into a posterior jointed abdomen and an anterior portion the cephalo- thorax. The entire body is covered by a hard calcarious shell the exoskeleton which is flexible at the joints where movement may take place. II. APPENDAGES. Note that all of the appendages are jointed that they are attached in pairs to the ventral surface of the body and that they vary much in sz'ze and form. Make a drawing of the crayfish as seen from the dorsal side. [54] GENERAL BIOLOGY III. APERTURES. Make out the following apertures in the body wall : 1. The mouth seen under the anterior part of the cephalothorax after separating from one another the crowded appendages. 2. Anus, a much elongated slit upon the lower side of the ter- minal piece of the abdomen, the telson. 3. Genital openings on the basal joints of the legs: (a) In the male on the delicate papilla on the last appendage of the cephalo- thorax (one on the right and one on the left) ; (b) in the female an opening with a valve-like edge on the antepenultimate appendage of the cephalothorax (one on the right and one on the left). 4. Auditory organs : A small opening on the appendage (anten- nule) just under each eye stalk. 5. Green glands : A large opening on the ventral side of the first joint of the next following appendage (antenna) on each side. B. ABDOMEN. This is made up of six segments or somites bearing appendages and a terminal, seventh piece, the telson, which is subdivided by a transverse hinge and bears the anus. Carefully examine the third abdominal somite. The following surfaces are found upon it: (a) Tergum, the dorsal arched portion overlapped anteriorly by the preceding tergum. (b) Sternum, the ventral portion between the appendages, composed of a transverse bar and a more calcified cuticle where movements take place in bending the abdomen, (c) Pleuron, the downward projecting portion on each side, overlapped in front by the pleuron of the preceding segment. The appendages are attached to the body by soft flexible cuticular parts of the exo- skeleton. Each abdominal appendage consists of the following parts : a. Protopo-dite : This is the proximal part of the appendage and is divided into a long joint, and a small ring-like piece by which it is attached. It bears distally two parts, b. Endopodite : This is the part nearer the middle line. c. Exopodite: The portion farther from the middle line. C. CEPHALOTHORAX. a. The large shield-like part of the exoskeleton covering the cephalothorax above and on the sides is the carapace, which is pro- longed in front into the frontal spine or rostrum. (55} GENERAL BIOLOGY b. A groove, the cervical suture runs across the carapace and marks off the head from the thorax. c. On the ventral side, the region between the appendages is very narrow; the anterior appendages project forward and not downward as do the more posterior ones. d. The locomotor appendages are attached to the thorax ; the posterior pair are upon a movable somite while all the others arise from a fused single mass continuous with the head. e. The free lateral part of the carapace, above the appendages, is the branchiostegite. Raise its edge and see that it covers the gills. f . Respiratory organs : Remove one of the branchiostegites, study the gills under water and observe: Six of them are attached to the appendages, podobranchiae ; eleven of them are attached to the soft cuticle joining the appendages to the body arthrobranchiae. At the anterior end of the branchial cavity a canal leads forward toward the mouth and in this lies the flat part of the second maxilla called the scaphognathite. D. APPENDAGES. Starting at the posterior end carefully remove all of the ap- pendages from one side with all the basal parts of each, see which are alike and then draw one of each kind or set (15 figures for the female, 15 for the male) keeping all of the small ones in water in watch glasses. I. ABDOMINAL APPENDAGES OR SWIMMERETS. Composed of a two jointed protopodite, and an exopodite and endopodite each with many joints ; found on all but the first and sixth somites (and the second also in the male) where the appen- dages are more or less modified. II. THORACIC APPENDAGES. There are five pairs of ambulatory, and three pairs of mastica- tory appendages (the maxillipedes.) a. The posterior pairs of ambulatory appendages have the fol- lowing seven joints: (i) Coxopodite, the short and very thick basal joint. (2) Basipodite, a very small and conical joint. (3) Ischiopodite, cylindrical and with a groove around it. (4) Mero- podite, very much longer than the last. (5) Carpopodite, about half as long as the last. (6) Propodite, slender and long. (7) Dactylopodite, the short, pointed terminal piece. Of these (i) and [56] GENERAL BIOLOGY (2) probably correspond to the protopodite of the abdominal ap- pendages, and the other five to the endopodite, as may be seen by comparing all of the other appendages with the third maxilliped. b. The next pair of appendages have in addition a branchia and epipodite upon the coxopodite extending up into the branchial chamber. c. The third and fourth appendages (counting forward) differ in having the propodite produced opposite the dactylopodite to form a pair of forceps. d. In the large anterior pair of ambulatory appendages, the chelae, the forceps is greatly enlarged and the basipodite and ischio- podite are united into one piece. e. The third or posterior maxilliped should be carefully studied. Note: (i) The large basal part, protopodite, bears a long five jointed endopodite and a slender many jointed external expedite, besides a curved lamella, epipodite, lying in the branchial chamber and bearing a branchia. (2) The protopodites and endopodites make up together a seven jointed organ like the ambulatory ap- pendages. f. The second maxilliped differs from the last mentioned, chiefly in the size of the endopodite. g. In the first maxilliped the endopodite is short and flat, the protopodite two jointed and foliaceous, the epipodite has no gill. III. CEPHALIC APPENDAGES. These are the maxillae, mandibles and antennae. (a) Second or post maxillae; the endopodite is not jointed, while the two parts of the protopodite are subdivided or cleft ; the large oval plate, scaphognathite, acting to bail water out of the branchial chamber, represents the epipodite and probably also the exopodite. (b) First maxilla; this is very small and lies close to the mandible. It is divided into three parts representing the coxopodite, basipodite and endopodite. (c) Mandibles ; each has a strong basal part bear- ing a two jointed palp or endopodite. (d) Post antenna (antenna proper) ; each has a two jointed protopodite with the opening of the green glands on a tubercle on the proximal joint, the scale-like plate is the exopodite and the long, filiform, many jointed part is the endopodite. (e) First antenna (antennula) ; here the pro- topodite has three joints and bears a long many jointed endopodite, and a similar exopodite, while upon its large proximal joint is the opening of the auditory organ, surrounded by hairs. [57] GENERAL BIOLOGY Compare your drawings of the different kinds of appendages, labelling homologous parts with the same name; the 19 pairs may be regarded as modifications of such a one as the third maxillipede. E. INTERNAL ORGANS. Pin the crayfish down under water, dorsal side up, and carefully remove the carapace bit by bit with strong forceps, commencing at the free posterior border. I. CIRCULATORY SYSTEM. 1. Heart. Posterior to the cervical suture, a median chamber is laid bare, the pericardial sinus, within which lies the polygonal, flat heart which has six openings into the pericardinal sinus, two on the dorsal surface, two on the lateral surfaces, and two on the ventral surface. 2. Arteries. Running anteriorly from the heart are : (a) the opthalmic artery in the mid-line and lateral to this, (b) a pair of antennary arteries, (c) a pair of hepatic arteries; posterior to the heart are: (d) the median abdominal artery from which (e) the sternal artery runs to the ventral side just posterior to the heart. Draw the heart and arteries. II. REPRODUCTIVE ORGANS. Carefully remove the heart to expose the reproductive organs. (a) Testes. In the male; these form a Y-shaped mass with the smallest of the three lobes passing back along the median line. (b) Vas deferens. Cut away the thorac wall on one side and trace the much convoluted tube from the union of the posterior and anterior lobes of the testes down to the external genital opening on the posterior ambulatory appendage on that side, (c) Ovary. In a female specimen the larger reddish ovaries have the same general form and position as the testes in the male, (d) Oviducts. These are short and go directly down from the ovary to the open- ings on the third, or middle, ambulatory appendages. Make a drawing of your dissection, showing all these organs in place. [58] GENERAL BIOLOGY III. DIGESTIVE TRACT. (a) Carefully remove the anterior part of the carapace and notice the very large sac-like stomach anterior to the heart. Pass a probe into it through the mouth and short oesophagus, (b) Dis- sect away the exoskeleton and muscles and follow the intestine from the stomach to the anus. Immediately posterior to the stom- ach is the "mid gut" having a short dorsal diverticulum on it. The remainder of the intestine is the "hind gut." (c) The digestive gland (the so-called "liver") forms a yellow mass opening by a duct on each side of the mid gut. Wash away its contents if the duct cannot otherwise be found, (d) Remove the stomach and cut it open along one side (under water) and note a large round (cardiac), and a narrow posterior (pyloric) portion. The chitinous lining forms in the cardiac portion three conspicuous tooth-like thickenings, the so-called "gastric mill." In the pyloric region ridges, set with hairs, reduce the lumen of the stomach to a nar- row slit. IV. EXCRETORY ORGANS. 1. Green Glands. In front of the stomach is a pair of sac-like structures, the green glands, or nephridia; each consists of a ventral glandular part and a dorsal saccular portion, the latter opens to the exterior by a duct. 2. External Openings. On the basal joint of each antenna observe a papilla with the external opening of the green gland at its summit. Draw a side mew of the digestive tract and excretory organs. IV. NERVOUS SYSTEM. Remove the muscles of the abdomen until the nerve cord is seen along the ventral wall of the body, (a) Note the relation of the ganglionic swellings to the somites, (b) Follow the cord into the thorax; here it enters a canal, the roof of which must be broken off bit by bit with forceps to show the nerve cord. Note the number of ganglia in the thorax, (c) The cord divides at the oesphagus into a right and left half which meet again at the brain. The brain, or supraoesophageal ganglion, lies just posterior to the eye stalks, close to the exoskeleton, and sends a large nerve into each of the eye stalks. Make a drawing of the nervous system. [59] GENERAL BIOLOGY RANA, The Frog. (Chordata, Vertebrata, Amphibia.) Read'. Parker and Parker, Practical Zoology, pp. 1-228; or Holmes, The Biology of the Frog, pp. 1-358. A. GENERAL STRUCTURE. I. EXTERNAL CHARACTERS. Note the smooth moist skin over the entire animal ; the absence of exoskeleton; the head, trunk, two pairs of limbs; the absence of a tail and of a neck. a. The head. Observe: 1. The eyes are prominent and have lids ; the ears are marked out by a modified part of the skin, membrana tympani posterior to the eyes ; the two anterior nares, or nostrils ; the position of the mouth opening; the soft flexible throat and hard parts of the endo- skeleton felt on the dorsal side of the head. Observe the move- ments of the throat in respiration. 2. After the frog has been killed with chloroform (see below II, i, a.) pass a bristle far into the anterior nares and one into the ear through a hole cut in the membrana tympani; on opening the mouth the bristles will indicate its communications with the nostrils and tympanic cavity. The second bristle appears in the Eustachian recess at the side of the posterior part of the mouth. In the male a small opening anterior to this recess leads into the buccal sac which can be distended, by means of a small blow- pipe. Turn the fleshy tongue forward and notice its mode of attach- ment. Note the slit of the glottis and the posterior opening of the mouth into the oesophagus ; pass a bristle into the former and a large probe into the latter. There are thus two median openings from the mouth cavity and six paired openings in the male frog; four in the female. Note the small teeth. b. The Trunk. This tapers towards the posterior end where the cloacal aperture is seen near the dorsal surface. Beneath the skin the hard endo- skeleton can be felt on the dorsal side and on the anterior part of the ventral side. c. The limbs. i. The anterior pair divided each into three regions, brachium [60] GENERAL BIOLOGY (upper arm), antebrachium (fore arm), manus (hand) ; the latter with four digits, the innermost of which bears a swollen cushion in the male. 2. The much longer posterior pair each divided into three re- gions, femur (thigh), crus (shank), pes (foot), the latter with five long digits connected by a web. There is a large firm promi- nence on the inner side of the ankle; callosites are found under the joints of both pes and manus. Draw entire frog and label parts named above. II. INTERNAL CHARACTERS. i. GENERAL. a. Place the frog under a bell-jar with a sponge saturated with chloroform; when dead pin out under water on its back. b. Cut through the skin along the median ventral line from the posterior end to the jaw (raising the skin from the body and not cutting deep) ; cut transversely at each end of first cut and turn aside the two large flaps thus made. c. On the flap of skin on each side is seen a large vein near the shoulder, the musculo'-cutaneous vein. The muscular walls of the abdomen are covered by a thin, shining connective tissue, sheath, the eponeurosis, through which in the median region is seen the rachis abdominis passing from the pelvis to the sternum and somewhat divided by transverse lines into segments or myotomes. Through this muscle is seen the dark blood of the an- terior abdominal vein in the median line. d. With a pair of forceps raise the body wall and carefully cut it through by a slit to the right of the median line ; continue this cut from pelvis to sternum and make transverse cuts as in the skin so as to throw back a flap of body wall on each side; the left one should show the anterior abdominal vein on its exposed sur- face. e. With forceps raise the sternum and carefully cut off the fibrous bands seen passing to soft organs dorsal to it ; with strong scissors cut through the sternum and other hard parts on the median line carefully holding it up away from the soft parts dorsal to it. Turn each half outward and pin firmly; pin the anterior limbs out at full length. f. The liver is conspicuous, forming a large brown mass with the pericardial sac just anterior to it. [61] GENERAL BIOLOGY g. Anterior to the heart note the broad flat transverse mylo- hyoid muscle through which can be seen the long first-vertebral nerve or hypoglossal. Note also the hard protuberant larynx and on each side of this a small soft body, the thyroid gland. 2. CIRCULATORY SYSTEM. Carefully cut away the membranous pericardium to expose the heart ; then with great care clean off bit by bit the tissue covering the vessels at the anterior end of the heart. a. Heart. (i) Note the firm conical posterior portion of the heart, the ventricle. (2) The cylindrical truncus arteriosus arises from the right side of the base or anterior end of the ventricle and passes obliquely forward to divide into two large branches. (3) The atrium forms a thin walled sac dorsal to the truncus and anterior to the ventricle (it is divided internally into two auricles). (4) The sinus venosus can be seen by carefully raising the ventricle to one side ; it forms a thin sac dorsal to the ventricle and atrium and receives three large veins (two anterior or superior venae and one large posterior or inferior vena cava. ) Two pulmonary veins open into the left auricle by a single opening. b. Pulsations of the heart. Observe: (i) A regular sequence of contraction and dilation. (2) The atrium contracts, then the ventricle, and immediately after the truncus. (3) On raising the ventricle, the sinus venosus can be seen to contract before the atrium. The contraction proceeds in the same order as that followed by the blood in passing through the heart. c. Arteries (the efferent vessels). The blood is carried through the common truncus arteriosus which gives rise to a right and left subdivision. Each of these divides into three branches derived from three embryonic arches : 1 i ) The most anterior branch or arch, the carotid, bears near its point of origin a pinkish glandular enlargement, the carotid gland. The common carotid divides into (a) the external carotid — situated near the median line but distributed to the superficial tissues of the head, and (b) the internal carotid which passes into the cranial cavity to supply the brain and sense organs (eye, in- ternal ear). (2) The middle or systematic arch unites with its fellow on the [62] GENERAL BIOLOGY opposite side to form the dorsal aorta. Trace the systemic arches dorsally around the oesophagus to their point of union. The dorsal aorta extends to the posterior end of the body cavity where it di- vides into two branches, the right and left iliac arteries to the hind legs. From each systemic arch a subclavian artery passes to the front leg. Named in order, beginning with the most anterior, the following branches arise from the dorsal aorta: (a) coeliaco- mesenteric (to liver, stomach and intestine), (b) ovarian (or spermatic) to gonads, (c) renal (to kidneys). (3) The posterior or pulmonary arch runs to the lungs giving off on its way a large cutaneous branch which carries blood to the skin for aeration when the animal is submerged. d. Veins (the afferent vessels). The blood is returned to the heart through five main trunks, right and left anterior venae cavae, a median posterior vena cava, and right and left pulmonary veins. The venae cavae are joined together at the heart to form the sinus venosus, which opens into the right auricle. (1) Each anterior vena cava is formed by the confluence of three veins: (a) external jugular, from face and throat; (b) in- ternal jugular, from cranial cavity; (c) axillary formed by union of the subclavian from the arm, and the musculo-cutaneous from the skin and abdominal muscles. (2) The posterior vena cava arises by the union of several large branches coming from each kidney (renal vein) ; anteriorly it re- ceives a large vein from each lobe of the liver (hepatic veins). The anterior-abdominal vein (unpaired) arises from the right and left iliac veins from the legs and runs to the liver and heart. (3) Portal veins (veins arising from and ending in capillaries) : Blood from the hind limbs is carried to the kidneys by the renal- portal veins which enter the kidneys along their external borders. The hepatic-portal vein carries blood from the stomach and in- testine to the liver. (4) The right and left pulmonary veins open into the left auricle through a common opening. Make a diagram of the heart and vascular trunks. Before leaving the laboratory open the skull and spinal canal as follows : Cut the skin along the median dorsal line and reflect it. With forceps pick off the muscles from the vertebrae. Open the neural canal by cutting into the membrane just posterior to the [63] GENERAL BIOLOGY skull, and bit by bit pick off the roof of the brain cavity with strong forceps. Tag your specimen with your name and put it into a jar of preserving fluid until the next exercise. 3. DIGESTIVE SYSTEM. (Preserved Specimen.) Posterior to the heart note: (i) The liver with its larger left lobe divded into two parts ; on raising the posterior border, the gall bladder is seen as a greenish sac on the right side; also the hepatic-portal vein, which enters the left lobe of the liver. The stomach, an elongated white body on the left side under the pos- terior edge of the liver. (3) A convoluted tube, the intestine pass- ing from the stomach to the right and then posteriorly to finally enter the pelvic cavity as an expanded rectum. It is slung by a delicate membranous fold of peritoneum, the mesentery, which is full of blood vessels. (4) The fat masses, long slender yellow masses on each side in the dorsal part of the body cavity anterior to the reproductive glands. Cut off all the dorsal part of the liver with strong scissors, cut open the body wall in the pelvic region without injuring the rectum. ( I ) the cloaca is now exposed ; a probe may be run through it into the rectum. (2) Uncoil the intestine and fasten to one side to ex- pose the spleen (a small red body near dorsal part of mesentery). (3) The pancreas is also seen as a pale-colored compact mass in the mesentery between the stomach, liver and small intestine. The bile duct from the gall bladder passes through the pancreas to open into the small intestine. (4) The oesophagus is a short straight tube ; pass a probe from the mouth into the stomach. Make a drawing of your dissection to show all of these parts. 4. URINO-GENITAL SYSTEM. (Preserved Specimen.) Remove the stomach, liver, mesentery and connected organs. (i) Posterior to the fat masses lie the reproductive glands, in the male yellow oval testes; in the female, folded or lobed, yellow ovaries (when the eggs are nearly ready for laying, each is a large sphere, light on one side and dark on the other, and the lobes of the ovary are so distended by great masses of ova as to fill most of the body cavity). (2) Sexual ducts: in the male, each testis sends numerous small thread-like ducts, vasa-efferentia, into the kidney lying just posterior and dorsal to it. In the female the oviduct is a long convoluted tube opening into the cloaca posteriorly and passing forward on each side to open by a funnel into the body [64] GENERAL BIOLOGY cavity near the oesophagus. It has no connection with the ovary. (3) The kidneys are elongated, red masses close to the vertebral column ; on the ventral surface of each is an elongated yellowish body the adrenal body. Entering the kidneys on their external side are the renal-portal veins; leaving the kidneys on their mesial side are the branches of the inferior vena cava. (4) Ureter, a whitish duct on each side passing from the outer side of the kidney into the cloaca. (In the male this serves also as a vas deferens.) (5) The urinary bladder, a large bi-lobed sac ventral to the rectum (it can be inflated through the cloaca by means of a blow-pipe). Make a diagram of the urino-genital system of your specimen. 5. THE NERVOUS SYSTEM. (Preserved Specimen.) a. DISSECTION. Finish the exposure of the brain and spinal cord. With strong forceps pick off bit by bit the roof of the skull, and remove the dorsal part of the vertebral arches in the same way. A delicate pigmented membrane (pia mater) covers the brain and the spinal cord but may be concealed in the latter region by soft substance (coagulation products after death) that can be washed away with a stream of water from a pipette. b. THE BRAIN. If this is not injured in exposing it, note : 1. Rhinencephalon or anterior part made up of two olfa\ctory lobes extending anteriorly from a common median part as two cylindrical so-called olfactory nerves to branch inside the nasal chambers. 2. Prosencephalon or cerebrum composed of two large masses, the cerebral hemispheres, separated by a median groove, pheres. Upon it is a very small pineal gland and below this a cen- 3. Diencephalon, a mass between and posterior to the hemis- tral cavity, the third ventricle, bounded on the sides by masses called optic thalami. 4. Mesencephalon, showing on the dorsal side a pair of large, rounded, hollow bodies, the optic lobes. 5. Mentencephalon or cerebellum, a small mass extending across the anterior edge of a large triangular cavity, the fourth ventricle. 6. Myelencephalon or medulla oblongata, forms the remainder [65] GENERAL BIOLOGY of the brain posteriorly and contains the fourth ventricle which is covered by a vascular part of the pia mater. c. CRANIAL NERVES. Ten pairs of nerves arise from the brain : 1. Olfactory, a direct anterior continuation of the olfactory lobe. 2. Optic, also part of the original neural tube, arises from the optic chiasma on the ventral side of the diencephalon. 3. Oculomotor, arises from the ventral surface of the mesen- cephalon just posterior to the optic chiasma; distributed to some of the eye muscles. 4. Patheticus, arises from the dorsal part of the brain just pos- terior to the optic lobes ; distributed to one of the eye muscles. 5. Trigeminal, arises from the side of the anterior part of the medulla; distributed to the scalp, face and jaw. 6. Abducens, arises near ventral fissure from anterior portion of medulla; distributed to one eye muscle. 7. Facial, arises from the side of the medulla near the origin of the 5th with which it leaves the cranial cavity by a single orifice; distributed to the facial area. 8. Auditory, arises from the side of the medulla with the 7th; sensory nerve to the ear. 9. Glossopharyngeal, arises with the loth, just posterior to the 7th and 8th ; distributed to the tongue and pharynx. 10. Vagus (pneumogastric), arises in conjunction with the 9th ; distributed to larynx, heart, stomach and lungs. d. SPINAL CORD OR MYELON. 1. Forming the continuation of the medulla oblongata poste- riorly, it rapidly tapers at about the fifth or sixth vertebra to form a slender filament. On its dorsal surface is a median line, the dorsal fissure. 2. On each side ten spinal nerves arise from the cord. By care- fully raising the cord a little, towards the posterior end, and search- ing with a pocket lens, the nerves are seen to arise each by two roots, one dorsal, one ventral. Make a sketch of the central nervous sytem and cranial nerves as thus exposed. [66] GENERAL BIOLOGY e. REMOVAL OF CENTRAL NERVOUS SYSTEM. 1. Cut the olfactory nerves away from the skull, gently turn the brain back cutting all the nerves close to the skull and thus remove as entire as possible the brain and spinal cord. Place in a dish of water and study the ventral side with a pocket lens. 2. Optic cJiiasm or commissure, a transverse elevation at the posterior end of the cerebral hemispheres continued up on the sides of the brain towards the optic lobes as the optic tracts, and giving rise in the other direction to the optic nerves, (cut off in removing the brain). 3. Tuber cinereum, a rounded somewhat two-lobed elevation posterior to the chiasm, continued ventrally into the conical in- fundibulum, which bears at its lower end a small conical mass, the pituitary body or hypophysis cerebri. 4. Crura cerebri, the large nerve bundles, extending anteriorly on each side from the medulla toward the cerebral hemispheres. 5. Ventral fissure, a median longitudinal groove along the ven- tral side of the medulla and spinal cord. Draw the ventral surface of the brain and spinal cord. f. PERIPHERAL NERVES. a. Spinal Trunks. In the dorsal wall of the body cavity ob- serve : (i) Sciatic plexus: A number of large nerves, on each side of the dorsal aorta, connected by branches and ending posteriorly in the sciatic (leg) nerve, while anteriorly it is formed from the 7th, 8th, and 9th spinal nerves. (2) Anterior to the sciatic plexus, three pairs of small spinal nerves, the 6th, 5th, and 4th pass ob- liquely outward and posteriorly along the wall of the body cavity. (3) Brachial plexus, formed from the union of the 2nd and 3rd spinal nerves ; it goes to the arm. b. Sympathetic System. Raise the dorsal "aorta and notice the two slender longitudinal sympathetic trunks dorsal to it, one on each side. ( i ) Each trunk has numerous enlargements or ganglia giving off fine nerves. (2) Large lateral trunks connect these ganglia with the spinal nerves. (3) Periganglionic glands; white masses of unknown function, surrounding the spinal nerves where they issue from the spaces between the transverse processes of the vertebrae. Draw these peripheral nerves. [67] GENERAL BIOLOGY III. MUSEUM SPECIMENS. Observe in the Museum, south wing, numerous preparations of organ systems of different vertebrates, and compare them with corresponding organ systems of the frog. B. THE SKELETON. In connection with a dried prepared skeleton, study a fresh skeleton, boiled for a few minutes after removing the skin and viscera. I. GENERAL ARRANGEMENT OF PARTS OF THE SKELETON. a. The main axis consists of the vertebral column continued anteriorly as the central part (brain case) of the skull. b. Connected with the main axis are the supporting parts of the appendages, and the lateral parts of the skull. 1. The anterior appendages consist of a free limb (containing a humerus, radio-ulna, carpus and digits, supported by a shoulder girdle or pectoral arch. 2. The posterior appendages consist of a free limb (containing a femur, tibio- fibula, tarsus and digits) connected with the spinal column by the pelvic girdle. C. HISTOLOGY. I. EPITHELIUM. a. Columnar epithelium: Gently scrape the inner surface of a frog's intestine that has been preserved in Miiller's fluid. The fragments removed, under a high power, are seen to be composed of elongated cells each with a nucleus and having one end more pointed than the other. b. Ciliated epithelium: Cut off a bit of the mucous membrane from the tongue or roof of the mouth of a freshly killed frog, mount in physiological salt solution and examine under a high power. Note the appearance on the free edge due to the cilia ; as the cilia become less active individual ones can be distinguished. Scrape off some of the epithelium and examine under a high power in physiological salt solution ; note the shape of the individual cells with cilia at one end. Draw both kinds of epithelium. [68] GENERAL BIOLOGY II. MUSCLE. a. Tease out a bit of injected frog's muscle preserved in alcohol, (i) It is composed of elongated fibres, some of which may be split up somewhat into fibrilae. (2) Numerous blood capillaries are found among the fibres. b. Examine with a high power : ( I ) Each fibre shows alternate darker and lighter bands, (2) A delicate sarcolemma or structure- less membrane envelopes each fibre and can be easily seen at places where the fibres are broken or twisted. e. Tease out fresh muscle in salt solution and examine with high power to note the above points ; treat with acetic acid and observe the oval nuclei in the fibre. Draw. III. NERVE. a. Nerve fibres : Tease out a bit of fresh nerve in salt solution and examine with a high power. Note: (i) Well defined fibres, each with a double contour, together with white fibrous tissue make up the mass of the nerve. (2) Each fibre has a highly re- fractive border (medullary sheath) and a central homogeneous axis cylinder, well seen in torn specimens where also the very delicate, innermost membrane (primitive sheath), may be some- times made out. b. Ganglion cells: Examine prepared specimens of ganglion cells that have been stained to make out the structure of the cells. Draw a nerve fibre and a ganglion cell. IV. CARTILAGE. Dissect out the tip of the delicate xiphisternal cartilage of a fresh frog, or slice a bit of the cartilage from the head of the femur with a razor; mount in salt solution and study under the high power. Note : ( I ) Large rounded cartilage cells scattered through a nearly invisible and structureless matrix which forms a refractive halo about each cell. (2) A distinct nucleus (or two) in each cell. (3) After some time the cells contract and thus a space is formed between the cell and the matrix. Draw. V. BONE. Examine with low power a section of bone (mammalian bone). Observe : ( i ) Haversian canals, rounded spaces often filled with air or dirt and then appearing black. (2) Lamellae, concentric lay- ers about each haversian canal. (3) Lacunae, oval black spaces be- [69] GENERAL BIOLOGY tween the lamellae. (4) Canaliculi, minute dark lines radiating from the lacunae. (5) Other lamellae are to be found not arranged about canals, but filling in spaces not occupied by such systems. Draw. VI. CONNECTIVE TISSUE. a. White fibrous tissue: (i) Tease out a bit of fresh tendon in water; with a high power it is seen to be made up of fine wavy fibres in bundles ; each fibre has faint outlines and does not branch. (2) Treat with acetic acid; most of the fibres swell and become invisible, but a few (yellow elastic fibres) and some elongated granular connective tissue cells remain visible. b. Yellow elastic fibres : Tease out in acetic acid some of the tissue immediately under the f rob's skin. Under a high power note : Branched fibres with well defined outlines ; these may not be found until several specimens have been examined. Draw both white and yellow fibres. D. EMBRYOLOGY. I. Early Cleavage Stages. Examine entire frog's eggs divided into two, four, eight and sixteen cells. Note in the two-cell stage that a grayish or slate colored area, crescentric in form, is present on one side of the egg, and that it is, as a rule, bisected by the fi*rst plane of cleavage. Note in the four-cell stage the relative dis- tribution of the pigment on the anterior and posterior sides of the egg. Note in the eight-cell stage the relative sizes of the upper and lower cells, also the distribution of the pigment in the cells, and the location of the grey crescent. Note in the sixteen-cell stage the position of the planes of the fourth cleavage in the upper and lower cells. Examine a section of one of the early cleavage stages ; note the nuclei surrounded by pigment. Draw. II. Later Cleavage Stages. Examine two of the later cleavage stages (Blastula stages). Note the comparative sizes of the cells in the upper and in the lower hemispheres of the embryo. Examine a section of a late cleavage stage. Note the large cleavage cavity ; the thinness of the roof, and the thickness of the floor. Draw. III. Gastrula Stages. Study surface views of three gastrula stages showing: (i)The beginning of the dorsal lip of the blasto- pore, (2) the backward-growth of the dorsal lip, and the appear- ance of the lateral lips, and (3) the formation of the ventral lip. [70] GENERAL BIOLOGY In the latter the blastopore is circular in outline and the yolk plug fills up its opening. Study a longitudinal section of a gastrula stage. Compare with the section of the blastula and note all differences. Observe especially: a. The lifting up of the floor of the segmentation cavity. b. The slit-like archenteron, opening behind the dorsal lip of the blastopore. c. The condition of the cells in the dorsal lip itself. Draw to large scale showing ectodern, mesoderm and endoderm. IV. Neural Plate. Study sections of three stages in the forma- tion of the neural plate: (i) Stage with the neural plate widely open, (2) Sides of the neural plate rolling in, (3) Neural plate completely closed to form the neutral tube (brain and spinal cord). Draw. V. Tadpole. In cross sections of a young tadpole study and draw : 1. Origin of eye-vesicle from the fore brain, and development of the eye. 2. Cross section through the ear vesicles and hind brain, show- ing the gill region, with the heart beneath. 3. Cross section through the middle of the embryo to show neural tube and crest, notochord, aorta, pronephros, somites and gut. Draw and label. VI. Museum Specimens. Observe in the Museum, south wing, numerous preparations illustrating the development of vertebrates. [71] PART II ECOLOGY Read: Semper, K., Animal Life; or all of the following'. Gadow, The Wanderings of Animals; Needham, General Biology, pp. 3-55, 368-433 ; Phillips, Habits of the Honey Bee. The following directions for laboratory work are general in character since they are intended to apply to various specimens collected in the field or brought from the Museum and Vivarium. Several specimens, illustrating different kinds of adaptations, etc., will be assigned, one after another, to each member of the class. Keep laboratory records for each specimen, and then the results of observations on the topics proposed. A. RELATIONS TO INORGANIC ENVIRONMENT. I. HABITAT. Is the specimen a marine (Halobios), fresh-water (Limnobios) or terrestrial (Geobios) form? What are the evidences upon which your conclusion is based? 1. If aquatic, is it a bottom form (Benthos) or a top form (Plankton) ? Give evidence for your conclusion. 2. If terrestrial, is it fitted for life in arid or swampy regions, or for subterranean, arboreal, or aerial life? Give reasons for your answer. 3. Are there any evidences that this species or its ancestors have ever changed habitat? If so, what are they? 4. Draw the specimen, devoting particular attention to those adaptations which have relation to the habitat. II. CLIMATE. 1. Temperature. Does the organism show any particular adap- tations to heat or cold ? In what conditions does it pass the winter ? The summer? 2. Moisture. Does it show adaptations for the prevention of the loss of moisture, or to protect it against too great moisture? 3. Winds. What adaptation, if any, does it show to winds? [72] GENERAL BIOLOGY 4. Light. Is it a form which seeks or avoids strong light, and what adaptations does it show in this connection? III. MOVEMENT. 1. Is the animal free-moving or sedentary? (a) If free moving is it passively carried by winds or currents, or does it move active- ly ? Do the organs of locomotion indicate that it is fitted for swim- ming, walking, running, creeping, leaping, burrowing, or flying? Draw one or more of the locomotor organs, (b) If sedentary is it free or attached? Show by drawings the means of attachment. Are there any rudiments of locomotor organs? Are sedentary animals descended from free-moving ones, or vice versa? 2. Does this species undergo migrations? If so describe them. In the specimen assigned you by what means is the dispersal of the species assured, and what are the barriers to such dispersal? IV. GEOGRAPHICAL DISTRIBUTION. i. To what Zoogeographical Region of the earth is the animal native ? B. RELATIONS TO ORGANIC ENVIRONMENT. I. FOOD. Animals are monophagous or polyphagous, depending upon whether they live upon a single kind of food or on several kinds ; they are carnivorous, herbivorous, or omnivorous depending upon whether they eat flesh, plants, or both. 1. Correlation of Food and Structures. Determine by means of the organs of prehension, the teeth, or the character of the mouth- parts of the specimen assigned you what is the nature of its food. Draw these characteristic structures. 2. Correlation of Food and Habits. Point out, if possible, the correlation between the food and the habits of the animal you are studying. 3. Storage of Food. Is this animal able to store up food in any form? If so describe the process. II. MEANS OF DEFENSE AND OFFENSE. 1. Active. Is the animal you are studying able to defend itself- actively or not? If so, draw and describe some of the organs used for this purpose. 2. Passive. If it defends itself passively describe the methods and structures by which this is done. [73] GENERAL BIOLOGY III. INTERRELATIONS BETWEEN DIFFERENT SPECIES. If the species which you are studying is always associated with some other species, in which of the following groups does it belong? 1. Commensalism. The commensal alone benefits, but the host is not injured. In the case in hand, which is the commensal and which the host? Is the commensal permanently fixed to the host, or free to separate on occasions? 2. Symbiosis. The symbionts derive mutual benefit from asso- ciation. These also may be free or fixed. 3. Parasitism. The parasite benefits at the expense of the host. Is the parasite an endoparasite or an ectoparasite? Is it temporary or constant? IV. COOPERATION BETWEEN INDIVIDUALS OF THE SAME SPECIES. Associations of individuals of the same species fall under one or another of the following heads : a. Colonial forms without division of labor. b. Colonial forms with division of labor. c. Association of separate individuals without division of labor. d. Association of separate individuals with division of labor. 1. Does the form which you are studying belong in any of these groups ? What advantage, if any, is derived from association without division of labor? If physiological division of labor is present is it associated with structural diversity? If so, draw each of the types present, and determine their relations to one another. 2. Study a colony of bees, or of ants, and draw figures of the males, females and workers. Observe the varied activities of the members of the colony. If other members (castes, slaves) are present in the ant colony make a study of them also. Note the way in which the food is stored and the young are cared for. Study a section through honey comb, and if possible, observe the method in which it is formed. Observe and draw to scale worker cells, drone cells and queen cells, and if possible observe the kinds and relative quantities of food which are fed to the larval workers and queens. V. SEXUAL REPRODUCTION. i. Sex. In many plants and lower animals the sexes are united in the same individual (Hermaphroditism) ; in higher animals the sexes are generally separate (Gonochorism). To which class does your specimen belong? [74] GENERAL BIOLOGY 2. Primary and Secondary Sexual Characters. The ovaries and testes are primary sexual characters ; all other sexual charac- ters, which are dependent for their development upon the primary ones, are secondary sexual characters. Draw and compare the secondary characters which distinguish male and female, and, if possible, determine the significance of each. 3. Semination. Is semination internal or external? Draw the structures of the male and female which serve to bring the sperma- tozoa to the ova. 4. Types of Development. Where does the development of the embryo occur? Is the animal oviparous or viviparous? Do the embryos obtain their food by their own activities (larval develop- ments), or from the mother (foetal development)? 5. Care of Eggs and Young. In the species you are studying are the eggs and young cared for? If so, how? [75] PART III BIOGONY A. ONTOGENY. Development of the Individual. I. ASEXUAL REPRODUCTION. Occurs by Fission, Budding, Segmentation, and has been studied in the Protozoa and Protophyta, Hydra, etc. If time permits study in detail prepared slides showing the process of fission in Sten- ostoma. II. SEXUAL REPRODUCTION. a. Monogony; sexual reproduction with only one parent. 1. Parthenogenesis (virgin reproduction). Observe and draw water fleas (Daphnia) containing broods of young produced from unfertilized eggs. The same phenomenon may be seen in plant lice (Aphides.) 2. Paedogenesis (infant reproduction). Observe and draw stages in the development of unfertilized eggs in the larvae (sporo- cysts, rediae) of the tematode worm, Diplodiscus. b. Amphigony; sexual reproduction with two parents. i. Oogenesis and Spermatogenesis. Study prepared sections of ; (i) Ovotests (hermaphrodite gland) of the snail, Planorbis, and draw a portion of the section to show the ova, the spermatozoa, and the method of development of each. (2) Ovary of the frog, showing eggs of very different sizes. Note the enormous size of the nucleus (germinal vesicle). It is rilled with nuclear sap, in which are scattered nucleoli and fine threads of chromatin. Note the distribution of the yolk and pigment in the egg. Draw. (3) Testis of frog, showing mature spermatozoa, their heads attached in bundles to nurse cells and their tails extending into the lumen of the seminiferous tubule. Around the walls of the tubule are seen the following stages in the formation of spermatozoa : (a) Sperma- togonia, cells with clear nuclei, at periphery, (b) Spermatocytes I, large cells with chromatin in clumps, (c) Spermatocytes II, smaller cells with densely staining nuclei, (e) Spermatozoa, with progressively elongating nucleus and cell body. [76] GENERAL BIOLOGY 2. Maturation and Fertilization. The last two cell divisions in the oogenesis and spermatogenesis are known as the "maturation divisions" and lead to the reduction of the chromosomes in the ma- ture egg and sperm to half the usual number. When the egg is fer- tilized the normal number is again restored. Carefully study the maturation and fertilization of the egg of Ascaris megalocephala, with especial reference to the chromo- somes. Observe that in the maturation of the egg (also of the sperm) the number of chromosomes is reduced to two, and in the union of the egg and sperm the number is increased to four, the normal number. Draw eggs and sperm showing all of these points. 3. Cleavage. Observe that in the cleavage of the egg of Ascaris each chromosome is split longitudinally, so that each daughter nucleus receives two chromosomes from the egg and two from the sperm. Draw. Enumerate the evidences that the chromosomes contain the in- heritance factors. III. COMBINATIONS OF SEXUAL AND ASEXUAL REPRODUCTION. 1. Metagenesis. Alternation of asexual reproduction with sex- ual, as in hydromedusae. 2. Heterogeny. Alternation of monogonic reproduction with amphigonic, as in fluke worms. IV. HEREDITY. Germinal likeness or variation as contrasted with environmental. 1. Mendelian (alternative) inheritance. Study and draw Mu- seum exhibits illustrating this kind of inheritance. 2. Give Mendelian formulas and ratios to the third filial genera- tion (F3) for the offspring of (a) two homozygous parents, (b) two heterozygous parents, and (c) one homozygous and one hete- rozygous parent. Explain sex as a Mendelian character and show by formulas and ratios in which of these three groups it belongs. 3. Describe any cases of inheritance, known to you, which seem to be non-Mendelian, and show how they may be explained in accordance with Mendelian principles. 4. All members of the class are invited, but not required, to fill out a Family Record blank, giving details of their own heredity for the use of the Committee on Eugenics. [77] GENERAL BIOLOGY B. PHYLOGENY. Development of Races, Species and larger subdivisions. I. VARIETIES AND SPECIES. 1. Varieties. In a large series of individuals of the same species pick out and draw individuals which represent the mean and the extremes of variation. 2. Species. In a genus containing a large number of species, pick out and draw species which represent the mean and the ex- tremes of the series. II. HOMOLOGIES. a. Comparative Anatomy. 1. Draw and label corresponding parts of the limb skeletons of three vertebrates, having different modes of locomotion. 2. Draw and label corresponding teeth of three vertebrates, which eat different kinds of food. 3. Draw and label corresponding parts in the appendages of a lobster, or crayfish, and a crab. 4. Draw and label corresponding parts of the skeleton of a starfish and a sea-urchin. How are such likenesses (homologies) to be explained? b. Comparative Embryology. 1. Study and draw the adult and larva of an ascidian, and of a barnacle, and show how embryology throws light on phylogenetic relationships. 2. Compare the branchial clefts in an embroyo chick and shark, and indicate the phylogenetic significance of this resemblance. c. Paleontology. Study in the Museum the paleontological history of some family of animals, and trace its Evolution. III. EXPERIMENT. With the aid of books which will be assigned you, describe the principal races of some one domestic animal or cultivated plant and compare them with the original wild stock. IV. FACTORS OF EVOLUTION. Explain the origin of the peculiar structures of the specimen assigned you according to the following theories : ( I ) Lamarckism. (2) Darwinism. (3) Mutation. [78] GENERAL BIOLOGY Read on Ontogeny : Conklin, Heredity and Environment ; or Punnet, Mendelism ; or Walter, Genetics. Read on Phytogeny : Darwin, Origin of Species ; or Morgan, A Critique of the Theory of Evolution ; or Scott, The Theory of Evolution. [79]