Elementary exercises in physiology

Preface to the Fourth Edition

The purpose of instruction is to fix ideas and facts in the brain where they may be available when needed. Information conveyed to the brain through the eye is a very direct route, for a thing once seen is more easily remembered than any amount of verbal description of it. This is the important function of a laboratory course. An experiment carried through its various details understandingly can be visualized by the observer with less tax upon his mental powers than by any other method.

It has been the aim in this course to select experiments which are elementary but fundamental in the physiological processes of the animal body, in order that they may serve as a basis for advanced work and for a clearer conception of the importance of a knowledge of the normal mechanism in its relation to pathology and medicine rather than a consideration of physiology as an isolated subject.

The laboratory more than any other method trains the power of observation; cultivates skill and accuracy and emphasizes the fact that first hand knowledge is the most desirable.

In the preparation of this guide, numerous standard books and papers have been drawn upon, in addition to which a number of useful suggestions from assistants, past and present, have been incorporated.

P. A. F.

January 1921.

Proteins; Classification; Albumins; Xanthoproteic, Millon's, Piowtrowski 's Reaction ; Ferrocyanide and Sodium Sulphate Tests ; Indiffusibility of Albumin; Globulins; Serum Globulin; Myosinogen ; Vitellin ; Peptone pp. 18-24

Chapter II

Derived Proteins ; Acid and Alkali Albumins or Albuminates ; Syntonin ; Albuminates ; Metallic Albuminates ; Coagulated Proteins ; Proteoses or Albumoses ; Hemoglobins ; Glycoproteins ; Nucleoproteins ; Keratin, Elastin, Collagen, Gelatin Tests; Bone.

pp. 25-29 Chapter III

Carbohydrates; Monosaccharids ; Disaccharids; Polysaccharids ; Starch ; Glycogen ; Dextrin ; Cellulose pp. 29-32

Chapter IV

Dextrose; Trommer's Test; Fehling's Solution; Boettger's Test; Phenyl-hydrazine Test; Yeast Test; Barfoed' Reagent; Lactose; Koumiss; Kephyr ; Sucrose ; Maltose pp. 32-36

Chapter V

Fats ; Lipase ; Glycerin ; Fatty Acids ; Saponification ; Neutral Fats; Stearin; Palmitin; Olein; Emulsion pp. 36-38

Chapter VI Examination for Proteins and Carbohydrates pp. 38-39

Chapter VII Salivary Digestion ; Reaction ; Ptyalin ; Mucin ; Albumin ; ; Sulphocyanide of Potassium; Nitrites; Chlorides; Sulphates;

Starch ; Soluble Starch ; Erythrodextrin ; Achroodextrin ; Maltose ; Effect of Drugs on Salivary Digestion; Bread; Potato. . .pp. 39-42

Chapter XVI

Dissection of Frog's Heart; Pericardium; Ventricle; Auricle; Truncus Arteriosus ; Frenum ; Carotid Artery ; Pulmo-Cutaneous Artery; Aorta ; Venae Cavae; Sinus Venosus; Semi-lunar Valves: Spiral Valve ; Pylangium ; Synangium ; Vagus Nerve ; Glossopharyngeal Nerve ;Sartorius Muscle ; Adductor Magnus ; Adductor Longus; Rectus Internus Major (Gracilis) ; Rectus Interims Minor; Triceps Extensor Femoris ; Rectus Anticus Femoris ; Vastus Internus; Vastus Externus; Gluteus; Biceps; Semi-membranosus ; Pyriformis ; Semi-tendinosus ; Adductor Brevis ; Peetineus ; Ilio- *psoas; Quadratus Femoris; Obturator; Gastrocnemius; Tibialis Posticus ; Tibalis Anticus ; Extensor Cruris ; Peroneus ; Sciatic Nerve pp. . 71-77

Reflex Action; Effect of Mechanical Stimuli; Effect of Acids; Shock; Strychnine; Chloral Hydrate; Nerve Muscle Preparation; Galvani's Experiment ; Demarcation Currents pp. 82-83

Chapter XX

Inductorinm ; Primary and Secondary Coils; Electrical Field; Short Circuiting Key; Electrodes; Battery; Carbon; Zinc; Make and Break Shocks; Maximal and Minimal Shocks; Interrupted Shocks; Kathode; Anode; Electrolysis of Potassium Iodide; Break Extra-Current j Rheocord; Unipolar Excitation; Polarization of Electrodes pp. 84-91

Influence of Veratrine on the Contraction of Muscle ; Fatigue of Muscle; Tetanus pp. 103-105

Chapter XXVI

Influence of Temperature upon the Contraction of Muscle ; Influence of Load upon the Contraction of Muscle ; Isotonic ; Isometric ;

After Load ; Induction in Nerves ; Telephone Experiment ; Form and Volume of Contracting Muscle pp. 105-108

Artificial Scheme of the Blood Circulation ; Conversion of an Intermittent Flow into a Continuous Flow ; Relation of Peripheral Resistance to Blood Pressure ; Pulse ; Mean Pressure ; Gravity ; Lymph Circulation pp. 116-121

Chapter XXX

Blood Pressure in the Frog; Stannius' Experiments on the Frog's Heart; Cardiac Delay or Latent Period of Cardiac Muscle; Maximum Contractions Only ; Staircase Contractions of the Heart ; Location of Motor Centers in Frog's Heart; Effect of Temperature Upon Heart Beat; Isolated Apex; Intracardiac Inhibitory Center in Frog pp. 121-126

CHEMICAL PHYSIOLOGY

APPARATUS FOR THE LOCKER

1 dozen test tubes, 6 inch

1 dozen test tubes, 5 inch

1 minim pipette

1 Beaker, 10 oz.

1 Graduate, 30 mils

1 Graduate, 250 mils.

1 Flask

1 Funnel, 1% inch

1 Funnel, 3 inch

1 Watch glass

1 Evaporating dish, 8 oz.

1 Urinometer

1 Glass rod

1 Dialyzer

1 Thermometer

1 Crucible, 8 mils

1 Piece wire gauze

1 Piece absorbent cotton

1 Box matches

1 Test tube brush

1 Test tube rack

1 Test tube holder, wire

1 Tripod

1 Piece muslin

1 Pack filter papers, 3 inch

1 Pack filter papers, 6 inch

1 Sponge

1 Clay triangle

2 Tin cans

1 Copper water bath 1 Towel

Special apparatus, not found in the locker, may be obtained, when needed, by handing an order for it to one of the assistants.

EXPERIMENTAL PHYSIOLOGY

APPARATUS

1 Metal tray

1 Frog board with 4 clips

1 Wooden stand

Battery

1 Iron standard with 2 clamps

Induction coil

1 Femur clamp

Make and break key

1 Muscle lever

Short circuiting key

1 Strip of pins

Electrodes

5 Coils of wire

Kymograph

5 Sheets Kymograph paper

Saline solution

Special apparatus, not found in the locker, may be obtained, when needed, by handing an order for it to one of the assistants.

LIST OF REAGENTS

Ammonium oxalate solution: saturated.

Barfoed's reagent:

Cuprie acetate 13 grains

Distilled water 200 mils *

Acetic acid (38% ) 5 mils

Barium chloride solution: 2%

Baryta mixture:

Saturated solution of Barium nitrate 1 volnme

'Saturated solution of Barium hydrate 2 volumes

Basic lead acetate or liquor plumbi subacetatis

Lead acetate 17 grams

Lead oxide 10 grams

Distilled water 80 mils

Boil half an hour. Let co'ol and add enough previously boiled distilled water to make 100 mils.

Biuret reagent, (Gies):

10% -Sodium hydroxide 1000 mils

3% Cuprie sulphate 25 mils

Calcium chloride solution: 2 %

Copper sulphate solution: 1 %

Fehling's solution:

A. Pure copper sulphate 34.64 grams

Distilled water, to 500. mils

B. Sodio-potassium tartrate (Bochelle salts) 173. grams

Potassium hydroxide 125. grams

Distilled water, to •. 500. mils

Keep in separate bottles. When ready to use, mix equal parts of A. and B.

Hydrochloric acid: 0.2%. May be made by adding 6.5 mils of the concentrated acid to 1 liter of water.

Iodine solution: (may be diluted if necessary)

Iodine 1 gram

Iodide of potassium 2 grams

Distilled water 300 mils

* In accordance with the recommendation of the last edition of the Pharmacopoeia the term mil, the abbreviation of millimeter, — one-thousandth of a liter — is substituted for the term cubic centimeter.

Milton's reagent: Dissolve mercury in an equal weight of concentrated nitric acid; heat to solution; add two volumes of water. Decant the next day.

Potassium ferrocyanide solution: 5 %

Potassium hydroxide solution: 20% also 0.1%

Potassium sulphocyanide solution: 5%

Physiological saline solutions:

For cold-blooded animals:

Sodium chloride 6.5 grams

Water 1000. mils

For warm-blooded animals:

Sodium chloride 9. grams

Water 1000. mils

Schultze's reagent:

Anhydrous zinc chloride 25. parts

Potassium iodide 8. parts

Water 8.5 parts

Iodine crystals to saturation.

Sodium carbonate solution: 1 %

Sodium hydroxide solution: 20%

Stoke' s fluid:

Ferrous sulphate 2 grams

Tartaric acid 3 grams

Water 100 mils

Water of ammonia, enough to make slightly alkaline.

GENERAL DIRECTIONS

It is desirable that the physiologic work should be correlated so far as possible, i. e., the lectures, recitations and laboratory work should be given during the same term and have as close connection with each other as possible. If this is not practicable then the recitations or lectures should precede the work in the laboratory.

Quizzes on the work gone over may be regarded as a part of the laboratory course and should occur at frequent intervals.

Notes should be taken on the experiments as soon as they are performed; the manual is intended as a laboratory companion and the blank pages are provided for this purpose. Note taking is a portion of the laboratory work and the notes are to be submitted to the instructors for correction.

Unexcused absences, tardiness in beginning the work and leaving without permission before the end of the period will be noted, and the mark or standing of those concerned will be correspondingly reduced at the end of the term.

Many of the reagents which are to be frequently used will be placed on the student's desk on or before the periods in which they are needed, other reagents less frequently used will be placed on the general shelves and the student mast not carry these general reagents to his desk.

Any special reagent or apparatus, not included in the above, may be obtained when needed, by handing an order for it to one of the assistants.

Before beginning his experiments, the student should inventory his locker and check oif on the slip, furnished for the purpose, each piece of apparatus that is designated. The slip is then signed by the student and is held by the department as a receipt. At the end of the term the contents of each locker are examined and compared with the receipt. Any articles that are missing, broken or damaged are charged to the student.

Where there is any doubt as to the result of the experiment or any indefiniteness in the reaction consult with the assistant before taking up a new experiment.

FOODS

Any substance which may be utilized for nutritive purposes may be considered a food. The term proximate principles refers to organic substances which may be broken down into simpler constituents. Protein, fat and carbohydrates are examples. The inorganic substances, salts and water, are necessary in the metabolism associated with animal and plant life and may properly be considered in connection with food. These substances are present in suitable proportion in milk and eggs and for young animals living upon them exclusively they are considered a perfect food. Eggs, although a perfect food for the developing bird, contain an insufficient amount of carbohydrate for a mammal.

In most vegetable foods carbohydrates preponderate, while in animal foods, such as meat, the proteins predominate. A suitable diet requires that these substances should be mixed in proper proportion according to the type of animal : herbivora, omnivora or carnivora. In green foods there is a relatively small amount of nutriment and in order to provide for the large meals eaten by the herbivorous animals there must be a correspondingly large development of the alimentary tract in these animals.

In all animals a healthy and suitable diet must contain the proper amount and proportion of the various chemical substances and should be adapted to the climate, age, and amount of work done. The food must contain not only the necessary amount of chemical substances, but these must be present in digestible form. Some vegetables (peas, beans, etc.) contain even more protein than meat, but are less nutritious because less digestible, a certain amount passing through unused, in the feces.

Vitamines. Experiments have shown that if an animal is fed upon a mixture of pure protein, fat and carbohydrate with a proper admixture of salts and water that, no matter how well balanced the ration may be, it does not thrive. A young animal fed upon such a diet will not grow, but if a small amount of a natural food such as milk is added to the above mentioned diet, it will become thrifty and grow normally. This indicates that some substance, about which at present we have little knowledge, is absolutely essential for the diet and quite small amounts of it are usually sufficient. If these little known constituents are absent from a man 's diet he reacts in a similar way. He becomes afflicted with the so-called ' ' deficiency diseases," such as scurvy, rickets and Beri-iberi. The latter is a disease characterized by general malnutrition and neuritis or inflammation of the nerves followed by nerve-degeneration and paralysis. This disease has been prevalent in Japan where it was the custom of the natives to eat polished rice — rice grains deprived of their outer layer. The disease can also be produced in birds by feeding them upon polished rice, and both man and bird can be rapidly cured by adding the discarded polishings of the rice grains. The germ or embryo contained in the outer layer of the grain contains the little known substance and it is to this substance that the term vitamine or accessory food has been applied. Its chemical composition is, at present, unknown.

Vitamine is not confined to rice grains but is found in other vegetable and animal foods. The amount of vitamine varies considerably ; for example, when pigeons are fed upon polished rice, as much as 20 grams of meat daily must be added to prevent the occurrence of Beri-beri ; whereas 3 grams of egg yolk or half a gram of yeast is sufficient.

The best known of the vitamines or accessory foods are : Fat Soluble A — This is contained in most animal fats, and is especially abundant in butter and cod-liver oil. It is absent from vegetable fats, but is present in the green parts of vegetables. Animals cannot . produce it for themselves, and it is therefore necessary for nursing mothers to receive it in their food.

Water Soluble B : — The condition of Beri-beri is the specific result of the absence of this substance. Both A and B are essential for growth in young animals.

Water Soluble C : — This is contained in the juices of fruits (especially oranges and lemons) and vegetables (especially turnips). The absence of this substance in the food leads to scurvy.

Oleomargarine has now become quite a staple article of diet. It is made with beef fat as its chief basis. It is a good food and contains the fat-soluble accessory substance. Many of the present margarines are made mainly from vegetable oils (cotton-seed oil, etc.), which undergo a process of hardening or hydrogenation at a high temperature to render them solid at ordinary temperatures. Such margarines are destitute of the fat-soluble accessory, and although their calorific value is of the usual standard, they are of inferior value — especially for growing infants and children. Even if present in a fat which undergoes hydrogenation, the high temperature of the process destroys it.

PROTEINS

1. The proteins form the chief organic eonstiuents of the animal body, and occur in greater or less quantity in plants. Their composition is very complex and but little is known of their structure. All proteins contain carbon, hydrogen, oxygen and nitrogen ; most of them also contain sulphur ; several, phosphorus, and some, iron.

Proteins are almost all amorphous, non-volatile, non-diffusible, colorless, odorless and nearly tasteless solids. They vary in solubility. When burned or subject to dry distillation, they give off a disagreeable odor due to ammoniacal derivatives. Proteins are also distinguished by the ease with which they undergo chemical change under the influence of reagents, ferments, or variations in temperature. They all undergo the process of putrefaction. By boiling with dilute acids or alkalies, and also by the action of certain ferments, the proteins undergo hydrolysis, forming simpler compounds.

Classification. Because of incomplete knowledge of their structure, an accurate classification is difficult. The simplest method, perhaps, is according to their source. 1. Native proteins, which may be isolated from the organism without loss of their properties. 2. Derived proteins, which are obtained by the action of heat and reagents on native proteins.

Another classification is according to their composition. 1. Simple proteins. 2. Compound proteins.

Simple proteins are the most prominent solid constituent in muscle, glands, and blood serum, and to a greater or less extent in all tissues. The average percentage composition of simple proteins is: carbon, 50-55% ; hydrogen, 6.3%-7.3% ; nitrogen, 15%-18% ; oxygen, 21%-24% ; sulphur, 0.3 %-2.5%. Some contain phosphorus, 0.85%, and a few a trace of iron.

By the action of heat or certain reagents, soluble simple proteins become insoluble modifications by coagulation. A coagulated protein cannot return to its original condition, thereby differing from a precipitated protein. The temperature at which coagulation occurs, depends upon the nature of the protein present, the reaction of the solution, and the presence of neutral salts — e. g. an alkaline solution does not coagulate on boiling, a neutral solution will do so partially, an acid solution completely coagulates, provided the quantity of neutral salts present is not too small.

The chemical knowledge of proteins is slowly progressing and doubtless will become more complete as time goes on. The following classification should be regarded as provisional. It retains many of the familiar names, but attempts also to incorporate some of the new ideas. The classes of animal proteins are as follows (Halliburton) :

Protamines.

Phoso-proteins.

Histones.

Conjugated proteins.

Albumins.

(a) Gluco-proteins.

Globulins.

(b) Nucleo-proteins.

Sclero-proteins.

(c) Chromo-proteins,

Protamines. These substances are obtainable from the heads of the spermatozoa of certain fishes, where they occur in combination with nuclein. They are generally accepted as the simplest proteins in nature and give a typical protein reaction with the copper sulphate test. On hydrolytic decomposition they yield substances of smaller molecular weight analogous to the peptones which are called protones, and then they split up into ammo-acids. Diamino-acids are prominent among the decomposition products. The number of resulting amino-acids is small as compared with other proteins and this is confirmatory of their simple nature. The protamines differ in their composition according to their source and yield their products in different proportions.

Histones. These are substances which have been separated from blood corpuscles; globin, the protein constituent of hemoglobin, is a well-marked example. They yield a larger number of amino compounds than do the protamines, but diamino-acids are still relatively abundant. They are coagulable by heat, soluble in dilute acids and can be precipitated from such solutions by ammonia. This property of precipitation by ammonia is possessed by no other protein group.

Albumins. These are typical proteins and yield a number of cleavage products. The albumins enter into colloidal solution in water, in dilute saline solutions, and in saturated solutions of sodium chloride and magnesium sulphate. They are precipitated by saturating their solutions with ammonium sulphate. They are coagulated usually at a temperature of 70°-73°C. Serum albumin, egg-albumin and lact-albumin are examples.

Globulins. The globulins give the same general tests as albumins : they are coagulated by heat but differ from the albumins principally in their solublities.' In general, globulins are more readily salted out than albumins ; they may be precipitated and separated from albumins by saturation with sodium chloride, magnesium sulphate or by half saturation with ammonium sulphate. Typical globulins are insoluble and may be precipitated by removing the salt (as by dialysis) which keeps them in solution. Their coagulation by heat varies considerably as to temperature. The more common globulins are fibrinogen and serum-globulin in blood, eggglobulin in the white of egg, para-myosinogen in muscle, and crystallin in the crystalline lens. Certain proteins resulting from enzyme coagulation on globulins such as fibrin and 'myosin should also be included. The real distinction between globulins and albumins is that on hydrolysis globulins yield glycine and albumins do not.

Sclero -proteins. These form a heterogeneous group of substances formerly termed albuminoids. The prefix sclero- denotes the skeletal origin and often insoluble nature of the members of the group. The following are the principal proteins of this group :

Collagen, the substance comprising the white fibers of connective tissue. Some regard it as the anhydride of gelatin. Ossein, a similar substance derived from bone. Gelatin. This substance is obtained by boiling collagen with water. It possesses the property of setting into a jelly when the hot water cools. On digestion, like ordinary proteins, it is converted into peptone4ike substances and is readily absorbed. It cannot altogether take the place of proteins in a diet although it acts as a "protein sparing" food. Animals, in whose diet the sole nitrogenous source is gelatin, waste rapidly. This is due to the absence of the tyrosin and tryptophan radicals ; if these be added to the diet the animals thrive better.

Chondrin. This is a mixture of gelatin and mucoid material obtained by boiling cartilage.

Elastin. This is the substance of which the yellow or elastic fibers of connective tissue are composed. It is very insoluble. The sarcolemma of muscle fibers and certain basement membranes are composed of a similar substance.

Keratin, or horny material, is the substance found in the surface layers of the epidermis, in hairs, nails, hoofs, and horns. It is very insoluble, and differs from most other proteins chiefly in its high percentage of the sulphur-containing amino-acid called cystine. Neurokeratin, a similar substance, is found in neuroglia and nerve fibers.

Phospho-proteins. The principal members of this group are vitellin, from egg-yolk; casein, the principal protein in milk, and paracasein, the result of the action of rennin on casein. Phosphoric acid is represented in their decomposition products. They should not be confused with the nucleo-proteins which yield products (purdn and other bases) which are characteristic of nucleo-compounds. The phosphorus is contained within the protein molecules, and not in another molecular group united to the protein, as in the case of the nucleo-proteins. The phospho-proteins are of special value in the nutrition of young and embryonic animals.

Conjugated Proteins. These are complex substances in which the protein molecule is united to other organic materials, which are also usually of a complex nature. The second constituent of the compound is usually termed a prosthetic group. The conjugated proteins may be divided into : chromo-proteins, gluco-proteins, and nucleo-proteins.

Chromo-proteins are compounds of proteins with a pigment. Hemoglobin and its allies are examples.

Gluco-proteins are compounds of protein with a carbohydrate group. They include mucins and mucoids.

Nucleo-proteins are compounds of protein with a complex organic acid called nucleic acid which contains phosphorus They are found in both the nuclei and cytoplasm of cells. Physically they often simulate mucin.

In working through the experiments, a good general rule to follow — unless otherwise directed — is not to use the whole of the material or solution at once but only a small portion of it so that other tests may be tried if necessary.

2. Preparation of Egg- Albumin Solution. Break a small hole in the end of a fresh egg ; carefully pour out 10 mils of the white of the egg into a beaker. Let the yolk remain in the shell and reserve for later use. Add about 200 mils of distilled water to the beaker containing the egg-white. Stir thoroughly with a glass rod to break up the membranes and thus liberate the albumin. Filter through a piece of muslin. Any opalescence is due to the precipitation of globulins. Egg-white contains about 11%-12% of egg-albumin, together with small quantities of globulins, grape sugar, and mineral matter. The white of one egg will serve for a number of students.

A igood solution for laboratory use may also be prepared by dissolving 1 gram of dry albumin in 200 mils of distilled water.

3. Heat 5 mils of the albumin solution in a test tube to boiling. Notice the coagulation. Add a little nitric acid, the eoagulum may turn yellow but it does not dissolve.

4. Xanthoproteic reaction. To a little of the albumin solution in a test tube, add some strong nitric acid ; a precipitate is formed, white in color, which on being boiled, turns yellow. After cooling, add ammonia till alkaline ; the yellow color changes to orange. With weak solutions there may be no precipitate at all. If only traces of albumin are present, the yellow 'color with the nitric acid may fail to appear, but the addition of ammonia gives the final test with, perhaps, a yellow instead of an orange color.

5. To another portion of the solution add some of Millon's reagent; a white precipitate is formed, which on boiling, becomes brick-red in color.

6. Piowtrowski 's reaction, (also known as the biuret reaction). Add excess of strong solution of potassium hydroxide and then a drop or two of very dilute solution of cupric sulphate, When a violet color results. The reaction occurs more quickly if heat is applied, and the color deepens. 'Make a check test by using some water instead of the albumin solution. (Peptones and albumoses give a pink color when only a trace of copper sulphate is used.)

7. Acidify another portion strongly with acetic acid and add a few drops of 5% potassium ferrocyanide. A white precipitate is obtained. Peptones do not give this reaction. (Albumin is also precipitated by lead acetate, mercuric chloride ; picric acid ; strong acid, e. g., nitric; tannin; and strong alcohol).

8. Make some of the albumin solution strongly acid with acetic acid, add a few crystals of sodium sulphate and boil. All proteins except peptones are precipitated in this manner. The nitrate, after boiling, can be used for other tests (peptones), as the acid and sulphate do not decompose the solution.

9. Indiffusibility of Albumin. Place some of the solution in a dialyzer. The salts (crystalloids) diffuse readily. At the next exercise test for chlorides by adding a little silver nitrate solution to a portion of the diffusate. Apply to another portion of the diffusate any of the preceding tests for albumin. None will be found. Albumin belongs to the group of colloid bodies.

10. Globulins are proteins insoluble in water, but are soluble in dilute saline solutions. They are coagulated by heat and are precipitated by saturating their solution with magnesium sulphate or sodium chloride, and by the addition of an equal volume of saturated solution of ammonium sulphate. They comprise (a) serumglobulin of blood plasma, lactoglobulin of milk, myoglobulin, myosin, and musculin of muscle ; ( b ) myosinogen of muscle, a peculiar protein, having properties like both albumins and globulins; (c) fibrinogin, differing from other globulins in being precipitated from its solution by an equal volume of saturated solution of sodium chloride and by forming fibrin when acted on by the fibrin ferment; and (d) vitellin differs from globulin in not being "salted out" by sodium chloride. Ovavitellin of egg-yolk and crystallin of the lens of the eye are vitellins.

11. Take a small portion of the yolk in a test tube and shake thoroughly with Yz tube of ether ! ! for several minutes. Let settle. Carefully pour off the ether into evaporating dish and repeat several times till residue is colorless or nearly so.

(a) Let the substance in the dish evaporate till the odor of ether is all gone. What is the substance? Pour a little of it in water and note results, also after placing a drop on a piece of paper.

(b), Transfer the residue in test tube to watch glass and let dry thoroughly. Divide into two portions. Place portion (1) in a test tube, fill tube x /s full of water and shake thoroughly for five or ten minutes. Does the substance dissolve? Let settle, pour liquid through "filter" and test it for protein with the biuret test. Save this test and compare with next.

To the undissolved residue in the tube add a little 10% solution of sodium chloride and shake as before. Result ? Filter and apply the biuret test. Result ? Conclusions ?

12. Place portion (2) in a crucible, add a few drops of concentrated HN0 3 and carefully evaporate to dryness high over the flame, charring as little as possible. When dry, let cool. Add a few drops of concentrated HN0 3 again and repeat evaporation as above. Now gradually lower crucible into flame and heat until the most of the black substance disappears. Let cool, add a little concentrated HO and heat to boiling. Dilute with an equal volume of water and filter. Divide into four portions. To the first add some potassium ferrocyanide solution, and to the second some potassium sulphocyanide solution. A blue color in the former and a red color in the latter indicates iron.

To the third portion add a few drops of barium chloride and let stand. A white ppt. indicates sulphates.

To the fourth add a few drops of cone. HN0 3 and an equal volume of ammonium molybdate solution. Let stand. A yellow crystalline ppt. indicates phosphates.

13. Peptones. Peptones are proteins soluble in water, but not coagulable by heat. They are the result of protein digestion and are diffusible through animal membranes. Proteoses are substances intermediate in constitution between albumins and peptones.

14. Peptones differ from albumins as follows : They are not coagulated by heat ; they are not precipitated by adding sodium chloride ; they are not precipitated by acids or alkalies ; they are not precipitated by sodium sulphate ; they are not precipitated by potassium ferrocyanide ; they yield a pink color with Piowtrowski's test instead of a violet as given for albumin. Make a weak solution of peptone and apply Piowtrowski's test. (See plate I).

15. Like albumin the}^ are precipitated by the addition of tannic acid ; they are also precipitated by alcohol, but it must be remembered that all proteins are precipitated by alcohol, and that the absence of other proteins must be proved before deciding that the precipitate with alcohol is peptone.