Copyright N°„ COPYRIGHT DEPOSIT. A LABORATORY HANDBOOK FOR THh ANALYSIS OF MILK, BUTTER AND CHEESE. BY James Rittenhouse £vans, B. s •Member of The American Chemical Society, The Society of Chemical Industry, The American Waterworks Association. Etc. LIBRARY of CONGRESS Two Copies rteceiveu JUL 1 1905 Joyyrurm tuiry y^JU. t, t^os- COPY 8. • ~— ~ . — - «,, iirnivMi PREFACE TO SECOND EDITION. Since the publication of the first edition, the author has had an opportunity to test the following pages on a class of students abso- lutely unfamiliar with Milk and Butter analysis. The results were exceedingly gratifying, in that while access was obtainable to all the more complete works on the subject, the following notes seemed to afford the greatest help in actual laboratory work. As stated in the preface to the first edition, no attempt has been made to interpret the results, the object being to eliminate everything not absolutely necessary to the complete understand- ing of the actual work of a determination. By following this system, it is hoped that much of the lack of clearness caused by an exhaustive treatment of the subject has been overcome. The text itself has been carefully corrected and revised, and it is hoped that the present edition will contain few, if any, mistakes. During the past year, a rigid investigation was undertaken for the purpose of determining the comparative value of the various "milk testers/' with the result that the lactoscope, creainometer, pioscope, lactobutyrometer and the lactometer, when used alone, were found absolutely worthless in the testing of a milk. The investigation has shown, however, that real merit exists in the centrifugal machines of the Babcock type, when carefully and intelligently used. As a result, an appendix has been added, describing the Babcock tester and its method of use. Several tables have also been added, and additional instruc- tions given wherever it has been found necessary or advan- tageous. J. R. E. PREFACE TO FIRST EDITION. This little handbook is intended merely as a laboratory guide to the chemist who has to deal with the chemical analysis of milk and its products. No attempt has been made to discuss the re- sults, beyond giving, in the form of an Appendix, a copy of the definitions and standards decided upon by the Department of Agriculture. The methods given are well known, the only attempt at originality being in the way they are arranged in distinct steps, this form of arrangement being found, by the author, to be to some extent, an aid to clearness. No mention has been made of the various methods of testing milk by the aid of instruments such as the lactoscope, cream- ometer, etc., their unreliability being well known. J. R. E. CONTENTS ANALYSIS OF MILK. Specific Gravity— Page a — Hydrometer Method 1 1 h— By the Sp. Gr. Bottle 12 c— By the Westphal Balance 13 Total Solids 1 j Ash 13 Fat ,4 Solids Not Fat 15 Lactose— a — By Difference [5 b — Polarimetric Method 16 Total Proteids 16 Casein 17 Albumen 17 Acidity 18 Lactic Acid 18 ANALYSIS OF CREAM AND CONDENSED MILK Total S< ilids 20 Ash 20 Fat 20 Total Proteids 20 Lactose 20 Cane Sugar 20 DETECTION OF ADULTERATIONS IN MILK Addition of Water 21 Abstraction of Fat 21 Addition of Water and Abstraction of Fat 22 Addition of Skim Milk 22 Addition of Pasteurized or Sterilized Milk 22 Addition of Starch 22 Addition of Sugars 23 DETECTION OF PRESERVATIVES Borax and Boric Acid— a— Detection 23 b— Estimation 24 Formalin • • 2=5 Gelatine 25 Benzoic Acid 26 Salicylic Acid 26 Sodium Carbonate 27 B-Napthol Compounds 27 DETECTION OF COLORING MATTER Sulphonated-Azo-Dyes 28 Annato 29 Analine Orange 29 Caramel 2 9 ANALYSIS OF BUTTER Water 33 Fat 3 3 Solids Not Fat 33 Ash 34 Salt 34 rotal Proteids 34 Curd • ;•••• 34 DETECTION OF ADULTERATIONS Addition of Water 35 Addition of Process Butter 35 Detection of Oleomargarine • 35 Addition of Fats Other than Butter Fat 36 EXAMINATION OF THE FAT Volatile Fatty Acids 37 Soluble Fatty Acids 38 Insoluble Fatty Acids 39 Koettstorfer Saponification Number . 40 Valenta's Test 41 Specific Gravity 41 Refractory Index 41 Iodine Absorption 42 Per Cent, of Iodine Absorbed 44 Melting Point 44 DETECTION OF PRFSFRVAT1VES Boric Acid- a— Detection 45 b— Estimation 45 Other Preservatives 46 DETECTION OF COLORING MATTER Annato 46 Saffron 46 Analine Colors 46 ANALYSIS OF CHEESE Water 49 Fat 49 Proteids ' 50 Nitrogen 50 Separation of Fat for Examination 50 Adulterations 50 APPENDIX I Standards of Purity for Milk, Butter and Cheese Si APPENDIX II Babcock Method of Estimating Percentage of Fat in Milk 57 Analysis of MilK. 1 1 Analysis of Milk. DETERMINATION OF SPECIFIC GRAVITY, a — By the Hydrometer. r. Fill the Hydrometer tube with the carefully mixed milk, avoiding air-bubbles, to within four inches of the top. 2. Insert the Hydrometer (preferably that used by the N. Y. B. H.), taking care that the graduated tube is not wet above the mark to which it sinks, and that the Hydrometer does not touch the sides of the jar. 3. Read the top of the merniscus. N. B.— TheN. Y. B. H. hydrometer or lactometer reads in degrees, which must be converted by means of the following table to its corresponding value in terms of the specific gravity of water. 12 TABLE SHOWING SPECIFIC GRAVITIES CORRESPONDING TO DEGREES OF THE NEW YORK BOARD OF HEALTH LACTOMETER. Temperature, 6o° F. DfcGREfc. Sp. Gr. i »i (,i.'i i Sp. Gr. go 1.026 19 gi 1.02639 92 1.02668 93 1.02697 94 1.02726 95 1.02755 g6 1.02784 97 1.02813 98 1.02842 99 1. 0287 1 100 1.02900 10 1 1.02929 102 1.02958 103 1.02987 104 1. 03016 105 1.03045 106 1 .03074 107 1. 03103 108 1.03132 109 1.03161 no 1. 03190 in 1. 032 1 9 112 1.03248 113 1.03277 114 1.03306 115 1.03335 1 16 1.03364 117 1.03393 118 1.03422 119 1. 03451 120 1.03480 b — By the Specific Gravity bottle. 1. Fill the bottle to the mark with recently distilled water. 2. Weigh exactly, taking care that the buttle is perfectly dry on the outside. 3. Pour out the water and dry thoroughly. 4. Fill with milk to the mark, avoiding frothing as much as 13 possible, and dislodging any air bubbles clinging to the sides of the bottle. 5. Weigh accurately. 6. The weight of the water and flask, divided into the weight of the milk and flask, is equal to the specific gravity of the milk, at the temperature at which the operation is performed. c — By the Wcstphal Balance. 1. Weigh in air a piece of glass, or metal, suspended from the arm of the balance by means of a fine copper wire. 2. Weigh the same in distilled and recently boiled water. The water should be contained in a beaker placed on the floor of the balance. 3. Dry the piece of glass, or metal, thoroughly and weigh in milk, avoiding air-bubbles and frothing. Then : Let P = Weight in air. P t = Weight in water. P 3 = Weight in milk. D Specific gravity of milk. u P - P, NOTE— The specific gravity, as obtained at any Certain temperature, should be corrected to that qt 15. 5 Cent., or 6o° Fahr. This may be done by using Vieth's table, given in Blyth's "Foods: their Composition and Analysis," fifth edition, page 216. DETERMINATION OF WATER, TOTAL SOLIDS AND ASH. 1. Weigh out about 3 grams of milk into a flat platinum dish, previously weighed, not less than 5 to 10 cm. in diameter. 2. Evaporate to dryness on water bath. 14 3. Heat in water oven to constant weight. This will take about three or four hours. 4. Cool in dessicator and weigh. The weight of residue is equal to the weight of TOTAL SOLIDS in the weight of milk taken. The difference between this weight and the weight taken, is the weight of WATER. Calculate both weights to percentages. 5. Gently ignite residue at low redness, until ash is white, or nearly so, breaking up all lumps with a platinum wire. 6. Cool in dessicator and weigh. 7. Weight of final residue is equal to the weight of ASH, in the weight of milk taken. Calculate to percentage. DETERMINATION OF FAT. Adams' Method. 1. Roll up a strip of fat-free blotting paper into a cylinder, fasten it with a piece of fine wire and place in a weighing tube. Dry at 105 Cent., for one hour. 2. Cool and weigh. 3. Pour on the paper, from a pipette, holding the paper up- right and pouring on from the top, about 5 c.c. of milk. N. B. — It is better to apply the milk to the inside of the cylinder. 4. Replace the cylinder quickly in weighing-tube and weigh. 5. The increase in weight is equal to the weight of the milk taken. 6. Place cylinder in water oven at ioo° Cent., for one or two hours. 7. Place in Soxhlet extraction apparatus and extract for two and one-half hours with anhydrous Ether, or refined Gasoline. 8. Remove the cylinder from the extractor and vaporize the 15 Ether, or Gasoline, from the paper by aid of suction, or gentle heat. 0. Place in weighing-tube, when perfectly dry, and weigh. The weight thus obtained, minus the weight of cylinder and tube, obtained at (2) will give the weight of the SOLIDS NOT FAT. This weight subtracted from the weight of the Total Solids will give the weight of FAT. The weight of FAT may be determined directly, if, before beginning the ex- traction, the flask of the extraction apparatus is weighed in a perfectly dry condi- tion, and after the extraction of the fat the Ether extract is evaporated to dryness in it, the residue dried in the water oven to constant weight and the flask ar.d con- tents again weighed. The increase in weight is equal to the weight in FAT. Do not keep in water oven too iong, as the fat has a tendency to increase in weight. ESTIMATION OF LACTOSE BY DIFFERENCE. Blyth. 1. Proceed as if the Total Solids were to be found and ex- tract residue with Ether. 2. Extract the dry fat-free residue with weak, boiling, Alcohol. 3. Filter and evaporate the Alcohol extract to dryness in a platinum dish. 4. Heat in water oven to constant weight. 5. Cool in dessicator and weigh. 6. Ignite residue until all Carbon has been burned off and ash is white, or nearly so. 7. Cool in a dessicator and weigh. 8. The difference between these two weighings is the weight of the Lactose in the weight of milk taken. Calculate to per- centage. i6 POLARIMETRIC METHOD OF ESTIMATING LACTOSE IN MILK. Method of Dr. IV. Wiley. i. Take 60 c.c. of milk. 2. Add 10 c.c. of a solution of Mercuric Nitrate (made by dissolving Mercury in twice its weight of Nitric acid (sp. gr. 1.42) and diluting with four volumes of water; and dilute mixture to 100 c.c. 3. Shake and filter through a dry filter. 4. Observe rotation at once with polariscope. 5. Take the specific rotation of milk sugar as (a)d 52.5 degrees. Calculate the weight of anhydrous sugar by following formula : _ 100 X d 100 — (1.075 F X .8 P) .Q5 S ~~ 52.5 X 1 X Q K Sp. Gr. Where Q = Quantity of milk taken. F = Weight of fat in quantity Q. P =- Weight of proteids in quantity Q. 1 Length of tube. d = Angular rotation found. DETERMINATION OF TOTAL PROTEIDS. KjeldahVs Method. 1. Weigh out about 5 grams of milk into a Kjeldahl flask fitted with a balloon stopper. 2. Add 20 c.c. strong Sulphuric acid. 3. Add a small globule of Mercury. 4. Place flask, stoppered, in an inclined position and heat nearly to the boiling point. 5. Keep at this temperature for 15 minutes. 6. Increase heat until liquid boils freely. 17 7. Boil until liquid becomes clear, or at most, of a pale straw color. Remove from the flame, keeping in an upright position. 8. Add at once, crystals of Potassium Permanganate until liquid remains of a purple or green color after shaking. 9. Transfer liquid to a liter distillation flask containing about 200 c.c. of ammonia-free water, a few pieces of granulated Zinc and 25 c.c. of Potassium Sulphide solution (40 grams to the liter). 10. Shake until all are thoroughly mixed. 11. Add 50 c.c. of a saturated solution of Sodium Hydrate in ammonia-free water. 12. Connect flask with a 600 m.m. Liebig Condenser, the end of which dips into a solution of accurately standarized deci- normal Sulphuric Acid. 13. Distil at a moderate heat until all the ammonia has gone over. 14. Titrate the acid against a carefully standarized alkali so- lution, using Cochineal as an indicator, and determine the amount of standard acid used up by the distillate. Convert cor- responding amount of ammonia to Nitrogen and multiply this re- sult by 6.25 to convert to milk proteids. N. B.— Care must be used in this determination to prevent the breaking of the flasks. The distilling apparatus must be freed from ammonia by adding C. P. water to the reagents and distilling until clean, previous to the actual determina- tion. DETERMINATION OF CASEIN AND ALBUMEN. 1. Take about 100 c.c. of milk and weigh accurately. 2. Divide into three approximately equal portions. 3. Dilute the first portion 4 times and acidify with Acetic acid until the Casein coagulates. 4. Pass Carbon Di-oxide through the liquid and let stand until precipitate settles. i8 5. Siphon off the whey into the second portion and, if neces- sary, add more acid. 6. Pass more Carbon Di-oxide and again allow to settle. 7. Siphon on to the third portion and repeat the same treat- ment with it. 8. Collect all the Casein on a weighed filter. 9. Wash dry and weigh. Weight equals CASEIN. 10. Take the weigh and filtrate in a large beaker and raise to the boiling point. Boil ten minutes. 11. Collect the Albumen on a weighed filter. 12. Wash, dry in water oven and weigh. Weight equals ALBUMEN. ESTIMATION OF ACIDITY. 1. Take 100 c.c. of milk. 2. Titrate in beaker with ~ NaOH solution, using Phe- nolphthalein as an indicator. 3. Call each cubic centimeter of alkali used one degree, and report in degrees of acidity. DETERMINATION OF LACTIC ACID IN MILK, 1 . Take 5 grams, approximately, of milk. Weigh accurately. 1. Evaporate to dryness in platinum dish on water bath. 3. Extract fat with Carbon Di-sulphide. 4. Treat residue with an Alcoholic solution of Oxalic acid. 5. Filter and wash. 6. Add an excess of Hydrated Oxide of lead to filtrate. 7. Filter and wash. 8. Saturate filtrate with Hydrogen Sulphide. 9. Filter and wash. 10. Concentrate by evaporation. 11. Add Zinc Oxide and boil. 19 12. Filter and wash. 13. Evaporate filtrate to a small bulk, 14. Let stand for some time. 15. Filter off the crystals of Lactate of Zinc on a weighed filter. 16. Wash with Alcohol and weigh. Calculate the Lactic Acid. The composition of the Zinc Salt is 2 (C a H 8 O a ) Zn + 3 H a O. ANALYSIS OF CREAM AND CONDENSED MILK. ANALYSIS OF CREAM. As Cream contains the same constituents, exactly, as Milk, differing from the latter only in the proportion of the constituents to each other, its analysis clearly follows the same lines. The following points should, however, be noticed : 1st. Smaller quantities should in all cases be taken. 2d. The fat should be extracted for a longer period of time, say, three and one-half hours instead of two and one-half. ^d. If desired, the cream may be diluted a certain definite amount and the diluted solution used for the ana- lysis, correcting the results, of course, to correspond with the degree of dilution. 4th. The same adulterations are to be looked for as in milk. ANALYSIS OF CONDENSED MILK. The sample is to be thoroughly mixed and accurately weigh- ed, about 15 grams being taken, and then diluted with water so as to make a io# solution, all results being carefully corrected in accordance with the dilution. TOTAL SOLIDS— Use 10 c. c. of 10 J? solution. ASH— Ditto. FAT — Use for extraction, Petroleum Spirit, or a mixture of this with anhydrous Ether, ( i$ c /< of Ether). TOTAL PROTEIDS— 10 c.c. of a 50'v solution of milk are diluted with wafer, and 5 c.c. of a 6'v solution of Sulphate of Copper added. It is then almost neutralized with deci-normal NaOH solution and the precipitate filtered off on a weighed filter, washed, and the Nitrogen in filter and contents determined as in milk. The Nitrogen contained in a similar filter should be determined and subtracted. (Ritthausen's Process). LACTOSE— As in milk. CANE SUGAR— An approximation may be made by de- ducting the proteids, ash, fat and milk sugar from the total solids. For a more exact method, see Blyth's "Foods: their Composi- tion and Analysis," fifth edition, page 264. Blyth gives the following formulas for calculating the original composition of the milk and the degree of concentration. rat of original Ml k 5 — ttta — ( > 1 — t^amI - " Proteids of Condensed Milk. A , . . . , „.„ Ash of Cond. Milk 8.9 Ash ot original Milk M c .. c .. , , n , .,-rrf Non ratty Solids of Cond. Milk. Total Milk Solids of Cond. Milk Degree of Concentration ^ Mjl| . g^- - f ^ Mk] 21 DETECTION OF ADULTERATIONS IN MILK. THE ADDITION OF WATER. To detect the addition of water to milk, it is necessary to know the percentage of Solids and Fat. The Dept. of Agricul- ture, in this country, (See Appendix I) requires that the percen- tage of Fat in Whole Milk (obtained by the complete milking of one or more healthy cows) shall not fall below 3.25'^ nor the percentage of Solids not Fat, in such milk, below 8.5^ , nor the Total Solids below 12%. The probable amount of added water can be obtained by the following formula given by Blyth: S X 100 Percentage of Added Water X 100 — — «— — S Solids not Fat in Sample. THE ABSTRACTION OF FAT. This adulteration may be detected by observing the ratio of the percentage of Solids not Fat to the percentage of Fat, or by the falling of the percentage of Fat below the standard of 3.25^ . The ratio between the two should not be far from the ratio of 9 to 4: The probable percentage of abstracted Fat may be found by the followiug formula given by Blyth : 3.25 — f Percentage of Fat abstracted = 100 — — — — 3.25 f — - Percentage of Milk Fat in Sample. THE ADDITION OF WATER AND ABSTRAC- TION OF FAT. This adulteration is practiced in order to bring the specific gravity to a normal reading and make the physical testing in- struments read the same as for a good milk. It may be detected as above. THE ADDITION OF SEPARATED OR SKIM MILK. This is probably the most common form of adulteration and can only be detected by the falling of the percentage of the Fat below the standard. Of course, if the original composition of the milk is known, it is not hard to detect this fraud. THE ADDITION OF PASTEURIZED OR STERIL- IZED MILK. This is not a common form of adulteration. Faber claims that it may be detected by the small amount of soluble Albumen. Blyth gives the following test for a milk which has been heated : i. Add to 5 ex. of milk yi gram of Paraphenylene-diamine. 2. Add 2 drops of a \o% solution of Hydrogen Peroxide. 3. Prepare at the same time another tube of good fresh milk to use as a check. Compare colors and draw conclusions. Fresh Milk ..Blue. Pasteurized Milk Faint Blue. Sterilized Milk Colorless. N. B. — The amount of soluble Albumen in normal fresh milk runs between .41 per cent, and .45 per cent. THE ADDITION OF STARCH. 1. To 100 c.c. of sample add a solution of Iodine in a solution of Potassium Iodine. (A few drops are sufficient). 2. A blue color indicates Starch. 23 THE ADDITION OF SUGARS OTHER THAN LACTOSE. Cotton's Method. i. Take 10 c.c. of milk. 2. Add 5 grams of powdered Ammonium Molybdatei 3. Add 10 c.c. dilute Hydrochloric acid (1 — 10). 4. Make up tube of good fresh milk in same way. 5. Compare colors after heating both tubes at 8o° Cent., for a few moments. The tube containing sugar will become blue. DETECTION OF THE ADDITION OF PRESER VATIVES TO MILK. THE ADDITION OF BORAX AND BORAIC ACID. Detection. 1. Evaporate 10 c.c. of milk, previously made alkaline with Sodium Hydrate to dryness. 2. Char the residue. 3. Add a little water and boil. 4. Acidify with HCI. 5. Dip into the liquid a piece of Tumeric paper. 6. Dry over a flame, being careful not to char paper. 7. Moisten with Ammonium Hydrate. 8. A dark blue coloration indicates a borate. N. B. — The dark red coloration nearly always produced must not be taken for the test. 24 Estimation. R. T. Thompson's Method. i. Take ioo c.c. of milk and make distinctly alkaline with Sodium Hydrate. 2. Evaporate to dryness in a platinum dish. 3. Char thoroughly. 4. Add 20 c.c. of water. 5. Heat. 6. Add Hydrochloric acid, drop by drop, until only Carbon remains. 7 t . Transfer to a 100 c.c. flask, washing out dish with water into flask. 8. Add 0.5 gram dry Calcium Chloride. 0. Add a few drops of Phenolphthalein solution. 10. Run in a 10 % solution of Sodium Hydrate until a per- manent pink color is obtained. 11. Add 25 c.c. saturated lime water. 12. Make up to exactly 100 c.c. with water. 1 3. Shake well. 14. Filter through a dry filter into a so c.c. flask. 15. When filtrate has just reached the mark, remove from under funnel. 16. Add normal Sulphuric acid until the pink color disappears. 17. Add a few drops of a solution of Methyl Orange. 18. Then add the acid until the yellow color changes to pink. 19. Add very carefully 5 NaOH until the yellow just appears. 20. Boil. 21. Cool. 22. Add 30 % of the volume of Glycerine. That is, 30 % of the resulting mixture. 23. Titrate with S NaOH until a permanent pink color is ob- tained. 25 24. Read burette. Each cubic centimeter of the 5 NaOH solution used is equal to .0124 Boric acid or .007 ELO :J . Multiply weight thus found by two and calculate percentage. THE ADDITION OF FORMALIN. Hfy/icrs Met hod. 1. Take 10 c.c. of milk in a test tube. 2. Add one drop of a solution of Ferric Chloride. 3. Dilute to 30 c.c. 4. Pour concentrated Sulphuric acid down the side of the tube so as to form a layer at the bottom. 5. A violet ring at the junction of the two liquids indicates Formic Aldehyde. THE ADDITION OF GELATINE. Allen's Method. 1. Take 10 c.c. of the milk in a test tube. 2. Add 10 c.c. of the acid Mercuric Nitrate solution. 3. Shake. 4. Add 20 c.c. of water. 5. Shake again and let stand 5 minutes. 6. Filter. In presence of much Gelatine the filtrate will be cloudy. 7. Take 10 cc. of the filtrate and add 10 c.c. of an aqueous solution of Picric Acid. 8. A yellow precipitate will occur if much Gelatine be present. Smaller amounts can be detected by the cloudiness of the solution. N. B. — The acid Mercuric Nitrate is prepared by dissolving Mercury in twice its weight of Nitric Acid of 1.42 Sp. Gr. and diluting this solution to 2=; times its bulk with distilled water. 26 THE ADDITION OF BENZOIC ACID. BIyth. i. Take 2oo c.c. of milk. 2. Make alkaline with Barium Hydrate. 3. Concentrate to 50 c.c. 4. Add Calcium Sulphate, mix to a paste, and dry on water hath. 5. Powder, and moisten with dilute Sulphuric acid. 6. Extract with $0 >, Alcohol. 7. Neutralize the Alcohol extract with Barium Hydrate. 8. Evaporate to a small hulk'. 9. Acidulate with Sulphuric acid. 10. Extract with Ether. 1 i. Evaporate Ether in a flask, by aid of suction. \2. I )issolve residue from Ether, in water. 1 ;. Add a little Sodium Acetate. 14. Test for Benzoic acid with .1 solution of Ferric Chloride. Note— If the acid is to be determined, sublime the residue and weigh the sublimate thus obtained, checking the result with " Foods : their Composition and Analysis." DETECTION OF ANNATO, ANAE1NE ORANGE AND CARAMEL. Leach's Method. i. Warm about i so c.c. of milk in a casserole. 2. Add s c.c. Acetic acid. ;. Heat slowly to near the boiling point, stirring constantly. 4. Separate curd from whey by gathering together with stir- ring rod or by straining. 5. Press the curd free from the adhering liquid. 6. Transfer to a small flask. 7. Add 50c. c. of Ether and macerate over night, keeping flask tightly stoppered and shaking occasionally. S. I >ecant the Ether. Place in an evaporating dish and evaporate. SAVE THE CURD IN ANOTHER DISH IE ( I )] OPED. 0. Make the fatty residue alkaline with Sodium Hydrate. Warm and filter while warm. 20 io. Wash residue from filter with a stream of water. Dry filter. If colored orange, AN NATO is indicated. Confirm by- adding a drop of Stannous Chloride Solution. 1 he orange color turns to pink. ii. If curd is colored orange or yellow, ANALINE ORAM JE is indicated Confirm by treating a lump of the fat-free curd in a test-tube with a little cone. Hydrochloric acid. It curd turns pink', the presence of ANALINE ORANGE is assured. 12. If curd be colored a dull brown, CARAMEL is to be sus- pected. Heat a little in a test-tube with strong Hydrochloric acid. The acid will turn deep blue, while the curd remains if CARAMEL is present. brown Nc >!>:.— In normal milk the solution will turn blue.butthe curj remains blue. Analysis of Butter. 53 Analysis of Bitter, ESTIMATION OF WATER IN BUTTER. i. Weigh about 2 grams of butter into a weighed platinum dish, containing a little recently-ignited Asbestos. 2. Heat in water bath until weight is fairly constant. 3. Cool in dessicator. 4. Weigh. 5. Loss in weight is equal to weight of water in weight of butter taken. NOTE. — Do not keep too long in bath, as the weight will gradually increase after a time. ESTIMATION OF FAT AND SOLIDS NOT FAT. 1. Dry, and weigh, a fat-tree cylinder of blotting paper, as in milk. 2. Place about a gram of butter on the inside of the cylinder. 3. Weigh immediately. Increase in weight is equal to the weight of butter taken. 4. Place in water oven in horizontal position until the melted butter is absorbed by the paper, then, dry further until most of the water has been driven off. 5. Place cylinder in Soxhlet extraction apparatus and extract for y/2 hours with anhydrous Ether. 6. Evaporate Ether extract in weighed flask by aid of sue- 14 lion. The residue, after being dried to constant weight in water oven, will equal weight of Fat in quantity of milk taken. 7. Remove cylinder from extraction apparatus, dry and weigh. Increase in weight over original weight of cylinder will equal the weight of Solids not Fat Salt. ESTIMATION OF ASH IN BUTTER. 1. Carefully ignite residue from wafer determination until w lute, being careful not to go above low redness. 2. Cool in dessicator. ;. Weigh. 4. Weight of residue is equal to weight of Ash weight of Salt in the weight of milk taken. ESTIMATION OF SALT. 1. Shake up a weighed amount of butter, about 10 grams, in 1 separatory funnel, with hot water. 2. Titrate water extract with a standard solution of Silver Nitrate, using Potassium Chromate as an indicator. NOTE.— It is customary to call all butler which contains Irss tha oi salt " Fresh," and all butter containing more than 2 per cent., "Salt." ESTIMATION OF TOTAL PROTEIDS. Make a Nitrogen determination as in Milk. Multiply weight of Nitrogen found by 6.2s. ESTIMATION OF CURD. Subtract the Salt Ash from the Solids not Fat. 35 DETECTION OF ADULTERATIONS THE ADDITION OF WATER. Determine by estimating the percentage of water. If this runs over 16 % , the butter has either been adulterated with water, or has been manufactured by a faulty process. DETECTION OF "PROCESS' ' BUTTER. FOAM TEST. lour. Amer. Chem. Soc, igoo, 22,150. 1. Heat 2 or 5 grams of butter to be tested in a platinum dish. If real butter, it will foam abundantly; if "Process But- ter," or "Oleomargarine," it will bump and sputter like hot grease, without foaming. APPEARANCE TEST. Jour. Amer. Chem. Soc, /goo, jj.jjj. 1. Heat 100 grams, approximately, of butter at 50° Cent. 2. In real butter the curd will settle, leaving the fat in a clear layer above it. If "Process Butter.'" the supernatant fat will not be clear. MICROSCOPIC EXAMINATION Jour. Amer. Chem. Soc. igoo, 22,150. 1. Place a bit of the sample on a glass slide, cover it and press into a thin film with cover glass. Examine immediately with polarizer at a magnification of from ico to 200 diameters. When a Selenite plate is placed between the slide and lower Nicol, butter will give a uniformly colored, blue field; "Process Butter" will give a blue field, mottled with yellow. THE DETECTION OF "OLEOMARGARINE." IVATERHOUSE TEST. Jour. Amer. Ckem. Soc, igoi, 23,200. 1. Half till a 100 c.c. beaker with sweet milk. 36 2. Heat nearly to boiling. 3. Add 10 grams of sample butter. 4. Stir with small wooden paddle until fat is melted. 5. Place beaker in cold water and stir until the temperature has fallen below the congealing point of the tat. 6. At this point the fat, if "Oleomargarine," will collect into one lump by usine, the paddle in the rijj;ht way. Butter, however, will granulate and be impossible to collect. THE ADDITION OF FAT OTHER THAN BUTTER FAT. The detection of this adulteration consists in making several, or all, of the following standard tests, and drawing conclusions from the results as to whether the butter has been adulterated or not. Blyth gives the following table showing the average values tor the standard tests : BUTTER. Mean. Max. Min. Marg-. Mean. Reichert-Woolny Valenta 1. >. 1 Specific Gravity at C. 15-5° Refract. Zeiss at 30 Centjgrade Koettstorfer Value Insoluble Acids Per Cent. . Soluble Acids Per Cent Iodine Value Melting Point 28 87. s 5.0 34 33.5 24 IQ.C ■>, c. .86 so \>> 4V7 22T.5 85.5 7.0 •t." 40 1 35.o°C. }O.G°C. 105 95-5 trace ss Cocoa. O. 7.5-8 .S74 82-00 0-95 20-28°C. 37 EXAMINATION OF THE EAT DETERMINATION OF VOLATILE FATTY ACIDS. Reichert- Woolny Process modified by Leffman and Beam. i. Melt the butter and keep in a warm, dry place at 6o° Cent., for two or three hours until wa'ter and curd have entirely de- posited. 2. Pour off the supernatant fat and filter through a dry filter, using a hot water funnel. Save. 3. Wash out a distillation flask with water, Alcohol and Ether respectively. Heat for half an hour in oven. 4. Warm a pipette to about 60° Cent., and after mixing the melted fat thoroughly, deliver into the flask, by means of the pipette, exactly S-75 cc. of the fat. 5. Add 20 cc. of Glycerine Soda Solution. (Dissolve 100 grams of NaOH in iod cc. of distilled water. Let stand until clear. To 20 cc of this solution add 180 cc pure concentrated Glycerol). 6. Heat until the mixture becomes clear. 7. Add 1 35 cc. distilled water, recently boiled. N. B.— Add drop by drop al firsl to prevent foaming. 8. Add s cc dilute Sulphuric acid. 9. Cool, and add a few pieces of pumice sfone. ( Prepared by throwing it at a white heat into distilled water and keeping it under water. ) 10. Connect flask to a condenser. 38 ii. Distil, carefully at lust, so as to produce noc.c. of dis- tillate in as near 30 minutes as possible. 12. Filter the 1 10 c.c. of distillate through a dried filter. 13. Place 100 c.c. of filtrate in a 200 c.c. beaker. 14. Add 1 c.c. of an Alcoholic solution of Phenolphthalein. [5. Titrate with deci-normal NaOH. 16. Multiply result in cubic centimeters by 1.1. This will give the Reichert-Woolny number. N. B.— Run several blank-. DETERMINATION OF SOLUBLE AND INSOLU- BLE FATTY ACIDS. Official Mi 7 'hod. SOLUBLE ACIDS. N. 13. --Run at leasi two blanks 1. Take about 5 grams of sample fat, by means of warmed pipette, run into a clean saponification flask and weigh accu- rately, having previously obtained the weight of the flask empty. This will give weight of fat taken. 2. Add so c.c. of Alcoholic Potash solution, (40 grams of K( )H in one liter of 95 '/, re-distilled Alcohol.) Measure each time with same pipette and let drain thirty seconds. 3. Insert a soft cork and tie down. 4. Place on water bath until the fat is entirely saponified, shaking occasionally. 5. Cool. 6. Remove stopper and wash contents of flask into an Erlenmeyer flask of about 200 c.c. capacity with a little 95 '/ alcohol. J9 7. Place on water bath together with blanks until Alcohol has disappeared. 8. Titrate blanks with half-normal Hydrochloric acid, using PHenolphthalein as an indicator. 9. Then run into sample i c.c. more of the Hydrochloric acid than is necessary to neutralize the Potash in the blanks. 10. Connect flask with a reflux condenser and place on the water bath until the separated fatty acids form a clear stratum on the surface of the liquid. 1 i. Cool flask and contents in ice water. \2. After the fatty acids have solidified, pour the liquid con- tents of flask through a dry filter into a liter flask, care being taken not to break the cake. 13. Add 300 c.c. of water to the flask containing the cake and reinsert the condenser. 14. Heat on steam bath until the cake is thoroughly melted. Agitate at intervals. 15. When separation lias again taken place, cool in ice water and filter in same way as before into the flask containing the first filtrate. [6. Repeat this treatment of the cake a third time. 17. Make up the combined filtrates to one liter. 18. Take mo c.c. of this volume and titrate with deci-normal NaOH. 19. Multiply result by 10. This gives the cubic centimeters of deci-normal solution of alkali. Diminish by 5 to correct fol- ic. c. of half-normal acid added. Multiply final result by .0088. This will give the weight of Butyric acid in amount of fat taken. Calculate to percentage. INSOLUBLL: ACIDS. 20. Allow the flask, containing the cake, together with the filter, to drain and dry for twelve hours. 40 2i. Transfer cake and as much of the fatty acids as can be scraped ofi the filter to an evaporating dish, previously weighed. 22. The funnel and filter are then placed in an Erlenmeyer flask and the paper thoroughly washed with absolute Alcohol. 2?. The llask is then rinsed with the washings just obtained, into the dish. It is then washed ten times with pure Alcohol into the dish. 24. Evaporate to dryness on water bath. 25. Heat in water oven for three hours. 26. Cool in dessicator. 27. Weigh. 28. Weight of residue is equal to the weight of insoluble acids in weight of butter taken. Calculate to percentage. DETERMINATION OF SAPONIFICATION NUMBER. Koettstorfer. N. B.— Conduci at least two blanks. 1. Place from one to two grams of sample fat in a saponifica- tion flask. 2. Add 25 c.c. Alcoholic Potash solution. (Use same pipette each time and let drain thirty seconds). ]. Stopper. Heat on water bath until saponification is com- plete. " 4. Cool. 5. Remove cork and add 1 c.c. of Phenolphthalein solution. 6. Titrate blank and sample with half-normal HCI, reading the burette in cubic centimeters. 7. The Koettstorfer number is obtained by subtracting the number of cubic centimeters necessary to neutralize the alkali after saponification from the number necessary to neutralize the 41 Potash in the blank, multiply this by zS. 06 and dividing the pro- duct thus obtained by the weight in grams of the fat taken. VALENTA'S TEST. Chattaway, Pearmain and Moor, N. B. — Make several determinations. i. The butter fat, melted and filtered at room temperature, is further dried by being filtered through a dry filter. 2. Weigh out 2.75 grams of the fat into a stoppered test tube. 3. Add 3 c.c. of 99.5 % Acetic acid. 4. Place in a beaker of water and gradually warm, noting temperature until on shaking the tube a clear solution is obtained. Note temperature at this point carefully. DETERMINATION OF SPECIFIC GRAVITY. Proceed the same as in milk, tilling the flask, previously standardized with water, with fresh, melted, filtered fat at ioo° Cent. Keep in boiling water for 30 minutes and then stop- per, the glass stopper having been previously heated to ioo°Cent. Wipe dry and allow to cool. Weigh. Weight <»f flask Fat. Specific Gravity Weju;ht (lf tl;(sk Watel , N. I!. — The weight "t flask and water should be obtained in the same manner as the weight of flask Fat. DETERMINATION OF REFRACTORY INDEX. Zeiss' 1 Bitty to Rej Tactometer. The directions for this determination are given with each in- strument, or can be found described in full in Blyth's "Foods: their Composition and Analysis." 42 DETERMINATION OF THE IODINE ABSORP- TION NUMBER. OFFICIAL METHOD. (Bulletin No. {6, Revised Edition. U. S. Department of Agriculture. ) SOLUTIONS. Iodine Solution— Dissolve 26 grams of puie Iodine in 500 c.c. ol 95 ' , Alcohjl. Dissolve 30 grams of Mercuric Chloride in 500 c.c. os'' Alcohol. Filter the latter solution and mix the two. Let stand twelve hours before using. Deci-Normal Sodium Thiosulphate Solution— Dissolve i\.^ grams of C. P. Sodium Thiosulphate, freshly pulverized and dried between blotting paper, in distilled water and dilute to one liter at the temperature of the titration. Starch Paste— Roil one gram of Starch with 200 c.c. of water for ten minutes and cool to room temperature. Potassium Iodide Solution— Dissolve 150 grams of Potas- sium Iodide in water and make up to one liter. Solution of Potassium Di-Chromate — Dissolve 3.874 grams of C. P. Potassium Di-chromate in distilled water and make up to one liter at the temperature of the titration. STANDARDIZING TME SODIUM THIOSULPHATE SOLUTION. 1. Place 20 c.c. of the Potassium Di-chromate solution in a glass stoppered flask. 2. Add 10 c.c. of Potassium Iodide solution. 5. Add 5 c.c. strong Hydrochloric acid. 4. Pun in the Sodium solution from a burette until the yellow color has nearly disappeared. 5. Add a few drops of Starch paste, 43 6. Add with constant stirring, the Sodium solution, until the blue color has been destroyed. 7. The number of cubic centimeters of the Sodium solution used, multiplied by 5 is the equivalent to one gram of Iodine. Calculate the value of one cubic centimeter. DETERMINATION. 1. One gram of the butter fat, as prepared under determina- tion of "Volatile Acids,' 1 is weighed into a 300 c.c. stoppered flask. 2. Dissolve completely in 10 c.c. of Chloroform. 3. Add 30 c.c. of Iodine solution. 4. Let stand in a dark place, with occasional shakings, for three hours. 5. Add 100 c.c. distilled water. 6. Add 20 c.c. of Potassium Iodide solution. Wash down any particles of Iodine adhering to the glass with this solution. 7. Run in Sodium solution from burette until the yellow color is nearly gone. 8. Add a few drops of Starch paste. 9. Continue titration with constant shaking until blue color has disappeared for the space of 5 minutes. 10. Read burette. Note— At the time of adding the Iodine solution to the fat, two flasks of the same size should be prepared with all the above reagents as in determination, minus the fat. In every other respect the performance of the blanks should be identical with the determination. These blanks should be run every time the Io- dine solution is used. EXAMPLE BLANK. 30 c.c. of Iodine solution required 46.4 c.c. of Sodium Thio- Sulphate solution. 30 c.c. of Iodine solution required 46.8 c.c. of Sodium solution. Average c.c. Sodium solution equals 46.6 c.c. Per cent, of Iodine absorbed : 44 Weight of fat 1.047 grams. Quantity of Iodine solution 30.0 c.c. Sodium solution equivalent of Iodine solution. . . .46.6 c.c. Sodium solution equivalent to remaining Iodine- • 14.7 c.c. Sodium solution equivalent to Iodine absorbed- • • 31.9 c.c. 31.9 0.124 100 7 37-75 Iodine Number. 1.047 DETERMINATION OF MELTING POINT. Wilef s Method. 1. Prepare disks of Fat as follows : allow melted and filtered Fat to fall from a dropping tube on to a smooth piece of ice float- ing in distilled water. By submerging the ice, the disks can be floated on to a steel spatula previously cooled in ice water. 2. A test tube 50 cm. by 3.5 cm. is nearly tilled with a hot mixture of Alcohol and water, (prepared by boiling distilled water and os'' Alcohol separately, to remove any gases which they might contain. The hot water is then poured into the test tube until it is nearly half-full. The hot Alcohol is then poured on top of the water until the tube is nearly full, taking care not to mix the two to any extent. ) 3. Place the test tube in a tall beaker tilled with ice water until cold. 4. Drop a disk of Fat from the spatula into the Alcohol water. It will float about half-way between the top and bottom. 5. Place a delicate thermometer in the test tube, the bulb being about even with the disk. This thermometer should be gently moved from time to time. 6. Meat water in the beaker gently, stirring constantly. 7. When the disk of fat begins to shrivel, continue the heat- ing with extreme slowness, noting the temperature by the delicate thermometer, constantly, « 45 8. When disk has just taken the form of a sphere, read the thermometer. NOTE — Repeat the determinations several times. THE DEFECTION OE PRESERVATIVES IN BUTTER THE ADDITION OF BORIC ACID. Detection. i. Melt 5 grams of sample in dish. 2. Add 5 c.c. of water. 3. Acidify with Hydrochloric acid. 4. Mix while warm. 5. Allow water to separate and test for Borates as in milk. Estimation. ( Richmond and Harrison 's Method. ) 1. Weigh out 25 grams of sample in a beaker. 2. Add 25 c.c. of a solution containing 6 grams of milk sugar and 4 c.c. normal Sulphuric acid to 100 c.c. 3. Place in water oven until fat has just melted. Stir well. 4. Allow aqueous portion to settle well and draw off 20 c.c. 5. Add 1 c.c. of a solution of Phenolphthalein. 6. Bring to a boil. 7. Titrate with halt normal Sodium Hydrate until pink just appears. 8. Add 12 c.c. Glycerol and titrate to pink. 9. The difference between these two titrations, less the amount of alkali required by 12 c.c. of Glycerol, multiplied by 46 .0368, will give the amount of Boric acid in 20 c.c, and this mul- tiplied by 100 : Percentage of Water and divided by 20 will equal the Per cent, of Boric acid. DETECTION OF OTHER PRESERVATIVES. See "Preservatives," under Milk. DETECTION OE COLORING MATTER DETECTION OF ANNATTO AND SAFFRON, Cornwall's Method. 1. Take 5 grams of Fat in a wide tube. 2. Dissolve in 50 c.c. Ether. 3. Shake with 15 c.c. of a very dilute solution KOH. 4. Allow to stand a few hours. 5. Draw off aqueous layer. 6. Evaporate to dryness. 7. Test with Sulphuric acid. Color chants as followe : Annatto Blue to Green Brown. Saffron Blue to Brown. ANAL1NE COLORS. See Blyth's "Foods : their Composition and Analysis." Analysis of Cheese. 49 Analysis of Cheese. DETERMINATION OF WATER. i. Weigh out about 2 grams of cheese into a weighed plati- num dish, containing a little previously ignited Asbestos. 2. Heat in water oven for 12 hours. 3. Cool in dessicator and weigh. 4. Loss of weight is equal to weight of water in the weight of cheese taken. Weight of residue is equal to weight of Total Solids. DETERMINATION OF FAT. (U\ S. Dept. of Agr., Div. of Chem. Bui 36 revised, p. 36.) 1. Weigh out 2 grams of finely divided cheese into a Soxhlet extraction tube, on the bottom of which is a layer of Asbestos covered with a mixture ot anhydrous Cupric Sulphate and pure dry sand to the depth of 5 cm. 2. Extract with anhydrous Ether for five hours. 3. Remove .cheese from tube and grind in a mortar to a fine powder with a little pure sand. 4. Replace in extraction tube, washing out mortar with Ether into the tube. 5. Continue extraction for ten hours. 6. Evaporate Ether extract in weighed flask Dry residue in water bath to as constant a weight as possible and weigh. 7. Weight of re>idue is equal to the weight of Fat in the weight of cheese taken. So DETERMINATION OF PROTEIDS. Obtain by subtracting the weight of all the other constitu- ents from 100. This method is preferable to determining the Ni- trogen and then multiplying by 6.25 on account of varying per- centage of Nitrogen in cheese proteids. DETERMINATION OF NITROGEN. Determine, as in milk, by the Kjeldahl process, using 2 grams of cheese. SEPARATION OF FAT FOR EXAMINATION. (£/. 5. Dept. of Agr., Div. of Chem. Bui. ji.) 1. Cut 300 grams of cheese into fragments the size of a pea. 2. Treat in a flask', with 700 c.c. of KOH solution (50 grams per liter) at 20 Cent., shaking vigorously at intervals. 3. Collect the lumps of fat into as large a mass as possible by shaking to and fro. 4. Add cold water until fat is driven up the neck af the flask and can be removed by means of a spoon. 5. Wash with cold water to free from Potassium Hydrate. 6. Examine fat as in butter. DETECTION OF ADULTERATIONS. Besides the addition of other fats than butter fat and the adding of an excess of water, there are tew adulterations to be looked for. Preservatives (?) may be detected as in milk and butter, and coloring matters in the same way. The rind ol the cheese should be examined for mineral poisons, especially Lead and Arsenic compounds. Appendix I. United States Department of Agriculture, OPPICE OF TME SECRETARY CIRCULAR NO. 10. Standards of Purity for Food Products. 53 FOOD DEFINITIONS AND STANDARDS. I. ANIMAL PRODUCTS. B. Milk and Its Products. a. MILKS. Definition. Milk (whole milk) is the lacteal secretion obtained by the complete milking of one or more healthy cows, properly fed and kept, excluding that obtained within fifteen days before and five days after calving. Standard. Standard milk is milk containing not less than twelve (12) per cent, of total solids and not less than eight and one-half (8.5) per cent, of solids not fat, nor less than three and one-quarter (3.25) per cent, of milk fat. Definitions. 2. Blended Milk is milk modified in its composition so as to have a definite and stated percentage of one or more of its con- stituents. 3. Skim milk is milk' from which a part or all of the cream has been removed. Standard. Standard Skim Milk is skim milk containing not lessthan nine and one-quarter (9.25) per cent, of milk solids. 54 4. Buttermilk is the product that remains when hutter is re- moved from milk or cream in the process of churning. 5. Pasteurised milk is standard milk that has been heated be- low boiling but sufficiently to kill most of the active organisms present and immediately cooled to fifty degrees (50 ) Fahr. or lower to retard the development of their spores. 6. Sterilised milk is standard milk that has been heated at the temperature of boiling water or higher for a length of time suffi- cient to kill all organisms present. 7. Condensed milk is milk from which a considerable portion of water has been evaporated. 8. Sweetened condensed milk is milk from which a considerable portion of water has been evaporated and to which sugar (suc- rose ) lias been added. Standard. Standard condensed milk and standard sweetened condensed milk are condensed milk and sweetened condensed milk, respectively, containing not less than twenty-eight (28) per cent, of milk solids, of which not less than one-fourth is milk fat. 9. Condensed skim milk is skim milk from which a considera- ble portion of water has been evaporated. b. MILK FAT OR BUTTHR FAT. Definition. 1. Milk fat ox hutter fat is the fat of milk. Standard. Standard milk fat or hutter fat has a Reichert-Meissl number not less than twenty-four ( 24) and a specific gravity not less than 0.00s (40" C. / 40° C). 55 C. CREAM. Definition. i. Cream is that portion of milk, rich in butter fat, which rises to the surface of milk on standing, or is separated from it by centrifugal force. Standard. Standard cream is cream containing not less than eighteen ( 18) per cent, of milk fat. 2. Evaporated cream is cream from which a considerable por- tion of water has been evaporated. cl. BUTTER. Definition. i. Butter is the product obtained by gathering in any manner the fat of fresh or ripened milk or cream into a mass, which also contains a small portion of the other milk constituents, with or without salt. By acts of Congress approved August 2d, 1886, and May gth, 1902, butter may also contain additional coloring matter. Standard. Standard butter is butter containing not less than eighty-two and five-tenths (82.5) per cent, of butter fat. Definition. 2. Renovated or process butter is the product obtained by melt- ing butter and reworking, without the addition or use of chemi- cals or any substances except milk, cream, or salt. Standard. Standard renovated or process butter is renovated or process butter containing not more than sixteen ( 16) per cent, of water and at least eighty-two and five-tenths (82.5.) per cent, of butter fat. t. of C. 56 e. CHEESE. Definitions. i. Cheese is the solid and ripened product obtained by coagu- lating the casein of milk by means of rennet or acids, with or without the addition of ripening ferments and seasoning. By act of Congress, approved June 6, [896, cheese may also contain ad- ditional coloring matter. 2. Whole milk or full cream cheese is made from milk from which no portion of the fat has been removed. 3. Skim-cream cheese is cheese made from milk from which any portion of the fat has been removed. 4. (.ream cheese is cheese made from milk and cream, or milk containing not less than six (six) per cent, of fat. Standard. Standard whole-milk or lull-cream cheese is whole-milk or full- cream cheese containing in the water-free substance, not less than fifty (so) per cent, of butter fat. Appendix II The Babcock Method of Determining the Percentage of Fat in Milk. 59 The Babcock Method of Determining the Per centage of Fat in Milk. Of all the testing machines and instruments on the market for the purpose of determining approximately the percentage of fat in milk, the only ones which have any value whatsoever are those depending on the solution of the casein by means of con- centrated sulphuric acid and the final separation of the fat by cen- trifugal force. Of the machines operating on this principle, perhaps the Babcock tester is the best known and most widely used. The outfit consists of a set of bottles, in which the separation is made, a centrifugal machine, a 17.6 c.c. pipette and a 17.5 c.c graduate. The bottles are of about 35 c.c. capacity, with long, thin, graduated necks, the graduations reading directly in percentage of fat when 17.6 c.c. or 18 grams of milk are used. The test is con- ducted as follows : 1. Add 17.6 c.c. of milk to one of the bottles by means of the graduated pipette. 2. Add carefully, 17. S c.c. of concentrated sulphuric acid, by means of the graduated cylinder, adding the acid little by little and rotating the bottle so as to give a rotary motion to the liquid within. When the acid has been completely added and the liquid thoroughly mixed, it should be of a uniform dark brown or black- color. 3. Having filled an even number of bottles with milk and acid, place them while stiil hot in the centrifugal machine, being JUL 1 1905