LESSON XV COAGULATION OF MILK 1. Prepare a solution of rennet by extracting the fourth stomach of the sucking calf with glycerine. Clark's rennet, which is sold for making junkets, will do equally well. 2. Prepare a solution of pure caseinogen in the following way :---Saturate milk with magnesium sulphate by shaking it with excess of the powdered salt. Allow it to stand for a few hours, and then filter. The caseinogen and fat remain together on the filter. Save the filtrate, and label it A. Wash the precipitate on the filter with saturated solution of magnesium sulphate until the washings contain no albumin. Add water to the precipitate. The caseinogen dissolves, the fat being insoluble. In this way a solution of caseinogen in weak magnesium sulphate is obtained. So far the operations should be performed beforehand by the demonstrator. 3. To this solution add acetic acid. The caseinogen is precipitated; collect it on a filter; wash the acid away with distilled water. Dissolve the precipitate in lime water by grinding it up in a mortar with the lime water; filter, and an opalescent solution of caseinogen is obtained. 4. To a portion of this solution add a few drops of rennet extract. Put it in the water-bath at 40° C., and if the caseinogen has been thoroughly washed no coagulation will occur. 5. Treat another portion in the same way, adding, however, a few drops of 0.5-per-cent. phosphoric acid as well as the rennet. Warm to 40° C. Coagulation-that is, formation of casein from caseinogen-usually occurs in a few minutes. 6. Examine the filtrate A (see above). Saturate a portion with sodium chloride. A small amount of precipitate of a proteid comes down. This is the so-called lacto-globulin. This contains only a trace of true globulin: it is mostly caseinogen previously left in solution, together with calcium sulphate. 7. Heat another portion of A to 77°, acidifying faintly with a few drops of 2-per-cent. acetic acid. Lactalbumin is coagulated at this temperature. 8. Ringer's method of showing the conversion of caseinogen into casein :Milk is strongly acidified with acetic acid. This precipitates the caseinogen and entangled fat. The precipitate is collected on a filter, thoroughly washed with distilled water, and ground up in a mortar with calcium carbonate. The mixture is thrown into excess of distilled water. The fat rises to the top; the excess of calcium carbonate falls to the bottom. The intermediate fluid contains the caseinogen in solution; it is usually very opalescent. Take some of this solution and divide it into three parts, A, B, and C. To A add rennet. To B add a few drops of 10-per-cent. solution of calcium chloride. To C add both rennet and calcium chloride. Put all three in the water-bath at 40° C. A clot of casein forms in C, but not in A and B. (See Chemical Physiology and Pathology,' pp. 580-585.) LESSON XVI FLOUR A. Prepare some gluten by washing a quantity of flour in a muslin bag (see Lesson IV.). It is sticky and elastic. Treat small portions as follows:— 1. Add 10-per-cent. sodium chloride solution; no gluten dissolves. The liquid may become slightly turbid if all the starch has not been washed away from the gluten. 2. Grind up some with alcohol in a mortar. Filter. The filtrate is opalescent, it having dissolved out a proteose from the gluten. The residue is not sticky, and is called gluten-fibrin. Test the filtrate as follows:-Add distilled water; this gives a white precipitate. Boiling gives no precipitate. A trace of copper sulphate and caustic potash give a pink colour (biuret reaction). B. Flour has been extracted with 10-per-cent. sodium chloride solution. After twenty-four hours the supernatant fluid is siphoned off. This extract has the following characters : 1. Reaction is neutral. 2. Iodine gives a reddish-brown colour due to erythro-granulose. 3. Copper sulphate and potash give a violet colour. 4. Magnesium sulphate added to saturation gives a dense precipitate of globulin and proteose. 5. Saturation with sodium chloride also gives a precipitate. This is less dense, and consists chiefly of globulin. Filter. The filtrate gives a precipitate of proteose with acetic acid. 6. Boil the original liquid. Globulin is coagulated. Filter. The filtrate contains the proteose, and gives the following tests: (a) Biuret reaction. (B) Saturation with magnesium sulphate or sodium chloride gives a precipitate; the precipitate with sodium chloride is, however, not very dense unless acetic acid be added also. (y) Acetic acid gives a precipitate. (8) Nitric acid gives a precipitate in the cold, which dissolves on heating and reappears on cooling. C. Examine the residue of the flour after extraction with 10-per-cent. sodium chloride solution. Wash it in a muslin bag and observe that very little gluten is obtainable, as the proteids, which are the precursors of gluten, have been previously extracted by the salt solution. (For Vegetable Proteids see Chemical Physiology and Pathology,' pp. 131-137.) LESSON XVII THE ALBUMOSES 1. Witte's peptone contains very little true peptone, but consists chiefly of albumoses, which are soluble, like peptone, in neutral saline solutions. 2. Make a solution of this substance in 10-per-cent. sodium chloride solution, and filter. Very little residue is left on the filter. This consists of dysalbumose, an insoluble form of hetero-albumose, formed during the process of preparing the substance. If hot saline solution is used instead of cold as a solvent, this amount of insoluble residue is increased, hetero-albumose being to a slight extent precipitated by heat. 3. The solution gives the following tests : (a) Biuret reaction (due both to peptone and albumoses). (b) A drop of nitric acid gives a precipitate, which dissolves upon heating and reappears on cooling. (This is due to the albumoses present.) (c) It does not coagulate on heating. Otherwise it gives the ordinary proteid reactions. - 4. For the separation of the albumoses and peptone proceed as follows:(a) Saturate the solution with ammonium sulphate, and filter. The filtrate contains the peptone and the precipitate the albumoses. The peptone is not precipitated by nitric acid, nor by most of the reagents that precipitate other proteids. It is precipitated completely by alcohol, tannin, and potassiomercuric iodide; imperfectly by phospho-tungstic and phospho-molybdic acid. It gives the biuret reaction, but in the presence of ammonium sulphate a large excess of caustic potash is necessary. (b) Dialyse another portion of the solution; hetero-albumose is precipitated. (c) Saturate another portion of the solution with sodium chloride after faintly acidulating with acetic acid. Proto-albumose and hetero-albumose are precipitated. Filter. The filtrate contains the deutero-albumose and peptone. The proto- and hetero-albumose may be redissolved by adding distilled water, and may be separated from each other by dialysis (see b). Deutero-albumose may be separated from the peptone by saturation with ammonium sulphate, or by the addition of a crystal of phosphoric acid. These reagents precipitate the deutero-albumose, but not the peptone. Deutero-albumose gives the nitric-acid reaction (see 3, b) characteristic of the albumoses only in the presence of excess of salt. If the salt is removed by dialysis, nitric acid then causes no precipitate. (See Chemical Physiology and Pathology,' pp. 128–131 and 645–648.) LESSON XVIII DIGESTION 1. Examine the comparative digestive power of the glycerine extracts of two stomachs. Take in two test-tubes an equal small weighed quantity of fibrin stained with carmine. Add to each 10 c.c. of 0.2 per cent. hydrochloric acid. Add to one a measured quantity of one glycerine extract, and to the other an equal quantity of the other glycerine extract. As the fibrin is digested the carmine is set free, and colours the liquid; that which is more deeply stained is that which contains the more active preparation of pepsin. 2. Pancreatic digestion. A finely divided ox-pancreas has been allowed to digest at 40° C. for twenty-four to thirty-six hours in a litre of 1 per cent. sodium carbonate to which the white of an egg has been added every ten hours. Note the odour, due to putrefaction. Another preparation has been similarly made except that thymol has been added to prevent decomposition. These should be got ready by the demonstrator. 3. Filter some of the extract and examine for leucine and tyrosine as follows: - (a) To some of the liquid add Millon's reagent and filter off the precipitated proteid. Boil the filtrate. The presence of tyrosine is indicated by a red colour. If tyrosine is abundant the red colour appears without boiling. (b) Boil another portion of the filtrate; filter off the proteid thus coagulated; reduce the filtrate to a small bulk by evaporation on the water-bath at the boiling temperature. Examine a drop microscopically for crystals of leucine and tyrosine. Treat the remainder with excess of alcohol to precipitate the albumoses and peptones and again filter. Concentrate the filtrate on the water-bath till it becomes sticky from the presence of leucine. Examine some of the concentrated fluid with the microscope; leucine will be found in crystalline spheroidal clumps. 4. Examine microscopic specimens of leucine and tyrosine which have been prepared by the demonstrator. The first of these additional exercises on digestion illustrates the principle of Grützner's method of comparing the digestive powers of solutions. In the original method the amount of carmine set free is estimated by an artificial scale consisting of ten solutions of carmine of different known strengths. The carmine solution for staining the fibrin is prepared by dissolving 1 gramme of carmine in about 1 c.c. of ammonia; to this 400 c.c. of water |