THE BIOCHEMISTRY OF MILK 145 



then titrate drop by drop, touching the glass rod 

 to the indicator paper. Let the precipitate settle 

 for 5 minutes. Filter it through cloth as follows: 

 place a piece of cloth over a beaker; depress 

 the middle. Pour in the suspension, slowly. 

 When the flow slows down, shape the cloth into 

 a bag, and squeeze as much of the excess liquid 

 into the beaker as possible. 



Add one marble chip to the filtrate in the 

 beaker, mark the level of the liquid with a wax 

 pencil, and boil gently over the flame of a bunsen 

 burner until the volume of liquid is reduced to 

 a little less than half. (The marble chip will pre- 

 vent bumping during the boiling.) 



Meanwhile, continue working with the casein. 

 With the precipitate still in the cloth, press out 

 excess moisture with paper towels. When this is 

 done, transfer the precipitate to a beaker, and 

 add sufficient alcohol to cover it. (Caution: 

 Highly inflammable.) Break up the particles of 

 casein with a glass rod. Let the casein settle 

 and pour off the liquid. Casein does not dissolve 

 in alcohol, but washing it in this way removes 

 impurities which do. Press out the excess alco- 

 hol from the precipitate in the beaker with a 

 paper towel. Repeat the alcohol washing. Press 

 out again and set it aside to dry. At the end 

 of the afternoon transfer the casein to a card- 

 board container. 



Filter out the coagulated proteins using the 

 suction funnel. Put a paper filter into the funnel, 

 wet it down with a little water, turn on the 

 aspirator, and connect the suction hose to the 

 flask. Pour in the solution containing the coagu- 

 lated protein. When the liquid has all passed 

 through, disconnect the suction hose or release 

 pressure by letting air enter. (Do not release 

 suction by turning off" the aspirator, since this 

 will cause water from the line to back up into 

 the flask.) Pour the filtrate into a beaker. 

 Wash the coagulated protein on the filter pad 

 by pouring alcohol over it and sucking it dry. 

 Do this twice; then remove the material from 

 the filter into a cardboard container with a glass 

 rod or spatula. Label. 



Rinse out the beakers and suction flask before 

 using them in subsequent steps. 



Evaporate the solution further. Use a very 

 gentle flame and stir constantly with a glass rod, 

 lest the liquid boil out of the beaker. (In heating 

 liquids avoid at any time having your face near 

 the mouth of the beaker or tube so that acci- 

 dental spurting will not result in injury. And 

 don't forget also to be your neighbor's keeper.) 

 Continue evaporation until a white precipitate 

 of calcium phosphate appears. (The volume of 

 the liquid will probably be about 20 ml.) Cool 

 to room temperature. Filter with suction. Set 

 aside the filtrate. Wash the precipitate three 

 times with small portions (about 5 ml) of cold 

 water. Suck dry. Transfer to another card- 

 board container. 



Evaporate the filtrate over a very gentle 

 flame until it becomes syrupy, or to a volume 

 of about 10 ml. Stir constantly and remove 

 the flame if necessary so that neither excessive 

 foaming nor charring of the sugar occurs. Add 

 this syrup to about 6 ml (about two inches) of 

 acetone in a test tube. (Caution: Highly in- 

 flammable.) Stopper, and shake to disperse 

 contents. Label the test tube with your name 

 and place it in the refrigerator until next 

 week. 



Make a dialysis sac out of cellophane tubing. 

 Obtain about 9 inches of tubing. Don't bend it 

 sharply while it is dry or it will develop leaks. 

 Wet it thoroughly with water, and open it by 

 running water through it. Tie a sturdy knot to 

 seal off' one end, and knot it again for good 

 measure. With a pipet introduce 5 ml of milk 

 into the sac. (In using the pipet, hold it between 

 the thumb and middle finger; the index finger 

 over the end acts as a valve.) Carefully tie a 

 knot in the top part of the bag. Put it in a large 

 test tube. Add water to the level of the milk. 

 Cover with a stopper, label, and leave it in the 

 refrigerator. 



Digestion of casein 



It requires many hours of boiling in strong 

 acid or alkali to hydrolyze a protein molecule. 

 Living systems accomplish the same thing in 

 relatively neutral solution, and at relatively low 



