10 



A. P. H. A. MILK ANALYSIS 



Procedure No. 2. Where large 

 quantities of agar are to be prepared 

 the following procedure has been found 

 useful. Prepare two separate solutions: 



Mixture A 



Beef extract 0.3 per cent of total 

 quantity of medium to be made. 



Peptone 0.5 per cent of total quan- 

 tity of medium to be made. 



Distilled water 40 per cent of total 

 quantity of medium to be made. 



Place in a kettle. Weigh kettle 

 with contents. Heat on stove to boil- 

 ing, and boil five minutes. If abso- 

 lutely necessary to adjust reaction 

 (see Reaction) do so at this point and 

 boil again. Make up with hot dis- 

 tilled water that lost by evaporation. 

 Do this by weight. Filter through 

 paper or paper pulp in a Buchner 

 funnel (see below). 



Mixture B 



Agar oven dried 1.2 per cent (market 

 1.5 per cent) of total quantity of 

 medium to be made. Soak and wash 

 under tap in sieve. Weigh before 

 and after soaking to determine quantity 

 of water absorbed. Distilled water 

 60 per cent of total quantity of medium 

 to be made, minus that absorbed by 

 the agar during the washing. 



Mix A and B (agar not yet melted). 

 Heat mixture over stove, stirring at 

 frequent intervals until agar is entirely 

 melted. Then boil and stir constantly 

 for 20 minutes. Make up by weight 

 water lost by evaporation by adding hot 

 distilled water. Keep kettle of agar 

 in chamber of flowing steam while pre- 

 paring funnel for filtering. 



Filter through cotton until clear. 

 For 10 liter amounts it is suggested 

 that either a Sharpies centrifuge or a 

 nine inch Buchner funnel with a 

 suction pump be used. The ordinary 

 filtration pump attached to a water 



faucet producing about 11 inches of 

 vacuum gives good results. 



Prepare paper pulp by soaking scraps 

 of ordinary filter paper for 36 to 48 

 hours in a large wide-mouthed bottle. 

 The paper and water should be in the 

 ratio of six sheets of soft absorb- 

 ent filter paper (20 by 20 inches) to 

 2}i liters of hot water. Moisten the 

 paper and tear it into fragments about 

 yi \.o yi inches square. Shake vig- 

 orously at intervals to make the 

 suspension fine and uniform. When 

 ready to prepare the nine inch funnel, 

 take 400 to 500 cc. of the paper pulp 

 and dilute it with about three liters 

 of very hot water. Cut a piece of 

 surgeon's lint (or cotton flannel) to 

 fit the bottom of the funnel exactly. 

 Rinse the funnel with hot water. 

 Place in it the lint with the fleecy 

 side uppermost. Pour in the hot paper 

 pulp suspension carefully so as to cover 

 the lint with an even layer about }/% 

 to y^, inch thick. Over this lay a disk 

 of filter paper. Place a four liter 

 suction flask under the funnel and 

 apply the suction to draw the water 

 into the filtration flask until the pulp 

 is firm, yet somewhat moist. The 

 agar will not go through too dry a filter. 



The funnel and the paper pulp 

 must be hot when the agar is poured in 

 carefully and slowly, striking the disk 

 of filter paper which prevents the 

 breaking of the surface of the paper 

 pulp. Discard the first 100 cc. of 

 agar which come through as they 

 contain some of the water from the 

 pulp. 



Even in the first filtration the agar 

 should come through very clear. Keep 

 the remainder of the unfiltered agar 

 hot in flowing steam while the first part 

 is running through the filter. 



Ordinarily the temperature of the 

 agar in the funnel is 80° to 85° C. but 



