404 APPENDIX. 



preparation after this is the same in both cases. Boil the mixture thoroughly 

 over a naked flame for about an hour (great care being required at first to 

 prevent the mixture boiling over in consequence of the large amount of 

 air contained in the agar-agar, which is, however, gradually boiled out). As 

 a small quantity of the agar is dissolved, the boiling point is gradually raised 

 until the temperature of the mixture is about 105 C., instead of 100 C., and 

 the complete solution of the agar-agar is more readily brought about. As 

 soon as the whole of the agar is dissolved, which is usually in about an hour, 

 the mass should be filtered as quickly as possible through a very hot filter. 

 To Make Glycerine Agar. After filtration of the agar jelly, add from 5 

 to 8 per cent, of glycerine, i.e., 50 to 80 cc. per 1,000, sterilize and put into 

 test tubes. After sterilizing, allow to cool in the oblique position in order 

 to obtain a larger surface. This also applies to the other agar-agar media. 



For the preparation of Agar- Agar and Gelatine (see p. 392.) 

 Koch's Sterilized Blood Serum. 



A large stoppered glass jar, well washed with soap and warm water, is 

 sterilized either by being wrapped up in paper and heated for an hour at a 

 temperature of 150 C., or by being rinsed with a I to 1,000 solution of 

 bichloride of mercury, and then with absolute alcohol to remove the mercury 

 salt. The stopper is greased with pure vaseline. When the animal from 

 which the blood is to be collected has been stabbed the first few drops of 

 blood that come should be rejected, and that which follows must be col- 

 lected as carefully as possible. The jar is then placed in cold water until 

 the blood coagulates, removed to the laboratory and placed in an ice box ; 

 and the first few drops of coloured serum that soon make their appearance on 

 the surface of the clot are carefully removed with a pipette. It is then left 

 for from twenty-four to thirty-six hours, at the end of which time a quantity 

 of clear serum will be found to have been expressed. In the meantime a 

 pipette has been prepared by exposing it to the action of boiling water for 

 ten or fifteen minutes, or by thoroughly cleaning it with hot water and 

 rinsing with bichloride of mercury and alcohol and then passing it through 

 a flame for the purpose of driving off the alcohol. This pipette is used to 

 transfer the clear serum from the space around the clot to the sterilized 

 plugged test tubes, care being taken that the serum is not allowed to smear 

 the sides of these tubes. The test tubes, with the contained clear serum, are 

 placed for an hour in a water bath, which is kept at a temperature of 58 

 to 60 C. every day for a week. Each day the serum becomes clearer, and 

 a small precipitate of grey powdery material collects at the bottom and a 

 thin film of cholesterin forms on the surface. This water bath may be 

 constructed very cheaply. It is made of tin, is about twenty-seven inches 

 high and four inches in diameter, with a collar projecting about half an 

 inch. Over this collar a piece of iron netting serves both as a lid and as a 

 support to the thermometer, which is fixed in a cork passing through a hole 

 in the centre ; or a bulb thermometer may be used. This is supported by a 

 second piece of wire gauze resting at some little distance about three- 

 quarters of an inch from the bottom of the bath. When the water has 

 reached a temperature of 58 C. it is easily maintained there by means of a 

 small spirit flame. In place of this simple apparatus any of the small incu- 

 bating chambers sold by the chemical apparatus makers may be used for this 

 discontinuous sterilization at comparatively low temperature. Special serum 



