6o4 LABORATORY EXERCISES 



Solidified Blood-scrum. — As this medium is rather difficult to obtain and prepare, 

 being one of the most difficult to make in culture work, the plant mycologist must 

 in general obtain blood-serum from the animal bacteriologist. Near Philadelphia it 

 can be purchased from the laboratory of H. K. Mulford & Co., Glenolden, Pa. The 

 solidified serum may be used either plain or with the addition of grape sugar. 



Fresh Serum How Obtained. — Procure blood by a sterile method from a horse, 

 or a cow, and stand it aside in a cool place, breaking the clot from the side of the jar 

 until the amber-colored serum rises to the surface, when it is to be drawn off. It 

 is then filtered and measured off. To 3 parts of this serum, i part of bouillon, pre- 

 pared in the ordinary way, is to be added. The mixture of bouillon and serum is 

 then to be filled into the sterile test-tubes, care being taken to slant the tubes. By 

 being placed in a steam sterilizer and sterilized by intermittent sterilization for three 

 days, care being taken to leave off the cover of the sterilizer, the serum will be found 

 to be white and quite hard and ready for use. If the lid of the sterilizer is kept on 

 and the heat becomes too great, bubbles will form in the serum and thus spoil the 

 usefulness of the hardened serum. 



Before filling the tubes, care must be taken that the mixed serum and bouillon 

 are thoroughly neutralized by NaOH. As blood-serum is rarely used in mycologic 

 work, the above notes are given merely for reference. The teacher will probably 

 find it convenient to omit this part of Lesson 10 entirely. 



LESSON 11 



Nutrient Gelatin.- — To 1000 c.c. of sterile peptonized beef-bouillon add 100 grams 

 of best quality gelatin. Soak two hours at room temperature, then steam five 

 minutes, cool, titrate, add the necessary alkali, steam thirty minutes, filter through 

 filter paper, wash with sterile boiling hot water, tube at once, and heat in the steamer 

 on three successive days fifteen minutes, ten minutes and five minutes respectively 

 at ioo°C. Do not autoclave, and carefully avoid long heating in the steamer. 

 Have all glassware sterile, the fluids sterile and sufficiently boiled to begin with. 

 The very best English, French or German gelatins should be used. +10 or +15 is 

 a good degree of alkalinity for many purposes. 



Sugar Gelatin. 



Water, c.c 600 



Peptone, grams 6 



Salt, grams 3 



Beef extract, grams 3 



Glucose, grams 6 



Gelatin, grams 60 



The gelatin is added as the mixture in water is brought to a boil. The mixture 

 is cooled down to 60° below the coagulating point of albumen and the white of two 

 eggs for every ioqo c.c. of water added. It is then brought to a boil, the albumen 

 coagulates and clarifies the medium. The fluid is then filtered through filter paper 

 previously wetted with boiling water. A funnel with wire support for filter paper is 

 to be preferred for ease in filtering. 



