196 Cultivation of Micro-organisms 



autoclave, after which the two solutions are poured together and 

 boiled until all of the albumins are precipitated. The coagulation 

 of the albumins of the meat-infusion serves to clarify the agar-agar, 

 but it must be filtered through gauze and cotton. 



If agar-agar is to be made with beef-extract, the bouillon should 

 be made first and filtered when cold, to exclude the uratic salts which 

 otherwise precipitate in the agar-agar when cold and form an un- 

 sightly cloud. 



The finished agar-agar should be a colorless, nearly transparent, , 

 firm jelly. It is dispensed in tubes like the gelatin and bouillon, 

 sterilized by steam, either by the intermittent process or in the auto- 

 clave, and after the last sterilization, before cooling, each tube is 

 inclined against a slight elevation, so as to permit the jelly to solidify 

 obhquely and afford an extensive flat surface for the culture. 



After the agar-agar jelly solidifies it retracts so that a Uttle water 

 collects at the lower part of the tube. This should not be removed, 

 as it keeps the jelly moist, and also distinctly influences the character 

 of the growth of the bacteria. 



Glycerin Agar-agar. — Certain bacteria among which is the 

 tubercle bacillus, will not grow upon agar-agar prepared as described 

 above, but will do so if 3 to 7 per cent, of glycerin be added after 

 filtration. This fact was discovered by Roux and Nocard. 



Blood agar-agar was recommended by R. Pfeiffer for the culti- 

 vation of the influenza bacillus and consisted of ordinary agar-agar 

 whose surface is coated with a little blood secured under aseptic 

 precautions from the finger-tip, ear-lobule, etc., of man, or from the 

 vein of one of the lower animals. Some bacteriologists prepare a 

 hemoglobin agar-agar by spreading a little powdered hemoglobin 

 upon the surface of the agar-agar. As powdered hemoglobin is 

 not sterile, the medium must be sterilized after its addition. 



As employed at the present time for the cultivation of the meningo- 

 coccus, influenza bacillus and other fastidious micro-organisms, the 

 blood corpuscles are hemolyzed and added to the medium just 

 before use. For the micro-organisms mentioned a dextrose-hemo- 

 globin — agar-agar seems to be most appropriate and is prepared as 

 follows: 



Ordinary meat juice agar-agar is prepared and sterilized. A one per cent, 

 aqueous solution of dextrose is made in distilled water in a flask and also sterilized. 

 Human blood is taken under aseptic precautions from a vein of the forearm 

 caught in a sterile flask, containing sterile glass beads with which it is defibrinated 

 by shaking and then decanted into a second flask containing twice the volume 

 of sterile distilled water. At the end of twenty-four hours the corpuscles are 

 usually laked and the hemoglobin in solution, the corpuscular bodies sedimenting. 

 The greatest pains must be taken to keep the blood and water mixture sterile. 



The agar-agar is now melted and cooled to less than 5o°C. The flask is 

 cautiously opened and one per cent, of the dextrose solution added (if the 

 sterilization of the agar-agar is to be made in the Arnold apparatus, the dextrose 

 addition may have been made beforehand) and then an addition of one per cent, 

 of the hemoglobin solution is made. When all are mixed, the medium may be 

 iilled under aseptic precautions into tubes or Petri dishes as the work requires. 



