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(2.) The organism must be isolated and obtained as 

 an absolutely pure culture. 



(3.) The pure culture of the organism when intro- 

 duced into susceptible animals must produce the disease. 



(4.) In the disease thus produced the organism must 

 be found distributed the same as in the natural disease. 



To these four requirements, a fifth may be added, 

 namely: That the chemical products of the organism must 

 produce the characteristic symptoms and effects of that 

 disease. 



The demonstration of the constant presence of an 

 organism in a disease is accomplished by hanging-drop 

 examination, stained cover-glass preparation, or by stain- 

 ing sections of tissues and organs. Frequently the direct 

 detection of the organism is difficult owing either to its 

 scarcity or to the absence of definite characteristics. In 

 such cases artificial, culture or animal experiment will 

 prove the presence of the organism. 



The mere fact that an organism is constantly present 

 in a given disease does not prove that it is the cause of 

 that disease. It certainly is strong presumptive evidence 

 that the organism does bear a causal relation to that dis- 

 ease, but at the same time the possibility must be admitted 

 that it may be an accompaniment, or even a consequent 

 of ihat disease. To complete the chain of evidence it is 

 necessary, therefore, to obtain the organism in a pure 

 culture, and, inoculation of animals with such cultures 

 must reproduce the disease. 



The isolation of the organism and the preparation of 

 pure cultures is accomplished by the gelatin plate method 

 or its modifications. The isolated colony which develops 

 on a plate is derived from a single cell, and is, therefore, a 

 pure culture. Transplantations from the colony, when 

 properly made, into tubes of gelatin, agar, or bouillon, in 

 turn are pure cultures. Subsequent transplantations from 

 tube to tube can be made as often as may be desired, or as 

 may be necesssary. Each growth thus obtained is called 



