LESSON VIII. 



ISOLATION OF BACTERIA IN PURE CULTURES. 



It will be fortunate if after inoculation of nutrient substances and culture of the 

 inoculated media the organisms grown should prove from their gross and minute 

 characteristics to be of a single type. This may be expected only in instances where 

 the efforts of the operator have from the first been directed, in the collection of the 

 infected material, to the acquisition of but a single species, or in culture to permit 

 conditions favoring the development of but a single type of organism; in the best 

 directed attempts it is not uncommon, and in ordinary investigation of much of the 

 material likely to be submitted for analysis it is invariable that there should occur 

 several, perhaps a great variety of growths upon the media. It may naturally be 

 . inferred as impossible to study with any success the characteristics outlined in the 

 preceding two chapters for any bacterium so long as it is confused with others. Hence 

 arises in all bacteriologic study, whether of pathogenic or non -pathogenic forms, the 

 necessity of Koch's second postulate: "The organism to be studied must be obtained 

 in pure culture." 



The methods available for the isolation of organisms in pure culture arrange 

 themselves readily into two groups: (a) mechanical procedures, and (6) methods based 

 upon physiologic peculiarities of the bacterium sought. 



(A) MECHANICAL METHODS. 



i. Plating Methods. When it is known from previous experience or may be 

 reasonably inferred that a given material to be submitted to, bacteriologic analysis 

 contains a number of different organisms the exact nature of which is not known 

 (for which reason it is impracticable to select a special method for isolation), efforts 

 to widely distribute the organisms, and the colonies resulting in culture from their 

 development, over an extended area, offer no little probability of success, so that with 

 the sterilized needle each growth may be picked up and transferred to a separate 

 container of sterile medium where it may develop alone. Any of the three modes of 

 plating may be employed, plates, Petri dishes, or Esmarch's tubes, selection of one 

 or other depending mainly upon the need of care to prevent contamination, but also 

 partly upon the medium of growth selected, the conditions of temperature and atmos- 

 phere to be used in the development of the plated culture, and upon the extent of 

 surface believed essential for proper dissemination of the bacteria. One would not 

 select the rolled tubes if a wide surface be desired; yet they offer the greatest chance 

 of remaining free from atmospheric contamination and are the most easily manipulated 

 in incubator and anaerobic conditions. Plates and dishes are much more e isily made 

 if gelatine, rather than agar, be the medium used. Petri dishes are less liible to air 

 contamination than plates, and are more conveniently placed in the incubator or 

 anaerobic jar and more readily examined in the course of the culture. In any com- 



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