ISOLATION AND FURE-CULTURE METHODS 1 1 



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had long been accumulating. More than a century prior to the 

 dates mentioned, Bonnet and Spallanzani showed some apprecia- 

 tion of the principles of sterilization and they were more or less 

 successful in attempting to demonstrate that when the organisms 

 in any given nutrient medium are killed, an entrance of germs 

 from without is necessary in order that growth may subsequently 

 occur. Nevertheless, belief in the infelicitous idea of spontaneous 

 generation gained strength, and was not finally abandoned by some 

 prominent scientific men until after the great conquests made by 

 Pasteur and others in the fields of fermentation and disease. An 

 important era was marked by Cohn's demonstration that the spores 

 of many bacteria are particularly resistant to heat, and that it is 

 only after passing into the vegetative condition that boiling may 

 effectually kill such organisms. This led promptly to the adoption 

 of a discontinuous method of sterilization, and thus it became a 

 matter of easy manipulation to grow organisms in media rendered 

 absolutely sterile. 



It was in 1873 that Klebs described a "fractional" method of 

 isolating bacteria, and Lister five years later developed a " dilu- 

 tion " method. Viewed in the light of to-day these methods were 

 burdensome, yet they were not impossible in the hands of careful 

 workers. The methods adopted were necessarily extremely crude 

 in comparison with those employed to-day. The dilution process 

 was the surest practical method of isolating yeasts and bacteria. 

 This method consisted essentially in diluting to such extent, in the 

 beaker or other vessel of sterile water, a drop of any fluid con- 

 taining the organism so that a drop of the diluted material would 

 contain, perhaps, not more than a single cell or organism. This 

 dilution was, of course, based upon a tedious count made under the 

 microscope. If, then, drops of this water in which the organisms 

 or cells were suspended should be transferred one to each of sev- 

 eral tubes containing any desired medium, a separation might be 

 effected. Drops of the liquid containing the organisms might also 

 be spread on the surface of a sterile slice of potato, and, with growth, 

 separate colonies might appear. This was practically the status of 

 methods which had been developed for the isolation of microscopic 

 organisms, up to about 1881, which date marks the beginning of a 

 very distinct advance. 



