CHAPTER IV 



ISOLATION OF MICROORGANISMS 



Transfer. — This is perhaps the simplest process connected with the hand- 

 ling- of organisms in culture and should be practiced by the beginner until 

 the operations can be performed quickly and entirely without contaminations 

 before more complicated procedures are attempted. Essentially the process 

 consists in taking a small amount of mycelium or spores of fungi or a few 

 bacterial cells and moving them from one receptacle of medium to another. 

 The test tube containing the culture of the organism to be transferred and 

 a tube of medium are held about half an inch apart between the thumb and 

 fingers of the left hand with the thumb above. The plugs of both tubes are 

 loosened in turn Avith a rotary motion, but not withdrawn. The transfer needle, 

 loop, or platinum spatula, as the case may be, is grasped between the thumb 

 and first finger of the right hand, the platinum is heated to redness in a blue 

 flame and allowed to cool while still in the hand and without the heated por- 

 tion touching anything. The cotton plug of the tube containing the organism 

 to be transferred is grasped between the second and third finger of the right 

 hand and gently withdrawn, care being taken to create as few air currents 

 as possible. A needle or loop is inserted without touching the walls of the 

 tube and touched to the spores or to the vegetative material if it is slimy, or 

 a platinum spatula is used to cut away a little mycelium which should adhere 

 to it. The needle is then gently withdrawn, the plug replaced and the other 

 plug withdrawn, and the needle stroked along the surface of the agar (or the 

 mycelium dislodged from the spatula). The needle is then gently withdrawn, 

 the second plug replaced, and the needle (or spatula) flamed before laying 

 it down, to destroy any organisms still adhering to it. The plugs may then 

 be pushed in more firmly if necessary. In some laboratories it is customary 

 to flame and quickly extinguish the cotton plugs. 



Isolation. — The simplest procedure is similar to simple transfer in which 

 the needle or spatula is touched to the infective material and then transferred 

 to the surface of agar in a test tube or Petri dish. This method is only occa- 

 sionally successful, usuallj^ in those cases where the infective material is small 

 and uncontaminated by other organisms than the one which it is desired to 

 study. If contamination is only slight, a carefully executed transfer from 

 the colony Avliich is desired may result in securing a pure culture. Otherwise, 

 resort must be had to dilution and plating out. 



Isolations From Skin Lesions. — After cleansing the skin with 95% alcohol 

 followed by ether, the lesion is scraped with a sharp, full-bellied, sterile scalpel 

 or shaved with a sterile safety razor to the point where bloody serum just 

 begins to ooze. A sterile Petri dish is placed beneath to catch the scrapings, 



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