ISOLATION OF MICROORGANISMS 59 



order to set up as few air currents as possible, never to lift the cover of the 

 Petri dish higher than necessary to insert the test tube for pouring, and to 

 keep the test tubes plugged as much as possible. As soon as the agar is poured 

 from the tubes, they should be lowered into a dish of water and boiled as soon 

 as possible, both to kill the organism which may have adhered to the agar 

 still in the test tube and to clean them before the agar has a chance to dry on. 



Inhibitors. — To keep back the rapidly growing organisms and allow the 

 slower growing ones to develop, various substances may be added to the 

 medium first used in isolation. Advantage is often taken of the fact that some 

 groups of fungi grow at different hydrogen ion concentration from others. 

 In these methods, varying small amounts of lactic or other organic acids are 

 added to the medium to inhibit the growth of bacteria. Sometimes dyes are 

 also used for this purpose, e.g., "gentian violet" (probably methyl violet) 

 1:500,000 (Farley 1920) or to indicate the presence of a small colony before 

 it has developed sufficiently to be seen otherwise in order that it may be 

 fished before it has been overgrown by a more rapidly growing organism. 

 Indicators are very useful in this way if the organism one desires to isolate 

 produces acid or alkali in the medium. Usually these special methods have 

 been developed in connection Avith an intensive study of a single organism and 

 are rarely useful unless the presence of a given organism is strongly suspected. 

 Slight amounts of organic acids are useful, however, in keeping down rapid 

 bacterial growth while waiting for the more slowly growing fungi to develop. 



Similarly, the choice of suitable media may do much to favor selectively 

 the development of one organism while retarding another. No general rules 

 can be given for these choices since they are largely the result of wide experi- 

 ence and a shrewd guess as to the probable organism to be isolated. A thor- 

 ough knowledge of the physiology of the various groups of fungi will be 

 helpful, but in the present state of our knowledge generalization is very 

 difficult. 



Microcultures. — In the study of the life cycle of many organisms it be- 

 comes desirable to have a given spore or bit of mycelium under more or less 

 continuous observation with the microscope. One of the early methods which 

 has yielded much useful information is the hanging drop culture. A.n early 

 and inexpensive form, usually referred to as a Van Tieghem cell, consists of 

 a glass ring cemented to a microscopic slide with wax (made by melting to- 

 gether pure beeswax and vaseline). The top of the ring is coated with vase- 

 line. A drop of the culture medium or water is placed in the bottom of the 

 cell thus formed. Another smaller drop is placed upon a clean cover slip of 

 sufficient diameter to cover the ring. The inoculum is then placed in the 

 center of this drop, and the whole seized by forceps and quickly inverted, care 

 being taken that the drop does not spread too near the edge of the cover dur- 

 ing the process. The cover (with the drop of medium or water hanging from 

 it) is then lowered to the glass ring and pressed down gently until the soft 

 vaseline (petrolatum) seals it to the ring. Thus we have a small moist chamber 

 with the organism suspended in a drop from the cover. The drop placed in 



