214 CULTURE METHODS 



Transfers of spores are made to the tubes by means of a piece of stiff 

 platinum wire about two inches long, set in the end of a glass rod when 

 melted in a flame. The lower end of the rod and the wire are sterilized in a 

 flame and the point of the wire is touched to a single spore-bearing hypha 

 in a culture. The cotton plug is then carefully removed from a tube and 

 held between the fingers while the point of the wire is drawn over the sur- 

 face of the agar. It is best that the tube be held inverted while being inoc- 

 ulated so that dust may not enter. The plug is replaced quickly and the 

 inoculated tube is laid aside to await developments. If but a single spore- 

 bearing filament has been touched, the culture will probably be pure. Should 

 an impure culture develop, transfers may generally be made from it to 

 another tube. Tubes with cultures may be prevented from drying out too 

 rapidly by cutting off the tops of the cotton plugs and coating the ends of the 

 tubes with paraSin. 



Potato agar may be poured into Petri dishes and sterilized in the same 

 manner as the test tubes. Such dishes are excellent for studies upon the 

 bacteria as outlined in Sec. 100. 



204. Hanging-drop cultures. These are used for the study of germinating 

 spores, pollen grains, and other subjects. The spores are placed in a drop 

 of the culture fluid on a cover glass, which is then arranged so that the drop 

 hangs down from the lower side in a small moist chamber on a slide. The 

 chamber may be made of a ring of glass cemented on the slide with wax or 

 gold size (Van Tieghem cell), over which the cover glass fits and is sealed 

 with vaseline. A little distilled water in the bottom of the chamber will save 

 some evaporation from the drop of the culture fluid, 



A more temporary but also effective chamber may be made by cutting a 

 square hole about one half inch in diameter in the center of a piece of card- 

 board one inch wide, one and one-half inches long, and one eighth of an 

 inch or more thick. The cardboard is boiled and pressed closely on the slide. 

 The culture drop is then placed in the center of a cover glass an inch square 

 or slightly smaller. This is inverted over the hole in the cardboard so that the 

 culture drop hangs down in the center and the cover glass is then pressed 

 closely against its wet surface. Water is added from time to time to the 

 edge of the cardboard to keep it moist, and the slide when not being studied 

 may be placed in a moist chamber, which will hinder the cardboard from 

 drying rapidly. 



The culture fluids vary with the subject. Boiled decoctions of horse dung 

 are good for the germination of many fungal spores. Decoctions of decayed 

 wood are used for the spores of slime molds. Solutions of sugar (3-30% in 

 tap water) are employed in the germination of pollen grains (see Experiment 

 XLII) ; 1.5% of gelatin may sometimes be added to advantage to the sugar 

 solutions. Spores of mosses and ferns germinate readily in sweetened water. 



