REAGENTS AND PROCESSES 555 



forceps. The bulb is then heated in the flame of an alcohol lamp or Bunsen burner 

 to expand the air. The end of the neck is next quickly dipped into the nutrient 

 fluid, which is forced up the neck into the bulb as the air in this cools. When the 

 bulb is two-thirds full, the neck is withdrawn from the fluid and hermetically 

 sealed in a flame. In filling the bulb the greatest care must be taken to keep the 

 stock of nutrient medium from any source of contamination, if it has once been 

 sterilized. Chemical flasks with narrow necks serve well for a common receptacle. 

 These should be kept stoppered with a cotton plug, and to fill the small flasks 

 the plugs need only to be lifted slightly while the sterilized capillary neck of the 

 small flasks is thrust past the plug into the nutrient fluid. If the nutrient fluid 

 is freshly prepared, and has not yet been sterilized, the small flask may be filled, 

 sealed up in the flame, and sterilized in the steam sterilizer or in a vessel of boiling 

 water for an hour each day on three successive days. The nutrient fluid will keep 

 indefinitely in the little flasks, and when a drop is wanted for a drop culture, it is 

 only necessary to sterilize the end of the capillary neck in a flame, break off the 

 head with sterilized forceps, invert the flask, and place the palm of the hand over 

 the bulb. The heat of the hand will expand the air over the fluid and force the 

 latter down the neck. With a little practice just the desired amount of fluid can 

 be forced out by the heat of the hand. The hand must not be placed on the bulb 

 until the flask is inverted. If it is desired to make cultures within the little flasks, 

 snip off the end of the capillary neck as before, and thrust a long platinum needle, 

 the end of which has been in contact with the source of inoculation, down the neck 

 into the fluid. Then withdraw the needle and hermetically seal the neck in the 

 flame. When cultures are to be made in the flasks, these should be only one-third 

 filled by the nutrient medium; there will then be sufficient air in the flasks for the 

 success of the culture after the flasks have been inoculated and hermetically 

 sealed. 



Pollen grains may be made to germinate in hanging drops composed of 100 parts 

 of well-water, 3 to 30 parts of cane-sugar, and 1.5 parts of gelatine. This should 

 be made as needed, or it may be sterilized and kept indefinitely in the little flasks 

 just described. The amount of cane-sugar to give the best results varies with the 

 species of pollen, and can only be determined by experiment, but 3 parts will proba- 

 bly answer for most pollen-grains. 



Spores of ferns may be made to germinate on pieces of flower-pot which are kept 

 half submerged in water and are covered by a bell-jar. They should be set before 

 a north window. They should never be exposed to the direct light of the sun, since 

 in such a position the temperature under the bell-jar would become very great. 



Orchella (Orseille). Sections of tissues containing actinomyces may be stained 

 to advantage by an orchella stain prepared as follows : Orchella which has been left 

 in the open air until it is free from its ammonia is dissolved in a mixture of 20 mils 

 of absolute alcohol, 5 mils of concentrated acetic acid, and 40 mils of distilled water, 

 until the mixture has a dark-red appearance. Sections are left in the filtered so- 

 lution for one hour. They are then washed in alcohol, stained with gentian violet, 

 washed again in alcohol, placed for a short time in xylol, and mounted in Canada 

 balsam. By this treatment the fungus will be double-stained red and blue. 



Orseillin and Aniline Blue. The mycelium of the Peronosporeas may be stained 

 blue, and the cell- walls of the plant which the fungus is parasitizing may be stained 

 red at the same time by a combination of orseillin and aniline blue. Sections of 

 tissues containing the parasite are bleached in Javelle water, then washed in a 

 saturated solution of potassium hydrate in alcohol. The sections are placed 

 for staining in acetic acid, to which have been added a few drops of an aqueous 



