60 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES 



hour. The Hquid is decanted and filtered until fairly clear. To 100 

 ml, of this aqueous soil extract are added 900 ml. of water, 1 gram 

 of glucose, 0.5 gram of dipotassium phosphate, and 15 grams of agar. 



Isolation of Pathogenic Actinomycetes. Although such weak 

 media serve for the isolation of saprophytic actinomycetes, they are 

 not suitable for the pathogenic species. The strictly anaerobic 

 Actinomyces bovis may be isolated in deep agar shake cultures, 1 per 

 cent of glucose in veal infusion agar being used ; or the method men- 

 tioned previously may be used. Various authorities have recom- 

 mended the addition of blood serum or ascitic fluid, but this has not 

 been found to be advantageous for all strains. Brewer's thioglycol- 

 late medium with glucose is also suitable for A. bovis. The aerobic 

 species may be isolated readily on the same solid media as are used 

 for A. bovis. 



Single-cell Isolation. Although for most purposes isolation from 

 a mixed culture by plating is readily accomplished, occasions may 

 arise when other procedures are preferable. Where several molds are 

 closely crowded together on a plate, it is sometimes quite feasible 

 to pick off a spore head of a desired species with a sterilized wire 

 under the higher-powered lenses of a binocular dissecting microscope. 

 For certain types of work, as investigations of apparent mutation, 

 cultures known to have developed from a single spore are necessary. 

 Single spores may, of course, be picked up by the use of a micro- 

 manipulator (such as the Chambers instrument) more readily than 

 bacteria, but much simpler procedures are available. Sass ^^ has 

 described such a method which he has used for growing single-spore 

 strains of mushrooms. A spore suspension of proper density is 

 sprayed over the surface of a sterile agar plate, which is then incu- 

 bated for a time. The plate is then examined for well-isolated spores 

 which have germinated, the low-power microscope lens being used. 

 When such a spore is found, a small sterilized spatula with a minute 

 hole in it is placed over the spore and pressed into the agar in such 

 a way as to force a small cylinder of agar (bearing the spore on its 

 upper surface) through the hole. The growing spore may then be 

 easily removed with a sterile needle without danger of touching any 

 other spore. It is simpler and safer to dispense with the perforated 

 spatula, which may touch other spores when it is placed on the agar 

 surface, and to remove the selected spore with a minute spade-shaped 

 needle whose entire cutting edges can be kept in full microscopic 

 view during the entire procedure. 



Media for Yeast. In general, yeasts may be isolated and cultivated 

 on the same media as are used for molds. Most species tolerate 



