^0 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES 



preparations very readily, but the small amount of nutrient is un- 

 desirable in some cases. A piece of blotting paper or several thick- 

 nesses of filter paper are placed in the bottom of a Petri dish and 

 sterilized in an autoclave. Just before being used, the paper is wetted 

 with sterile water. The cover glasses are cleaned, placed in alcohol, 

 sterilized by flaming, and then placed on the surface of the paper. 

 A tube of the agar is melted in a water bath, cooled to between 43° 

 and 46° C, and inoculated rather heavily with spores of the mold to 

 be studied. With a wire loop one or two loopfuls of the agar are 

 deposited quickly on each cover glass and spread in a rather thin 

 film. The Petri dish may then be incubated. If it is placed in a 

 can, evaporation will be retarded. It is essential to maintain an 

 atmosphere nearly saturated with moisture. 



For preparations which are to be only temporary, agar is prefer- 

 able to liquid media, since when hardened it will "stay put" on the 

 cover slip. Agar cover slips placed on slides may be examined first, 

 culture side up, with the low- and medium-power lenses, then turned 

 over and mounted in a drop of Amann's fluid for more complete 

 study. Semi-permanent preparations may be made by sealing the 

 edges of the cover slip with asphaltum or other varnishes, first re- 

 moving all excess of the mounting fluid. More permanent mounts 

 may be made if glycerin jelly (1 part gelatin, 6 parts water by 

 weight; 1 per cent phenol added) is used instead of Amann's fluid. 

 This also requires sealing of the mount with asphaltum. 



Beautiful permanent preparations mounted in balsam may be 

 made from cover slip cultures, but for this liquid media are preferable 

 to agar media because it is almost impossible to obtain a stain which 

 does not color the agar rather deeply. A tube of the liquid medium 

 is inoculated, and then with a sterile pipet small quantities (about 

 0.01 ml.) are deposited on the cover slips in the Petri dish. The dish 

 must now be handled carefully so that the liquid does not run off on 

 to the filter paper. Czapek's or corn meal solution is suggested for 

 actinomycetes and for those molds which will grow on them. 



When sufficient growth has taken place the cover slips are removed 

 and thoroughly dried. If drying is not complete the film of mold 

 (or of agar if it has been used) will wash off in the staining opera- 

 tions. It is well to dry the cover slips on some sort of warm plate 

 and to let them continue to dry for about 5 minutes after they appear 

 to be dry. Since the mold is not easily wetted by water, an alcoholic 

 fixing solution is desirable. For ordinary purposes the formalin- 

 alcohol-acetic acid mixture commonly used for plant material is 

 satisfactory. It is made of 



