ISOLATION OF MICROORGANISMS 61 



removed and if care is taken to work in a relatively dust-free atmosphere, 

 often several examinations may be made before the colony becomes contami- 

 nated. The size of the colony is rather limited in this device. In 1927 he 

 proposed a more elaborate flask. It is essentially a flask of the Erlenmeyer 

 type, made in two pieces, a lower portion to hold the medium and a cover 

 fitting over it rather more closely than the usual Petri dish cover. The pieces 

 are held tog-ether by a metal plate underneath and a ring around the neck of 

 the flask connected by three springs which hook into the upper ring to provide 

 sufficient compression to prevent contamination. The flask is manipulated as 

 an ordinary flask, the neck being plugged with cotton. After the colony is 

 grown, the springs are unliooked from the ring and the top is lifted off. If 

 care is taken in the examination, little contamination results. So far as I am 

 aware, this type of flask has not been placed on the market. 



Sing^le Cell (Spore) Cultures.— Finally, there comes a time in the study 

 of many fungi when the results of a study of cultures made by the above- 

 mentioned methods are ambiguous, and it becomes desirable to work with 

 mycelium and spores produced by a single spore in order that problems of 

 sexuality or homothallism and heterothallism may be investigated, or that the 

 relationships of apparent stages in a life cycle may be studied and verified. 

 The problem has been variously met by different investigators, depending 

 somewhat on the size and nature of the spore to be isolated and the instru- 

 ments available for the work. If the spores are large and the hand is very 

 skillful, it may be possible to pick up a single spore on a needle under the 

 low power of the microscope and transfer it to a sterile tube or a hanging 

 drop. Various mechanical devices have been developed to aid in this work, 

 e.g., the old Barber spore picker or pipette or some of the modern micro- 

 manipulators used in microdissection studies. In these devices, motion is 

 secured by means of micrometer screws, and the spore is usually sucked into 

 the end of a tiny pipette made by drawing out a piece of glass tubing to an 

 inside diameter only slightly larger than the spore or cell to be isolated. This 

 is then discharged into a hanging drop or a sterile culture tube. For detailed 

 directions, those accompanying the instruments should be consulted. 



Ascospore Detection. — Since classification is based primarily upon the 

 spore forms resulting from the sexual act (caryogamy), every effort to secure 

 the sexual or perfect stage should be made before relegating an organism to 

 the large heterogeneous group known as the Fungi Imperfecta There is no 

 single method which is equally successful for all organisms, nor even for mem- 

 bers of a single group. 



Perhaps patience is the first requisite. Frequently one finds evidence of 

 sexuality by a diligent search of a colony 3-4 months old, after the agar has 

 begun to dr5\ This seems very useful in the filamentous yeasts whose im- 

 perfect stage is usually placed in the genus Monilia. Some media seem better 

 than others, but usually a careful search will show traces of sexuality on most 

 media upon which the organism has made a good growth. This method is 

 very tedious, especially among pathogens when the physician wishes a prompt 



