184 BACTERIOPHAGES 



this and other evidence these authors concluded that the dough- 

 nuts are serologically equivalent to the heads of phage particles 

 (Chapter VIII). 



Proflavine lysates were prepared ft'om T2-infected bacteria 

 labeled with either P'^- or S^'". The isolated doughnuts were 

 found to contain about -/s as much sulfur per particle as mature 

 phage, but less than Ve as much phosphorus. These observa- 

 tions are in agreement with the electron microscope studies in 

 suggesting that the doughnuts are empty phage heads, made of 

 protein and containing little or no nucleic acid. The dough- 

 nuts do not adsorb to bacteria, which is consistent with the 

 absence of tails. 



Further morphological study was facilitated by the develop- 

 ment of the "agar filtration method" of quantitative electron 

 microscopy (Kellenberger and Kellenberger, 1952; Kellenber- 

 ger and Arber, 1957). In this method a sample of crude lysate 

 is placed on a collodian membrane supported on a specially pre- 

 pared agar surface. Water and crystalloids pass into the agar, 

 while larger particles are deposited on the membrane. The 

 material is fixed with formaldehyde vapor and the membrane is 

 transferred to a specimen grid for electron microscopy. Count- 

 ing of particles per unit volume of lysate is achieved by calibra- 

 tion with latex spheres. The advantage of this method over 

 others is that no fractionation of the lysate is necessary before 

 preparation of specimens. 



Kellenberger and Sechaud (1957) applied this method to pre- 

 mature lysates of both normal and proflavine cultures of bac- 

 teria infected with T2 and T4. They found, in confirmation of 

 earlier work, that doughnuts appeared a few minutes before 

 phage particles in the lysates, and soon reached a constant num- 

 ber of about 50 per bacterium, whereas phage particles increased 

 for a longer period, to 150 per bacterium. Proflavine greatly 

 suppressed the formation of phage particles but did not afTect the 

 formation of doughnuts. This result is unexpected, because, if 

 proflavine acts mainly to prevent the conversion of doughnuts 

 into phage particles, some additional mechanism must be invoked 



