2 MICROSOMAL PARTICLES 



A. vinelandii examined in the electron microscope. Several speakers at this 

 conference have referred to similar granularity in sections of Escherichia coli as 

 ribonucleoprotein particles, but no clear identification of these granules as such 

 has yet been accomplished. Ribonucleoprotein particles, similar in size and 

 chemical composition to those from animal cells, yeast, and fungi [14], can, 

 however, be prepared from bacteria [15]. Several papers in this volume describe 

 the ribonucleoprotein of E. coli, and this paper will treat the preparation and 

 properties of ribonucleoprotein particles from A. vinelandii. 



RUPTURE OF BACTERIAL CELLS 



One of the aims of this work is to prepare subcellular structures having a 

 useful correspondence to structures that existed in the intact cell. We have no 

 single criterion to indicate when such a preparation has been accomplished, 

 but we use as supporting evidence reproducibility of the product when pre- 

 pared by several varying methods and we also invoke all the information about 

 the stability of isolated components. The stability observations are described 

 in a later section of this paper. Three different methods of cell rupture have 

 been found to permit isolation of indistinguishable particles, provided that the 

 cultures used were harvested at a similar stage of growth. 



The first method was physical grinding with number 320 mesh Carborundum 

 which had been washed with hydrochloric acid and rinsed with distilled water 

 until neutral. The cells had been previously washed with distilled water. The 

 packed cell paste was ground with 4 parts by weight Carborundum for approxi- 

 mately 15 minutes or until moist. An additional 2 parts of Carborundum was 

 added, and the cells were ground for approximately 5 minutes more. Visible 

 microscopic examination of the mixture revealed that approximately 95 per 

 cent of the cells were ruptured by the grinding method. The ground cells were 

 diluted with 8 times the original cell volume of the following buffer: 1.6 X 10" 3 

 M K2HPO4, 0.4 X 10" 3 M KH2PO4, and 5 X 10" 3 M MgS0 4 . This will be re- 

 ferred to as the RNP buffer. The supernate from centrifuging this mixture at 

 500g for 30 minutes is referred to as the crude extract. 



The second method of cell breakage employed cells grown in the presence 

 of 2 M glycerol. The cells were collected by low-speed centrifugation, and the 

 pellet was diluted into 8 volumes of the RNP buffer to rupture and produce 

 the crude extract. 



In the third method of cell breakage, A. vinelandii protoplasts were ruptured 

 by osmotic shock. Weibull [16] showed that Bacillus megatherium, when 

 treated with lysozyme in sucrose solutions of high osmotic pressure, changed 

 to a spherical form which is readily ruptured by lowering the osmotic pressure. 

 We have avoided the use of sucrose in view of our findings on the instability 

 of the isolated particles in dilute sucrose solutions. The A. vinelandii cells 

 were washed in the RNP buffer, and suspended in 1.5 XlO" 3 M EDTA at a 

 dilution such that the optical density at 660 m\\ was approximately 0.75. These 

 solutions had been previously osmotically adjusted with glycerol or with Carbo- 



