ISOLATION AND PROPERTIES OF WALLS 



cal Structure of the wall as it occurs in the intact cell. Con- 

 sequently, more refined and less drastic methods for isolat- 

 ing walls have been evolved, and mechanical disintegration 

 of cells and tissues has become the universal starting point. 



Isolation of Cell Walls 



The isolation of microbial structures as homogeneous 

 morphological entities has resulted from the application 

 of the methods of biochemistry, biophysics, and electron 

 microscopy. Weibull ^ was one of the first to use a combi- 

 nation of such methods for the isolation of a bacterial struc- 

 ture when he separated and characterized flagella from 

 Proteus vulgaris. Although mechanical methods have been 

 available for the disintegration of microorganisms for some 

 time, they were not applied to the problem of isolating wall 

 structures until Mudd, Polevitsky, Anderson, and Cham- 

 bers ^ showed by electron microscopy that sonic disintegra- 

 tion of bacteria left a resistant wall. Dawson ^ later 

 demonstrated the complete separation of cytoplasm from 

 the wall of Staphylococcus aureus by disintegrating the 

 cells with glass beads. It thus became apparent to several 

 of us (Mitchell and Moyle,io Salton and Horne,^^ Salton ^2) 

 that such procedures could be used in conjunction with 

 differential centrifugation to obtain homogeneous prepara- 

 tions which could be submitted to the techniques of analyti- 

 cal chemistry for the elucidation of their nature. 



The methods for isolating microbial cell walls follow 

 well-known recipes, and as we are all familiar with what 

 good and bad cooks can do with recipes we need not dis- 

 cuss the isolation procedures in any detail. Cells may be 

 disintegrated and deprived of their cytoplasm by one of 

 the following three methods: 



1. Mechanical disintegration (disruption by violent agita- 

 tion with beads,^'^^'^^ sonic and ultrasonic disintegra- 



