This study examined endotoxin fractions from Escherichia coli O78K80 using electron microscopy after negative staining with phosphotungstate. The researchers compared 'free endotoxin' secreted into culture supernatant with conventional cell-wall-derived endotoxin. Free endotoxin preparations contained rod-like particles approximately 295 × 65 Ångströms, representing native lipopolysaccharide-protein complexes with estimated molecular weight of one million Daltons. Phenol-water extraction caused these complexes to aggregate into chain-like structures, suggesting that protein removal triggers degradation and polymerization. Aqueous ether extraction produced a mixture of rods and chains. A non-toxic polysaccharide fraction (native protoplasmic polysaccharide) was not visible under electron microscopy but contained cell-wall-derived fragments characterized by ridged structures 14-30 Ångströms wide. These fragments, likely lipoprotein in nature, were occasionally detected in toxic fractions and crude preparations. The findings support the hypothesis that mild extraction methods preserve native endotoxin structure, while chemical treatments cause degradation and aggregation.

Key findings

• Free endotoxin from E. coli culture supernatant consists of native rod-like particles (295 × 65 Å) representing lipopolysaccharide-protein complexes with molecular weight ~1 million Daltons
• Phenol-water extraction causes aggregation of endotoxin complexes into chain-like structures due to protein removal and degradation
• Aqueous ether extraction produces heterogeneous mixtures of rods and chains, containing more structural diversity than free endotoxin
• Cell-wall-derived fragments with characteristic ridged structures (14-30 Å wide) were detected in multiple fraction types
• The native endotoxin structure is preserved by mild extraction methods but degraded by chemical treatments like phenol extraction