A new rapid method of glycolate test by diethyl ether extraction, which isapplicable to a small amount of bacterial cells of less than one milligram

The glycolate test is a method to discriminate N-acyl groups of muramyl res idue in peptidoglycan of bacterial cell walls by color reaction without pur ification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentr ated H2SO4. Instead of the previous column methods for quantitative analysi s, a qualitative method by solvent works was developed to simplify and to m iniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hyd rolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction und er several conditions, the ability of removing various interfering compound s, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of "Micromonospora nigra" JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid . The solvent method was applied to 20 strains of typical actinomycete spec ies whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for pos sible applications to other fields.