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SUMMARY : The phosphorus-containing compounds of certain Gram-positive and Gram-negative organisms were estimated by a modification of the Schmidt & Thannhauser (1945) procedure. Another method (Mitchell & Moyle, 1950, 1951 a, b, 1954) in which pentosenucleic acid (PNA) was estimated from the optical absorption of the ' PNA fraction ' at 260 mp. was found to be inaccurate, because of the presence of ultraviolet-absorbing impurities. A more accurate method for estimating PNA has now been developed, in which the nucleotides are separated from impurities by paper electrophoresis. The results thus obtained show that non-nucleotide phos- phorus-containing compounds are present in the 'PNA fraction' of both Gram- positive and Gram-negative organisms. There is, therefore, no evidence to connect these compounds with the Gram staining properties of Gram-positive micro- organisms (cf. Mitchell & Moyle, 1950, 1951a, b, 1954). It has been shown that magnesium ribonucleate, in association with certain proteins and possibly polysaccharides, is the complex responsible for the retention of the Gram stain in Gram-positive micro-organisms (Henry & Stacey, 1943, 1946; Henry, Stacey & Teece, 1945; Jones, Muggleton & Stacey, 1950). These conclusions, which have been largely confirmed by other workers (Bar- tholomew & Umbreit, 1944) have since been questioned by Mitchell & Moyle (1950, 1951a, by 1954) who claimed that Gram-positive organisms contain a particular phosphoric ester fraction (XSP) which is absent from Gram- negative species and that this 'XSP.factor ' is responsible for the retentionof the stain in the Gram reaction. In view of these claims, it was decided to re- investigate the problem and in particular to analyse various micro-organisms for their XSP content. METHODS Organsh used and methods of cultivation. The following organisms were used : (a) Gram-positive; baker's yeast (Distillers Co.), BaciZZus subtilis, Clostridium welchii, Sarcina lutea, Streptococcus faecalis (NCTC, 370) ; (b) Gram-negative; Aerobacter aerogenes (NCTC 8172), A. cloacae (NCTC 8813), Escherichia coli and Klebsiella pneumoniae (NCTC 3279). They were grown in a medium containing (yo, w/v):O.5, Lab Lemco; 1, Oxoid bacteriological peptone; 1, glucose; 0.5, sodium chloride. The solution of Lab Lemco, peptone and sodium chloride was adjusted to pH 9, boiled for 5-10 min. and the resulting precipitate filtered off. The filtrate was adjusted to pH 7-5 and the solution autoclaved at 10 1b.lsq.h. for 15 min. Sterile glucose solution was then added aseptically. The complete medium was inoculated with an

The Phosphorus-containing Compounds of Gram-positive and Gram-negative Organisms in Relation to the Gram Staining Reaction