The gram-staining reaction of disintegrated micro-organisms.

Abstract

SUMMARY: Disintegration of yeast and Clostridium welchii gave cell envelopes which stained Gram-negative by a modified Jensen's Gram-staining procedure (procedure l), but stained in part Gram-positive by a modification of the Gram stain in which the films were dried after application of the iodine and before application of the decolorizing agent (90% v/v ethanol; procedure 2). Procedure 2 appeared to give a typical Gram reaction in that a series of organisms gave identical results when stained by the two procedures. Disintegrated Gram-negative organisms gave only Gram-negative material when stained by procedure 2. The Gram-positive material could be removed from the yeast envelopes by extraction at pH 8, but attempts to 'replate' the envelopes with the extract were unsuccessful. The results suggest that the Gram-staining reaction of yeast and C. welchii is due in part to a specific staining component and in part to a factor dependent upon the intact nature of the cells. The latter may be associated with the stability or orientation of the staining component or with some membrane effect. A number of workers have noted that when Gram-positive organisms are broken they become Gram-negative (Benians, 1912, 1919; Burke & Barnes, 1929). These results have been taken as support for the theory that the Gram-staining reaction is due to the presence of a membrane in Grampositive organisms which is impermeable either to the dye + iodine complex or. to .the decolorizing agent. This effect of breaking cells may be equally well explained as being due to the degradation or disaggregation of a Gram-positive complex when the cell membrane is ruptured. It is not certain that Grampositive organisms always become Gram-negative when broken. Thus Burke & Barnes (1929) observed certain Gram-positive structures in preparations of broken yeast cells, and Kemp (1931) showed that broken yeast cells showed Gram-positive structures when stained by a particular modification of the Gram stain. The results reported in the present paper show that although certain Grampositive organisms which had been disintegrated by shaking with glass balls stained Gram-negative by Jensen's modification of the Gram method, a change in this procedure as simple as just allowing the slide to dry before applying the decolorizing agent resulted in the distintegrated organism staining Grampositive. METHODS Organisms. The following were used : Saccharomyces cerevisiae (baker's yeast, Distillers Co., Ltd. DCL) ; Clostridium welchii ; Sarcina lutea ; Escherichia coli ; Axotobacter chroococcum (NCIB 8003) ; Caryophanon sp. (kindly supplied by Dr T. Gibson).. S. lutea, C. welchii and E. coli were grown at 37 in a Lemco