THE CHEMICAL SIGNIFICANCE OF THE GRAM TEST FOR BACTERIA A. S. W. HASLETT London, England
THE
GRAMstaining test for bacteria, which was first introduced by Christian Gram in 1883, and for more than 50 years remained as one of a number of empirical tests used in routine rnicrohiological identification, was lifted into sudden significance by the clinical success of penicillin in 1941. It was Sir Alexander Fleming who, in his original work on penicillin more than 10 years earlier, had first shown that those bacteria, the growth of which it effectively inhibited, were in all cases Grampositive, while in general those against which it was ineffective were Gram-negative. This observation aroused little interest at the time, although Sir Alexander himself made regular use of it in the differential culture of bacteria. However, after Sir Howard Florey and his fellow-workers a t Oxford University had demonstrated the clinical importance of this distinction in relation to penicillin therapy, curiosity was naturally aroused as to its physical significance. Among those who first attacked the problem were the Birmingham bacteriologist, Dr. H. Henry, and Professor M. Stacey, Professor of Chemistry in the University of Birmingham: First Stage. By 1943, when penicillin itself was still the main center of interest, they were able to publish their first evidence suggesting that the staining quality characteristic of the Gram-positive bacterium might be associated with the presence of magnesium ribonucleate within the organism. Now, after three further years of research, they have presented to the Royal Society Of London a chain of evidence1which appears to be beyond doubt or argument. Additional details have also been given by Professor Stacey in his recent Tilden Lecture to the Chemical Society (as yet unpublished). The first stage of their work was the discovery that if either C1. welchii or yeast cells were suspended in 2 per cent bile salt in normal saline, an extract was obtained which appeared to be in some vay associated with the original staining quality of the cells. This was substantiated both by a reduction in dry weight, and the disappearance of the original Gram-positive quality. Further, it was found that after treatment with a reducing agent, the cytoskeletons could be induced to reahsorb from the extract whatever had been taken from them. They again appeared Gram-positive although, as Professor Stacey expressed it in his Tilden Lecture, "looking as if they had been knocked about a bit." Even more encouraging, the extracts from C1. welchii and from yeast cells proved to be interchangeable for this purpose. This suggested, not only that they con-
tained the same, or closely related, substances, but also that there might exist a similarity in the chemical strocture of that pa.rt of the cytoskeletons with which combination occurred. The range of the investigation was accordingly widened by the inclusion of certain anaerobic bacteria: aerobic spores, including B. anthracis; and certain strains of streptococci. At the sametime, a more intensive study was made of the resulting bile salt extracts. Fractional separation of the latter led to a series of products, one of which was identified as magnesium ribonucleate, on the double evidence of an absorption band a t 2,600.A. and the characteristic degradation products obtained from it. Finally, came the demonstration that magnesium ribonucleate prepared from commercial sources was equally capable of restoring the Gram-positive quality to bacteria from which it had been removed. Foundation For Further Research. Akhough it is too early as yet to assess the general significance of magnesium ribonucleate in the economy of Gram-positive bacteria, a solid foundation has thus been provided for future' research. Apart from the elegant demonstration by Henry and Stacey, there is the nolv considerable body of evidence that resistance to penicillin is connected with the production of penicillinase, the penicillin-destroying enzyme which was first isolated from Gram-negative bacteria by Abraham and Chain in 1940 (9). I t is a tempting assumption that some connection will be established bet,ween ability to produce penicillinase on the one hand, and absence from the organism of magnesium ribonucleate on the other. But, as lately pointed out by Selbie ,a i t by no means follows that the same mechanism can necessarily be invoked to explain the phenomenon of acquired resistance to penicillin on the part of Gram-positive hacteria, which also was first reported by the Oxford worker^.^ Finally, it may be noted that the significance of the Gram test is further increased by the fact that other antibiotics besides penicillin are "Gram sensitive." Although this fact must appear for the moment as an added complication, it also provides many more angles from which the problem can be attacked. LITERATURE CITED Proc. Rw.Soc. B., 133, No. 873, December 3, 1946. . ' Nature. 146, 837.
Vvitish Medieat Bulletin, 4, No. 4 , 1946.
