GRAM STAINS MECHANISM 



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GRAM STAINS MECHANISM 



the interpretation of the findings has 

 been concisely presented by Dubos, R. 

 J., The Bacterial Cell. Harvard Univ. 

 Press, 1945, 460 pp. The Gram + bac- 

 teria differ from the Gram — ones in 

 being more acidic and perhaps in pos- 

 session of lipids with higher content of 

 unsaturated acids. Their Gram posi- 

 tiveness depends on intactness of their 

 cell walls, for erosions of the walls make 

 them Gram negative. When the outer 

 layer of the cell walls is removed by 

 extraction with bile salts they become 

 Gram negative. The Gram positive 

 property can be restored by "replating" 

 the bacteria with the extract of the 

 outer layer. The outer layer appar- 

 ently contains a protein ribonucleate 

 complex, for Gram positive organisms 

 can be made Gram negative by action 

 of the enzyme, ribonuclease. The 

 quality of the cell membrane conditions 

 not only the entry and retention of 

 stains but the whole manner of life of 

 the cells. See Cell Membrane, Acid 

 Fast Bacilli, and Dead Cells. 

 Gram Stains Mechanism — Written by 

 James W. Bartholomew, Dept. of 

 Bacteriology, University of Southern 

 California, Los Angeles 7, Calif. July 

 9, 1951 — The literature concerning the 

 mechanism of the Gram stain is 

 voluminous and makes very interesting 

 reading although the exact mechanism 

 is still unknown. The laboratory tech- 

 nician need not be too concerned with 

 the mechanism. It is important for 

 the technician to realize that the tech- 

 nique involved is differential due to 

 relative rather than absolute differences 

 in the organisms studied. Thus, re- 

 gardless of whether one uses the 

 Hucker, Kopeloff-Beerman, Weiss, or 

 other modification of the Gram pro- 

 cedure, the dependability of the results 

 obtained are greatly influenced by the 

 experience and skill of the operator. 

 One method should be adopted, and 

 practiced, until proficiency and self 

 assurance are obtained. 



Gram differentiation is mostly due to 

 one fact. Gram-positive cells resist 

 decoloration after treatment with dye 

 and iodine to a much greater extent 

 then Gram-negative cells. The cause 

 of this resistance has been attributed to 

 1) cell membrane permeability char- 

 acteristics, 2) the presence of certain 

 unique chemical compounds, 3) a low 

 isoelectric point of the cell protein, and 

 4) the presence of a morphological struc- 

 ture, the Gram-positive cortex. Each 

 of these theories, by itself, fails to ex- 

 plain certain experimental data. It is 

 certain that the mechanism of the Gram 

 stain is very complex and several of the 



above factors probably combine to give 

 the differentiation effect. Cell mem- 

 brane permeability is hard to ignore 

 since it is well known that rupturing 

 the cell wall immediately results in a 

 Gram-positive cell staining Gram-nega- 

 tively. The importance of a chemical 

 factor has been well established through 

 the easily demostrable dependence of 

 the Gram-positive state on the pres- 

 ence of magnesium ribonucleate in the 

 cell. The isoelectric point theory ap- 



Elies to the general staining phenomena, 

 ut on close inspection looses its sig- 

 nificance as a prime factor in the mecha- 

 nism of the Gram stain. The morpho- 

 logical concepts must await further 

 development in our knowledge of bac- 

 terial cell morphology, and better cell 

 sectioning methods, before the con- 

 tribution of morphological factors to 

 the Gram staining mechanism can be 

 determined. 



Currently, an appealing explanation 

 based on published literature could be 

 written as follows. Cell membrane 

 permeability to iodine in alcoholic solu- 

 tion is of first importance. Magnesium 

 ribonucleate and possibly other com- 

 pounds such as lipoproteins are im- 

 portant in that they influence the cell 

 membrane permeability. Gram-posi- 

 tive cells are less permeable to iodine 

 in alcoholic solution than are Gram- 

 negative cells. On performing the 

 Gram stain a reaction between the dye 

 and iodine, and also possibly the cell 

 protein, takes place within the cell. 

 The alcohol, which is added next, enters 

 the cells and dissociates the dj'^e-iodine 

 precipitate, or dye-iodine-cell protein 

 complex, which has been formed. Since 

 the alcoholic iodine permeates fastest 

 through the cell membrane of the Gram- 

 negative cells the precipitate or complex 

 are dissociated more rapidly in them 

 than in the Gram-positive cells. 

 Hence, the Gram-negative cells are the 

 first to declorize. The counter stain 

 also has a certain decolorization action 

 and it must be employed if a true Gram 

 stain differentiation is desired. 



While this concept of the Gram stain 

 mechanism has not been proven, it has 

 the happy quality of combining chemi- 

 cal concepts with cell membrane perme- 

 ability and it does not rule out possible 

 morphological factors. In the case of 

 the often observed Gram-positive 

 bodies within the cell, a more strictly 

 chemical concept would have to be used 

 to explain them. 



As is often the case, the segregation 

 of cells into two distinct groups, the 

 Gram-negative and the Gram-positive, 

 does not coincide with the state actually 



