28 STAINING REACTIONS OF BACTERIA 



readily permit the contained large, compound iodine molecule in alcoholic solution to 

 pass through it. 



6. Gram negative bacteria are of two types as regards cell membrane: (a) (Rep- 

 resented by M. neisseri) allow attachment and probable permeation of the dye ; but 

 from them iodine-dye precipitate is readily washed out by the decolorizer. These or- 

 ganisms approximate the gram positives so far as they contain the dye, but the ab- 

 sence of the necessary specific cell membrane does not permit the retention of the dye 

 when alcohol is applied, (b) (Represented by B. coli) allow no penetration of the dye 

 but only peripheral adsorption. 



7. Not all bacteria which fail to retain the stain are similar in structure. The ap- 

 parently permeable gram negatives (M. neisseri) probably have more in common 

 with the gram positives than with the gram negatives. 



Burke corroborated Benians' conclusions by demonstrating a reverse gram re- 

 action.' He found that an alcoholic solution of the iodine-dye complex stained the 

 gram negatives but not the gram positives, so that gram positives are characterized 

 by permitting the entrance of water-soluble dye but not the egress of the alcohol- 

 soluble iodine-dye complex. The opposite is true of gram negatives. 



Brudny,^ on the other hand, considered the gram positives more permeable to io- 

 dine so that in these organisms a deeper iodine-dye precipitation occurs which is less 

 accessible to the decolorizers. Other observers have laid stress on the chemical con- 

 stituents of the bacterial surface. Eisenberg found that ether extraction of staphy- 

 lococcus reverses its gram behavior, concluding that the lipoid-protein compounds on 

 the surface were the important factor; while Dreyer, Scott, and Walker were able to 

 turn B. coli into gram positive organisms by treating them with lecithin. 



Stearns and Stearns^ called attention to the fact that bacteria exhibit the ampho- 

 lytic character common to protein and tend to retain acid stains when in acid solu- 

 tion and basic stains when in alkaline solution. They found that gram positive bac- 

 teria can be rendered gram negative by increasing acidity, and that the reverse effect 

 is produced by alkalies ; at the iso-electric point there is little staining, and the so-called 

 "iso-electric range" is generally wider with gram negative than with gram positive 

 bacteria. Mordanting, they suggest, is due to a mild oxidation which increases acidity 

 and hence the affinity for basic dyes. They suggest further that gram positivity de- 

 pends on the presence in the compound bacterial lipoproteins of unsaturated fatty 

 acids which are partially oxidized by the mordant, intensifying the acid properties and 

 increasing affinity for basic dyes. The presence of unsaturated fatty acids has been 

 invoked by other observers to explain the phenomenon, but in a different way. Thus 

 Jobling and Petersen'' suggested that gram positivity depended on a high fatty acid 

 content and a high affinity for iodine; and Tamura found that the lipoid extract from 

 bacteria contains the element responsible for retention of the dye. Hottinger,^ on the 



'Burke, V.: loc. cit. 



= Brudny, V.: CcnlralU. f. Bakleriol., 21, 62. 1908. 



3 Stearns, A. E., and E. W.: op. ciL, 9, 463, 479, 491. 1925; 10, 13. 1925. 



■t Jobling, J. W., and Petersen, W. H.: loc. cit. 



sHottinger, R.: loc. oil. 



