32 STAINING REACTIONS OF BACTERIA 



stances somewhat uncertain, to the extent at least that they can hardly be the sole 

 factors concerned in acid fastness. Just as in the case of the gram stain, bacillary in- 

 tegrity is essential for acid fastness. It was pointed out by Koch and later by Benians, 

 and confirmed by others, that disintegrated tubercle bacilli lose the property, from 

 which it would appear that the fatty constituents of the tubercle bacillas are not per 

 se the cause of the staining reaction characteristic of the organism. 



Like gram positivity, acid fastness is not an absolutely constant characteristic but 

 is influenced by factors like age of culture and conditions of environment, and may be 

 entirely upset by treatment with chemical agents. It appears established that very 

 young tubercle bacilli may not stain at all by Ziehl's method (Krylow),' and from the 

 work of Wherry, Mellon, and many other observers there is strong evidence that 

 acid fast organisms go through a varied life-cycle during which their morphology is 

 profoundly altered and their staining characteristics modified. Bienstock and Gott- 

 stein were able to make ordinary bacteria acid fast by artificial Einfettimg through 

 growth on butter agar. Wherry^ was able to render acid fast saprophytes non-acid 

 fast by continual growth under conditions unfavorable to the synthesis of fats. 

 Ritchie^ has shown that tubercle bacilli treated with boiling xylene, boiling toluene, 

 boiling benzene, or Aronson's mixture (ether, alcohol, and HCl) lose their acid fast- 

 ness; and Browning and Gulbransen^ have shown that the property is rapidly re- 

 moved by treatment with CHCI3 to which minute amounts of HCl have been added 

 together with a small amount of C2H5OH to yield a permanent mixture. These obser- 

 vations support the view that the loss of the acid fast property is due to a dissociation 

 of the constituents of the bacillus which is readily effected by the combined action of 

 small amounts of HCl along with lipoid solvent like CHCI3. 



Any amount of extraction of tubercle bacilli with simple fat solvents leaves them 

 acid-fast, probably due to the fact that they continue to contain a small amount of 

 lipin. But if this last trace of lipin be removed by treatment with acid, acid fastness 

 disappears (Long).s 



It is not easy to harmonize completely the observations which have just been 

 cited and to present an entirely satisfactory theory of acid fastness. Three facts seem 

 to be established (Long) : (i) Disintegration of the cell destroys acid fastness. (2) Ex- 

 traction of the cell body with fat solvents until no more lipin is removed leaves the 

 bacilli acid fast provided they have not been mechanically disintegrated (as by tritu- 

 ration) during the process. (3) The wax of the tubercle bacillus is acid fast.^ It re- 

 mains to reconcile the two chief conflicting explanations: that the phenomenon is due 

 to the presence of an impermeable membrane and that it is due to the content of 

 acid fast wax. Both factors may — as in the case of the gram stain — be of importance. 

 Acid fastness appears ultimately to depend on the small amount of lipin held as an 

 emulsion in the protein substrate and not extractable by fat solvents. But since it is 



'Krylow, D. O.: Zlschr.f. Hyg. u. Infeklionskrankh., 70, 135. 1911-12. 



'Wherry, W. B.: /. Infect. Dis., 13, 144. 1913. 



3 Ritchie, W. T.: /. Path, b- Bad., 10, 334. 1905. 



■• Browning, C. H., and Gulbransen, R.: ibid., 27, 326. 1924. 



swells, H. G., DeWitt, L. M., and Long, E. R.: Chemistry of Tuberculosis. 1923. 



