VI. ESTIMATION' 23 



tioiKil Xorit-troaliHl liydi-oly/.cd (-isciii. 'Hicsc inodilicalioiis wore loiind to 

 pioinolc inoiv j»;r()\\ili of Arclubdclcr t<iil>ox!j<laiis in response to PABA than 

 had |)ic\ iously been obtained. 



Tlie Airlolxiclcr i^iihoxydans method for the determination of PAHA has 

 l)een applied by Landy el «/." and by Spink d al}^ to a study of PAPA pro- 

 duetion by sulfonamide-sensiti\'e and -resistant bacteria. 



A miert)biologieal method for PABA determination was proposed by 

 l>e\vis*'^ that depends upon the essential nature of PABA for LaclobaciUus 

 arabinosus.^^ The strain employed, the basal medium, and the cultural 

 details are quite similar to those outlined by Snell and Wright^^ for the 

 microbiological determination of nicotinic acid. It is essential that the acid- 

 hydrolyzed, \-itamin-free casein used as the main source of nitrogen be 

 vigorously Xorit-treated to remove traces of PABA or other contaminating, 

 microbiologically active material. The Lactobacillus ai-abinosus method is 

 an extremelj'- sensitive method for the determination of PABA with a re- 

 sponse range to the compound of about to 0.0005 y per 10 ml. 



Considerable difficulty has been experienced in a number of laboratories 

 in the use of this method for the determination of PABA because of excess 

 growth in the "blanks" (no added PABA). Although contamination of the 

 glassware and the medium with PABA appears to be the most plausible 

 explanation for the difficulty, it is now the conclusion of a number of in- 

 vestigators,-^^ including also the reviewers, that spontaneous mutants of 

 Lactobacillus arabinosus are produced with such frequency that selection 

 of these mutants readily occurs. For this reason most laboratories have 

 abandoned the use of the Lactobacillus arabinosus method for the determina- 

 tionofPABA. 



The Lactobacillus arabinosus method has been applied by Spink et al.^'" 

 to a study of PABA synthesis by sulfonamide-sensitive and -resistant strains 

 of staphylococci. 



A microbiological assay for PABA has been developed by Mitchell et al:'^ 

 and by Thompson d al.^° that depends upon the essential nature of PABA 

 for an x-ray induced mutant of Neurospora crassa discovered by Tatum 

 and Beadle.^' ■^- The latter investigators determined that a variety of com- 

 pounds related in structure to PAI5.\ do not promote growth of the mutant, 

 that both the mutant and the normal parent strain are inhil)ited by sulfanil- 



" J. C. Lewis, /. Biol. Chem. 146, 441 (1942). 

 36 H. Isbell, /. Biol. Chem. 144, 567 (1942). 

 " E. E. Snell and L. D. Wright, J. Biol. Chem. 139, 675 (1941). 



38 D. Pennington, Science 103, 397 (1946). 



39 H. K. Mitchell, E. R. Isbell, and R. C. Thompson, ./. Biol. Chem. 147, 485 (1943). 

 « R. C. Thompson, E. R. Isbell, and H. K. Mitchell, J. Biol. Chem. 148, 281 (1943). 

 " E. L. Tatum and G. W. Beadle, Proc. Natl. Acad. Set. U.S. 28, 234 (1942). 



- G. W. Beadle and E. L. Tatum, Proc. Xatl. Acad. Sci. U.S. 27, 499 (1941). 



