VII. ESTIMATION 4G1 



certain strains of chrvsomonads (photosynthetic phagotrophic flagellated 

 protozoa). These investigators expressed the hope that since chrysomonads 

 were able to ingest large particles thej'' would respond to all naturally 

 bound forms of vitamin B12 in a manner similar to that of rats or chicks 

 The chrysomonads are able to utilize intact proteins. Baile^*** described 

 experiments with the chrysomonad Poferiochromonas stipitata, showing 

 that in a variety of samples of natural materials the chrysomonad assay 

 gave consistently the lowest value for vitamin B12 . An ambitious and 

 practical study of the use of the chrysomonad Ochromonas malhamensis was 

 undertaken by Ford.'^ When this procedure was compared with the 

 Euglena gracilis and E. coli assays, it was found that factors A, B, C, and 

 pseudovitamin B12 possessed considerable activity for all organisms except 

 the ochromonad. The hope that the chrysomonads would be able to de- 

 termine natural or bound vitamin B12 unavailable to other microorganisms 

 as expressed by Hamilton et al. was not realized, since Ford found that 

 vitamin Bjo in sows' milk must first ])e liberated by a preliminary digestion 

 of the milk with papain. Comparing the vitamin B12 content of natural 

 materials as determined with Ochromonas and E. coli, Ford found that the 

 two methods gave similar values for cows' milk, beef liver extract, and fish 

 solubles; however, feces or urine gave significantly higher values by the 

 E. coli method. It appears that the chrysomonad Ochromonas malhamensis 

 assay for \dtamin B12 offers for the first time a method specific for the 

 cobalamins. The existence in nature of substances related to vitamin B12 

 which are inactive in animal nutrition but are active for the growth of 

 certain microorganisms presents obvious problems in the interpretation of 

 microbiological assays. These substances may be essentially absent from 

 certain materials, such as liver extract, in which case L. leichmannii or 

 E. coli may be used for the assay. However, if these substances are sub- 

 stantially present, as in the case of intestinal contents, the Ochromonas 

 assay should be used, unless the interfering substances are first removed 

 bj^ paper electrophoresis. The main disadvantages of the Ochromonas 

 method are the incubation time of 4 days and the general lack of familiarity 

 of most workers with this type of organism, 



c. Lactobacillus leichmannii (4797) 



Skeggs et al.,^^ finding the use of the L. lactis Dorner (8000) assay pro- 

 cedure "difficult and unpredictable," investigated L. leichmannii (4797) as 



3*'^ D. Baile, mimeograph distributed by the National Dairy Research Laboratories, 



Long Island, 1953. 

 3»« J. E. Ford, Brit. J Xatrition 7, 299 (1953). 

 39 H. R. Skeggs, J. W. Huff, L. D. Wright, and D. K. Bosshardt, J. Biul. Chem. 176, 



1459 (1948). 



