Deoxynucleic acid in some gametes and embryos 



by 



E. HOFF-J0RGENSEN 



Biokemisk Institut, Kobenhavns Universitet 



The sensitivity and specificity of the known chemical methods for the determination 

 of DNA seem to be insufficient for the estimation of the minute concentration of DNA 

 present in eggs and in embryos during the early stages of development. A microbio- 

 logical assay method, which is very sensitive and highly specific, has been used in the 

 investigations reported in this paper. 



ASSAY METHOD 



Principle. The lactic acid bacterium Thermo bacterium acidophilus R 26 Orla Jensen 

 (ATCG 1 1506) requires a deoxynucleoside as an essential growth factor. Neither 

 vitamin B 12 nor any other of many substances tested can replace the requirement for 

 a deoxynucleoside. This organism therefore can be used as a test organism for micro- 

 biological assays of deoxynucleosides and also of DNA after depolymerization of the 

 DNA. (Hoff-Jorgensen, 1952). 



Stock cultures are maintained in the following medium by weekly transfer : o • 1 g. 

 of cysteine and 05 g. of yeast extract (Difco) are dissolved in 100 ml. skimmed milk, 

 at pH 6-8. The milk medium is dispensed in 2 ml. quantities to test-tubes (100 x 

 10 mm.). About 01 g. of CaC0 3 is added to each tube. The tubes are plugged with 

 cotton, autoclaved at 120 C. for 10 min., inoculated with a wire loop, incubated 

 for 24 hr. at 37 C, and stored in a refrigerator. 



Inoculum medium. 50 ml. of the double-strength basal medium are mixed with 50 

 ml. of water. The minimum amount of peptone (e.g. about 5 mg. Difco per ml.) 

 which gives maximum growth is added. The medium is dispensed in 5 ml. quantities 

 to 15 ml. centrifuge tubes, each containing a glass bead. The tubes are plugged with 

 cotton, autoclaved at 120 C. for 10 min., and stored in a refrigerator. Fresh inoculum 

 medium is prepared every month. 



Inoculum. A small loopful of the stock milk culture is transferred to a tube contain- 

 ing 5 ml. of the inoculum medium. After incubation at 37 G. for 20-24 hr., the 

 cells are centrifuged, washed once with 10 ml. of sterile saline, and resuspended in 

 10 ml. of sterile saline. One small drop of this suspension is used to inoculate each 

 assay tube. 



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