PURE CULTURE STUDY OF SOIL BACTERIA 55 



conditions; actually, a number of strains may be obtained, which, on 

 separation, may often be mistaken for different species. 



The direct method of isolation suggested by Winogradsky 3 may also 

 be employed. This consists in adding a specific nutrient to a silica gel 

 plate, containing the necessary nutrients, and inoculating with particles 

 of soil. Only the organisms capable of acting upon the specific substrate 

 will develop on the plate in forms readily isolated. 



In the case of the majority of soil bacteria (especially the 

 heterotrophic forms), both aerobic or anaerobic, the plate method 

 will prove convenient for the isolation of the individual organisms 

 from colonies. The selective culture method in most cases and 

 the plate method in some cases yield only crude cultures of the 

 organisms. In the majority of cases these cultures are quite satisfac- 

 tory. 4 However, for biochemical studies and especially in the study 

 of life cycles of bacteria, it is advisable to obtain single-cell cultures of 

 the organisms. In most instances this is accomplished by the dilution 

 method, which may be accompanied by the transfer of a single cell by 

 the India ink 5 method or by means of a capillary pipette. 6 The first 

 consists in mixing a highly diluted culture with sterile India or China 

 ink, placing pin-point droplets of the mixture upon the surface of sterile 

 agar or gelatin, covering with sterile cover slips, examining with high- 

 power objective for the presence of a single cell, incubating, and finally 

 transferring or lifting the cover slip with adhering bacterial cell and 

 inoculating into sterile medium. The second method consists in 



3 Winogradsky, 1925 (p. 11). 



4 Lohnis, F. Studies upon the life cycles of the bacteria. I. Mem. Nat. Acad. 

 Sci. 16, 1921, No. 2, p. 39. 



6 Burri, R. Eine einfache Methode zur Reinziichtung von Bakterien unter 

 mikroskopischer Kontrolle des Ausgangs von der einzelnen Zelle. Centrbl. Bakt. 

 II, 20: 95-96. 1908; Das Tuschepunktverfahren. G. Fischer. Jena. 1909. 



6 Barber, M. A. On heredity in certain micro-organisms. Kansas Univ 

 Science Bull. 4: no. 1 48 p. 1907; Jour. Inf. Dis. 5: 379. 1908; 8: 348. 1911 

 The pipette method in the isolation of single microorganisms and in the inocula 

 tion of substances into living cells. Philippine Jour. Sci. B. 9: 307-360. 1914 

 The use of single cells method in obtaining pure cultures of anaerobes. Jour 

 Exp. Med. 32: 295. 1920; Shouten, S. L. Reinkulturen aus einer unter dem 

 Mikroskop isolierten Zelle. Ztschr. Wissensch. Mikrosk. 22: 10, 1907; 24: 258 

 1907; Hecker, F. A new model of double pipette holder and the technic for the 

 isolation of living organisms. Jour. Inf. Dis. 19: 305. 1916; Hort. Jour. Hyg 

 18: 361. 1920; Topley, W. W. C, Barnard, J. E., and Wilson, G. S. A new 

 method of obtaining cultures from single bacterial cells. Jour. Hyg. 20 : 221-226 

 1921; Malone, R. H., A simple apparatus for isolating single microorganisms 

 Jour. Path. Bact. 22: 222. 1918. 



