26 



THE ACTINOMYCETES, Vol. I 



strains was first suggested by Waksnian d 

 al. for obtaining highly potent antibiotic- 

 yielding strains from a particular antibi- 

 otic producing organism. It was also used by 

 Umezawa et al. (1949) for the isolation of 

 chloramphenicol-producing organisms. 



For the isolation and cultivation of mem- 

 bers of the genus Actinomyces, or the meso- 

 philic anaerobic forms, special methods have 

 to be employed. M. H. Gordon suggested 

 the use of ordinary nutrient broth to which 

 a few drops of fresh human blood have been 

 added. The material is inoculated into two 

 lots of blood broth, one of which is covered 

 by a layer of oil 1 cm deep. After incubation 

 for a few days at 37° C, the organism can be 

 seen growing, at the foot of the tube in 

 small white masses — like little puffballs. Ac- 

 cording to Gordon, growth occurs first in 

 the broth covered with oil; when other bac- 

 teria are also present the actinomyces may 

 appear first in the aerobic tube. 



Various other methods ha\'e been used for 

 the isolation of microaerophilic strains of 

 Actinomyces. The method proposed by Rose- 

 bury et al. (1944) for the isolation of this 

 organism from gingival scrapings was modi- 

 fied further by Ennever et al. (1949). A 

 tooth-bearing remo^'al appliance is used to 

 collect the plaque mass. The latter is re- 

 moved with a sterile, hooked blade and 

 placed in a micromortar containing O.Oo ml 

 of sterile saline with Triton A-20 at 1:200 

 dilution. The mass is triturated with a sterile 

 glass pestle, and one visible granule trans- 

 ferred by means of capillary pipette to 

 brain-heart infusion agar. Care is taken to 

 transfer as little saline as possible. The gran- 

 ule is streaked upon the agar, and plates are 

 incubated anaerobically in an atmosphere of 

 5 per cent COo for () days. 



Preserving Cultures of Actinomycetes 



The gi'eat variability of actinomycetes, 

 frequently accompanied by loss of desii-able 

 properties, makes it ne(;essary to dex'elop 



methods for the maintenance of cultures in 

 which the organisms will undergo the least 

 morphological or biochemical change. The 

 ability to form spores is an important desir- 

 able property to be conserved. 



Various methods are used for the preser- 

 xation of actinomycete cultures. Artificial 

 media, including both synthetic and organic, 

 have been used for maintaining cultures of 

 actinomycetes. As a rule, organic media have 

 been found more favorable than synthetic 

 media for maintaining the original morpho- 

 logical and cultural properties of actinomy- 

 cetes. 



R. Gordon of the Rutgers Institute of 

 Microbiology has reported her experiences 

 in comparing three methods for the preser- 

 vation of the culture collection of actinmy- 

 cetes at the Institute. 



1. Soil culture. Thirty-nine strains of 

 Streptomyces were added to sterile soil. All 

 of them were \iable a year after inoculation; 

 2 years after inoculation three of the 39 did 

 not grow; 3 years after inoculation, six of 

 the 39 did not grow. 



2. ISIineral oil. Twenty-four cultures of 

 Nocardia and Streptomyces were co^'ered with 

 sterile mineral oil. Three years later, 14 of 

 the cultures were viable and 10 were dead. 



3. Lyophilization. All strains of actino- 

 mycetes in the Institute's collection ha^'e 

 been lyophilized, and no difficulty in reviving 

 them has been experienced. In the regular 

 schedule of testing lyophilized cultures for 

 viability, 33 strains of actinomycetes have 

 grown after 5 years' storage; none have 

 failed to grow. 



Hartsell reported that the grisein-produc- 

 ing strain of S. griseus was ^-iable after 7 

 years' storage undcn- mincM'al oil and that 

 the streptomycin-producing strain of this 

 organism was \-iable after (> yeai's' storage 

 under the same conditions. 



Ilaynes et al. can-ied out extensive inves- 

 tigations on the preser\-ation of the collec- 

 tion of more than 2800 cultures at tlu^ Xorth- 



