24 



THE ACTIXOMYCETES, Vol. I 



modified by Giolitti and Bertani and others. 

 Excellent differentiation can thu.s be ob- 

 tained for the vegetative and sporulating 

 growth. 



Electron microscope methods. The electron 

 microscope has opened a new field for the 

 detailed study of the finer structure of cells 

 of actinomycetes. References to the results 

 thus obtained are presented in Chapters 5 

 and 6, and elsewhere throughout this vol- 

 ume. 



Plate Methods 



The plate methods are the oldest and still 

 most reliable procedures for determining the 

 nature and measuring the abundance of the 

 microbiological, including the actinomycete, 

 population of such materials as soils, com- 

 posts, water, and milk. In making micro- 

 biological counts of such materials, the 

 actinomycetes are usually included with the 

 bacteria. More recently, selective media fa- 

 voring the preferential development of 

 actinomycetes have been devised, as by ad- 

 dition of sodium propionate to the plate 

 (Crook et al., 1950) or by addition of anti- 

 biotics. Certain purification procedures, such 

 as the use of various concentrations of sul- 

 furic acid, have also been utilized (Fellinger, 

 1931). 



Beginning with the work of Hiltner and 

 Stormer in 1902, actinomycetes came to be 

 recognized as a separate group of organisms, 

 distinct from the bacteria and fungi, and 

 were frecjuently so reported. The samples of 

 soil, compost, or other material are usually 

 suspended in sterile tap water, and various 

 dilutions with sterile tap water subseciuently 

 made. These dilutions are then plated out 

 on suitable agar or gelatin media. The plates 

 are usually incubated at 28-30° C and exam- 

 ined after 2 to 7 days. It is sometimes diffi- 

 cult to differentiate between bacterial and 

 actinomycete colonies, unless one has had 

 considerable experience^ with actinomycetes 

 or unless one uses microscopic magnifications 



to distinguish l)etween the two types of colo- 

 nies. 



At first, organic media were used to enu- 

 merate the abundance of actinomycetes in 

 the soil. Alore recently, synthetic media have 

 been employed with only small amounts of 

 a protein or polypeptide added, such as 0.005 

 per cent peptone, 0.025 per cent powdered 

 egg albumin, or 0.02 per cent casein. This 

 tends to prevent development of the more 

 rapidly growing and spreading bacterial and 

 fungal colonies and allow development of 

 the more slowly growing actinomycetes. The 

 \'arious water dilutions are so adjusted as 

 to allow a final count of only about 30 to 

 100 colonies per ordinary Petri plate. 



If one is interested in obtaining a great 

 variety of actinomycetes for isolation pur- 

 poses, rather than in large numbers of colo- 

 nies, synthetic media are best for plating 

 purposes. With asparagine, glutamic acid, or 

 sodium nitrate as a source of nitrogen, and 

 glycerol, starch, glucose, or a salt of an or- 

 ganic acid, such as malate, as a source of 

 carbon, a relatively simple medium can be 

 prepared. Such a medium will favor the de- 

 velopment of actinomycetes rather than of 

 fungi and bacteria. The colonies of actino- 

 mycetes produced on such media are cjuite 

 characteristic and can easily be recognized. 



Numerous modifications of the plate 

 methods have been proposed, depending on 

 such factors as the treatment of the soil, the 

 nature of the medium used, and the tem- 

 perature and length of the incubation period 

 (Rao and Subrahmanyan, 1929). Some meth- 

 ods are devised for the purpose of excluding 

 most of the bacteria and fungi. In others, an 

 attempt is made to distinguish l)etween the 

 abundance of viable spores and mycelial 

 fragments of the actinomycetes. Skinner 

 made use of the fact that, when shaken in 

 suspension, vegetative mycelium of actino- 

 mycetes breaks into viable fragments, which 

 are killed when shaking is prolonged (Table 

 3). He observed further that spores do not 



