856 ORDER VIII. MYXOBACTERALES 



merits, so far not analj'zed, and their cultivation accordingly requires the addition of pep- 

 tone or yeast extract to the medium containing the specific polysaccharide which they at- 

 tack. The polysaccharide-decomposing myxobacteria are all mesophilic, strictly aerobic 

 organisms and develop best in neutral or slightly alkaline media. 



For the vast majority of the fruiting myxobacters, however, the nutrient requirements 

 have not yet been analyzed, and cultivation of these organisms still remains a highly em- 

 pirical affair. The technique of enrichment developed by the Krzemieniewskis {op. cit., 5, 

 1927) provides the best general method for obtaining them from natural sources. Soil is 

 placed on blotting paper in a large Petri dish and covered with pellets of sterilized rabbit 

 dung. The soil is moistened with water, and the plates are incubated at 26° to 30° C. After 

 5 to 10 days, fruiting bodies begin to form on the surface of the dung pellets, from which 

 they can be picked and transferred to agar media for purposes of purification. Singh (op. 

 cit., 1, 1947, 1) recommends enriching by the use of non-nutrient agar, on the surface of 

 which a few loopfuls of the cells of a suitable true bacterium {e.g., Aerobacter sp.) are 

 spread in the form of a disc about 1 inch in diameter; this disc is then inoculated with soil, 

 compost, or a soil suspension, and the plate is incubated at 25° C. for 1 to 3 weeks. The myxo- 

 bacters develop at the expense of the eubacterial cells and form fruiting bodies on the sur- 

 face of the agar. The technique of Singh is excellent for the enrichment of a few of the more 

 common members of the Myxococcaceae but does not yield the wide diversity of forms ob- 

 tainable by the Krzemieniewskis' method. 



Following these methods of enrichment, pure cultures of some species can be obtained 

 fairly readily by repeated transfer on solid media of various compositions. Dung extract 

 agar, potato extract agar and peptone agar either with or without the addition of carbo- 

 hydrates have proved satisfactory. Perhaps the best general method is the use of agar con- 

 taining a suspension of the cells of true bacteria. Many of the fruiting myxobacters are 

 capable of Ij'sing the cells of true bacteria and other microorganisms and of developing at 

 the expense of the materials so liberated (Beebe, Jour. Bact., 4-0, 1940, 155; Snieszko, Hitch- 

 ner and McAllister, Jour. Bact., 4^, 1941, 26). Although the vegetative development of 

 myxobacters on bacterial agar is usually somewhat scanty, vigorous fructification occurs, 

 whereas on many of the other complex media which have been employed, the ability to form 

 fruiting bodies may be greatly reduced or lost completely. The nature of the materials lib- 

 erated from lysed true bacteria which promote fructification has not been determined. 

 Bacterial agar has the additional advantage for purposes of purification that the growth of 

 true bacteria, fungi and protozoa, invariably present in the original enrichments, is much 

 slighter than on other complex media. 



By the use of the methods described in the preceding paragraph, certain fruiting myxo- 

 bacters, particularly representatives of the Myxococcaceae, are readily isolated and main- 

 tained in pure culture. There are many species, however, which have not yet been obtained 

 in pure culture despite considerable efforts by a number of investigators. For Chondromyces 

 crocatus, the claim has been made (Pinoy, Ann. Inst. Past., 35, 1921, 487; Kiihlwein, Arch, 

 f. Mikrobiol., 14, 1948, 678) that growth occurs only in "symbiotic association" with other 

 microorganisms; this may simply reflect the ability of the species in question to lyse other 

 microorganisms and to obtain, thereby, nutrients not furnished by conventional complex 

 media. 



No precise quantitative studies on the nutrition of the fruiting myxobacters have been 

 reported, apart from the work of the Krzemieniewskis {op. cit., 15, 1937, 11) on the cellu- 

 lose-decomposing Sorangium spp. and that of Noren (Svensk. Bot. Tidskr., 46, 1952, 324). 

 The widespread ability of these forms to destroy and to develop at the expense of true bac- 

 teria and fungi suggests that their chief source of food in nature may be the cells of other 

 microorganisms, even though growth on simpler media is sometimes possible with pure 

 cultures. 



Relationships of the myxobacters. Thaxter {op. cit., 17, 1892, 389), whose work laid 

 the foundations for our knowledge of the fruiting my.xobacters, regarded these organisms 



