Myxobacterias

Journal of Microbiological Methods 54 (2003) 21 – 27 www.elsevier.com/locate/jmicmeth

Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains
LiPing Zhang a,b,*, HaiYing Wang a,b, XiaoMei Fang a, Erko Stackebrandt c, YanBo Ding a,b
b a College of Life Science, Ministry of Education, Hebei University, Baoding, Hebei, People’sRepublic of China Branch of the Key Laboratory for Microbial Resources, Ministry of Education, Hebei University, Baoding, Hebei, People’s Republic of China c DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Brunswick, Germany

Received 8 October 2002; received in revised form 11 December 2002; accepted 11 December 2002

Abstract By using baiting techniques and differentpurification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced tosterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of somemyxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study. D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Myxobacteria; Isolation; Purification; Fruiting body

1. Introduction Myxobacteria, common in many soil types,reveal an astonishing and complex life cycle and are by far the most sophisticated prokaryotes (Li and Zhang, 1993; Dworkin and Kaiser, 1985; Shimkets, 1990). Moreover, many myxobacterial strains can produce
* Corresponding author. Branch of the Key Laboratory for Microbial Resources, Ministry of Education, Hebei University, Baoding, Hebei, People’s Republic of China. Tel.: +86-3125079696; fax:+86-312-5065533. E-mail address: zhlping@sina.263.com (L.P. Zhang).

secondary metabolites, most of which have practical applications, stimulating an even broader interest of biologists (Li et al., 1998; Reichenbach et al., 1988; Reichenbach and Hofle, 1989; Reichenbach and ¨ Dworkin, 1992; Beyer et al., 1999). The method of myxobacterial isolation and purification is different from that of otherbacteria because of their specific characteristics. Myxobacteria, which can move on solid medium because of slime production, easily carry contaminates, e.g., fungi and soil amoebae (Reichenbach and Dworkin, 1992). Since the first description of a myxobacterium, Myxococcus fulvus, (Thaxter, 1892), bacteriologists have tried to

0167-7012/03/$ - see front matter D 2003 Elsevier Science B.V. Allrights reserved. doi:10.1016/S0167-7012(02)00257-9

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Table 1 Isolation and purification scheme of myxobacteria Method of purification Transferring fruiting body Freezing and heating Purification with antibiotics Freezing, heating and multiple treatment with antibiotics Streak culture Transferring cover slip Inoculation with sediment growth Purification with crystal violeta Second baitingtechniquea Using micromanipulatora
a

Number of isolates 115 69 42 19

Number of pure culture 3 1 1 1

Proportion of pure culture (%) 2.6 1.5 2.4 5.3

Object of treatment Fruiting body Fruiting body Fruiting body Fruiting body

Period of purification 1 – 2 months 1 – 2 months 1 – 2 months 1 – 2 months

Type of strain Formation of large fruiting bodies Formation of mature fruiting...