Current protocols in microbiology

Isolation and classification of Bdellovibrio and like organisms.

PMID 22875568


Bdellovibrio and like organisms (BALOs) are obligate predators of Gram-negative bacteria. BALOs are isolated as plaques growing at the expense of their prey and are cultivated as two-member cultures. The growth cycle is composed of an extracellular attack phase and an intraperiplasmic elongation and replication phase. However, there are methods for obtaining host-independent (HI) mutants that grow without prey on rich media. BALOs are commonly found in the environment but generally constitute small populations; therefore, their isolation may require enrichment steps. Contamination by other bacteria during isolation necessitates efficient separation between the smaller BALO cells from the majority of larger bacteria. BALOs can also be directly detected and quantified in environmental samples using specific PCR. Synchronous cultures of both wild-type and HI derivatives can be obtained to study the different growth phases. These can be further separated by centrifugation. Classification is based on 16S rDNA analysis. Protocols relevant to these aspects of BALO detection, isolation, growth, classification, and quantitation are presented in this unit.