Estimating the Growth Rate of a Bacterial Species in a Complex Mixture by Hybridization of Genomic DNA

Abstract

> Abstract Advances in molecular techniques have enabled new approaches to identifying bacteria. However, once identified, there is no quantitative information on the in situ growth rate of the species, mainly because the technology has not been available. The quantitative incorporation of [methyl-3H]thymidine into dividing bacteria is coupled with a molecular (hybridization) method, to determine the growth rate of bacterial species in situ. The basis of this molecular method is a reverse gene probe-natural populations are labeled in situ with [methyl-3H]thymidine. The probe (3H-Tdr-DNA) is captured, using a hybridization procedure, on a positively charged nylon membrane on which is attached non-labeled target DNA. Two bacterial species, Bacillus cereus and Zoogloea ramigera, were used to demonstrate the principle in laboratory cultures and in a municipal activate sludge treatment process. The DNA of the dividing bacteria in activated sludge was radioactively labeled with [methyl-3H]thymidine, and the DNA of Z. ramigera was recovered using a DNA hybridization method. The recovered radioactively-labeled DNA was used to estimate the growth rate (0.03 x 10(9) cells . ml-1 . h-1) of Z. ramigera in situ. Simultaneously applying these two powerful molecular-based methods could potentially be used to study bacterial population dynamics in situ.