Estimation of the growth rate of mixed ruminal bacteria from short-term DNA radiolabeling

A method based on P-32-labeling of DNA in short-term incubations was developed for estimating the growth rate of mixed rumen bacteria. A freeze/thaw procedure was optimized to quantitatively disrupt mixed rumen bacteria and extract bacterial DNA. The preliminary enzymatic lysis step, with lysozyme rather than proteinase K, sodium lauroyl sarcosine, and, to a lesser extent, sodium dodecyl sulfate (SDS) strongly improved cell disruption and DNA recovery rates. Sodium deoxycholate, CHAPS or Triton X-100 had no significant effect. Increasing the number of cycles or lowering the freezing temperature from -20 degrees C to -50 degrees C had no effect on DNA extraction efficiency while setting the thawing temperature at +60 degrees C rather than +37 degrees C slightly increased DNA yield but also increased its contamination with RNA. The method finally selected led to the lysis of at least 93% of cells and to the extraction of 85% of bacterial DNA. The kinetics of in vitro P-32 incorporation into rumen bacteria DNA was then determined in batch incubations of strained rumen contents with no additional substrate. The curvilinear effects of the amount of P-32 and the incubation time (5-15 min) On the DNA radioactivity were investigated by applying a Doehlert experimental design and fitting a second order polynomial model to data. The DNA radioactivity was linearly related to time (p < 0.02) with other coefficients in the model being equal to zero(p > 0.20). The incorporation of P-32 into bacterial DNA was initiated approximately 70s after the start of incubation. Taking into account the accuracy of scintillation counting, 10-15min incubations, with 15 mu Ci P-32 and 10mL rumen contents per tube, appeared satisfactory for future studies. (C) 1998 Academic Press.