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

A method based on 32P-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 32P 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 32P 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 32P into bacterial DNA was initiated approximately 70 s after the start of incubation. Taking into account the accuracy of scintillation counting, 10-15 min incubations, with 15 microCi 32P and 10 mL rumen contents per tube, appeared satisfactory for future studies.