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 d eveloped for estimating the growth rate of mixed rumen bacteria. A fre eze/thaw procedure was optimized to quantitatively disrupt mixed rumen bacteria and extract bacterial DNA. The preliminary enzymatic lysis s tep, with lysozyme rather than proteinase K, sodium lauroyl sarcosine, and, to a lesser extent, sodium dodecyl sulfate (SDS) strongly improv ed cell disruption and DNA recovery rates. Sodium deoxycholate, CHAPS or Triton X-100 had no significant effect. Increasing the number of cy cles or lowering the freezing temperature from -20 degrees C to -50 de grees C had no effect on DNA extraction efficiency while setting the t hawing temperature at +60 degrees C rather than +37 degrees C slightly increased DNA yield but also increased its contamination with RNA. Th e method finally selected led to the lysis of at least 93% of cells an d to the extraction of 85% of bacterial DNA. The kinetics of in vitro P-32 incorporation into rumen bacteria DNA was then determined in batc h 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 D oehlert experimental design and fitting a second order polynomial mode l to data. The DNA radioactivity was linearly related to time (p < 0.0 2) with other coefficients in the model being equal to zero(p > 0.20). The incorporation of P-32 into bacterial DNA was initiated approximat ely 70s after the start of incubation. Taking into account the accurac y of scintillation counting, 10-15min incubations, with 15 mu Ci P-32 and 10mL rumen contents per tube, appeared satisfactory for future stu dies. (C) 1998 Academic Press.