To improve accuracy and precision of NDF disappearance in vitro and bacterial fatty acid (FA) quantification by GLC-mass spectroscopy, ground alfalfa hay was weighed directly (DIR) into a 50-mL tube or was weighed into an ANKOM (Macedon, NY) filter bag (BAG), sealed, and placed in tubes. Using a mixed model with random block (replication), 24-h alfalfa NDF disappearance in vitro was greater (31.0 vs. 23.7%; P = 0.03) vs. BAG when NDF was analyzed with filter bags; NDF analysis of DIR with filter bags was not different vs. the filter paper procedure (30.1% vs 31.4%, P = 0.78). At 24 h, BAG pH was higher vs. DIR (6.86 vs. 6.42; P < 0.01), suggesting incomplete mixing with inocula. With 50:50 alfalfa:corn, NDF degradation by filter bag was lower than with filter paper (17.0% vs. 41.2%; P < 0.01). To prevent artifact FA peaks with GLC, 3 methods were assessed to extract particulate-associated bacteria (PAB) for FA analysis: (1) lowering the pH to 2.0 using acid (AC); (2) cold shocking (CS) with 2 ice-cold saline washes; and (3) storing at 4°C for 24 h (4C). All 3 methods were factorialized and compared with no extraction (NO; i.e., fluid-phase bacteria). With NO, 74% of the peaks from 2 standards of bacterial FA were recovered, but CS decreased (P < 0.01) recovery to 54%. Combining 4C and AC had the highest recovery (85%; P = 0.44 vs. NO), whereas combining CS and 4C had the lowest (34%; P = 0.04 vs. CS). A Poisson regression was used to examine the recovery of 22 peaks not present in the 2 standards. Compared with AC, 20 unknown peaks remained with NO and decreased (P < 0.01) to 13 with CS. Either 2 low temperature steps (LT) or a single high temperature step (HT) for methylation were tested after incubation with ¹³C-labeled Ile, Leu, and Val. The recovery of known standard peaks was 83% (P = 0.38 between methods). With HT, we detected 19 unknown peaks, which LT increased (P = 0.04) to 21. Ten unknown peaks were enriched with ¹³C for both LT and HT. Feed should be placed directly in the tube during incubation, and filter papers ensured accurate NDF degradation for mixed feed. Neither methylation nor PAB extraction methods examined herein introduced artifact FA peaks.