## 3 Results
### Optimization of DNA Extraction  
Broth cultures and bacterial colonies for several different genera and species from our collection were tested for DNA liberation using (a) boiling, (b) sonication, (c) glass-bead beating, (d) enzymatic extraction [34], (e) lipid extraction with 1-ethyl-3-methylimidazolium acetate (C2 minOAc) [12], and (f) NaOH lysis with sodium acetate neutralization [35]. Bacteria species tested included _Enterococcus faecalis_ (three isolates), _Enterococcus ceorum_, _Escherichia coli_ (two isolates), _Escherichia fergusonii_, _Staphylococcus aureus_ (two isolates), _Staphylococcus gallinarum_, _Staphylococcus nepalensis_, _Staphylococcus simulans_, _Staphylococcus saprophyticus_, _Staphylococcus lentus_, _Staphylococcus squorum_, _Staphylococcus colntii_, _Streptococcus agalaciae_, and _Salmonella enterica_. Of the six methods tested, the boiling approach was the most reliable, as methods (b)-(f) were largely ineffective for many of the Gram-positive organisms. Particularly problematic for the boil method were the Enterococcus isolates, of which from multiple boil extractions only a few gave positive PCR for 16S rDNA amplification. NaOH lysis is a common method for bacterial lysis, especially for plasmid isolation [13], and procedures have been published for extraction of plant, human, bacterial, and yeast DNA for PCR using NaOH and heat [17; 19; 24; 36], so we investigated how to best process bacterial NaOH extracts for subsequent PCR. We first examined what the threshold concentration of NaOH is for inhibition of a standard qPCR reaction for 16S rDNA using \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Day** & **Minutes** & **Total CFU** & **Extraction** & **qPCR Positive** & **Avg Ct 16S** \\ \hline
17 & 3 & 4100 & Boil & Yes & ND \\
17 & 10 & 8825 & Boil & Yes & ND \\
20 & 15 & 12,205 & Boil & NS & \\
20 & 30 & 21,050 & Boil & NS & \\
21 & 20 & 4500 & Bead beating & NS & \\
27 & 20 & 35,200 & Sonication & NS & \\
29 & 20 & 4000 & Sonication & NS & \\
35 & 20 & 17,200 & Sonication & NS & \\
42 & 20 & 6000 & NaOH + sodium acetate & NS & \\
42 & 20 & 6600 & NaOH + MB & Yes & 23.9 \(\pm\) 0.03 \\
44 & 20 & 20,000 & Boil & NS & \\
44 & 20 & 29,300 & NaOH + MB & Yes & 22.0 \(\pm\) 0.1 \\
46 & 20 & 46,400 & Boil & NS & \\
46 & 20 & 20,000 & NaOH + C2 minOAc & Yes & 20.8 \(\pm\) 0.1 \\
48 & 20 & 16,000 & C2 minOAc & NS & \\
48 & 20 & 20,000 & NaOH + MB & Yes & 23.1 \(\pm\) 0.1 \\
50 & 20 & 8000 & NaOH + MB & Yes & 24.4 \(\pm\) 0.2 \\
50 & 20 & 12,000 & NaOH + MB & Yes & 20.5 \(\pm\) 2.4 \\
52 & 20 & 9000 & NaOH + MB & Yes & 23.2 \(\pm\) 0.6 \\
52 & 20 & 5000 & NaOH + MB & Yes & 23.3 \(\pm\) 0.6 \\
54 & 20 & 12,200 & NaOH + MB & Yes & 23.2 \(\pm\) 0.5 \\
54 & 20 & 12,500 & NaOH + MB & Yes & 17.4 \(\pm\) 2.0 \\
56 & 20 & 20,000 & NaOH + MB & Yes & 19.7 \(\pm\) 2.4 \\
56 & 20 & 52,900 & NaOH + MB & Yes & 21.8 \(\pm\) 0.3 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Air samples, bacterial counts, extraction method, and qPCR results during a 56-day lameness trial in spring 2021. Samples were collected using the impinger on the indicated day for a given sampling duration (minutes). For each sample, the resuspended pellet was divided for serial dilution for plate counts and for DNA extraction. Total CFU is the computed total viable bacteria in the original sample. Extraction indicates the method used (see text) for that sample where NaOH + MB is the NaOH extraction with magnetic bead capture and elution. qPCR positive indicates whether that extract produced a specific product for qPCR-HRM, where NS signifies no specific amplification based on HRM. Avg Ct 16S presents the average Ct (\(\pm\)SEM) from triplicate samples using 8F \(\times\) 936R 16S primers, where ND (not determined) indicates that species-specific qPCR primers were used. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H\({}_{2}\)O, which performed as expected. purified _S. agnetis_ DNA as the target.