Table 1.
Homogenized stool subsamples have less variance compared to non-homogenized stool subsamples.
Mean, standard deviation and variance values for each of the bacterial taxa detected via qPCR collected from four different subjects`stool subsamples that had been homogenized on liquid nitrogen or not homogenized. Levene's p-values are reported where a significant p-value denotes whether variance is significantly different between groups; the p value is italicized if the non-homogenized subsamples (underlined) have significantly higher variance compared to the homogenized subsamples. The averaged mean, standard deviation and variance between the four subjects is included in the right column (bolded text; large variance seen in non-homogenized subsamples).
Fig 1.
Bacterial taxa abundance differ in the inside compared to the outside microenvironments of stool.
A single stool was subsampled five times from the inside environment and five times from the outer environment, DNA was extracted and used to compare bacterial taxa via qPCR. Firmicutes and Bifidobacteria spp. were decreased in the outside microenvironment compared to the inside microenvironment of the stool. *, p = 0.03.
Fig 2.
The mean variances of bacterial taxa are lower in homogenized subsamples compared to non-homogenized stool subsamples.
The variance values were calculated for each of the bacterial taxa tested using qPCR from five subsamples where the stool was homogenized by crushing on liquid nitrogen into a fine powder and compared to stool not homogenized. The mean variance was calculated by taking the average of the variances determined for each bacteria taxa from the four subjects that were examined.
Fig 3.
Stool storage at room temperature alters the abundance of bacterial taxa.
Ten subsamples from the same stool were either stored at room temperature for 15 minutes or for 30 minutes, followed by DNA extraction and used to compare bacterial taxa via qPCR. Bacteroidetes detection decreased after 30 minutes at room temperature, whereas Firmicutes increased after 30 minutes. *, p > 0.05.
Fig 4.
Stool storage in a domestic frost-free freezer affects the abundance of bacterial taxa.
A homogenized stool sample was stored in a domestic freezer for 0, 3,7,14, and 30 days, DNA was extracted and used for qPCR to compare bacterial taxa abundance. All bacterial taxa tested showed some change in abundance by day 30. *, p < 0.05.
Fig 5.
Freeze-thawing stool up to four times does not affect bacterial taxa abundance.
A homogenized stool sample was subject to a series of up to five consecutive full freeze-thaw cycles, DNA was extracted and used for qPCR to compare bacteria taxa abundance. There were no changes of bacterial taxa abundance until the 5th freeze thaw cycle where Bacteroidetes were increased and Enterobactericeae decreased. *, p < 0.05.
Table 2.
RNAlater reduces DNA yields from stool samples.
DNA extracted from samples not stored in RNAlater was significantly greater (p<0.0001) than samples stored in RNAlater.
Fig 6.
Stool stored in nucleic acid stabilizer prior to processing did not protect against bacterial taxa changes.
Stool was either stored with or without RNAlater (Qiagen) prior to freezing and then processing stool samples. Detection of Firmicutes, Lactobacillus spp. and Bifidobacteria spp. was reduced after storage in RNAlater. *, p = 0.05.
Table 3.
List of primers used in this study.